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Journal articles on the topic '3C-seq'
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Tanizawa, Hideki, and Ken-ichi Noma. "Unravelling global genome organization by 3C-seq." Seminars in Cell & Developmental Biology 23, no. 2 (April 2012): 213–21. http://dx.doi.org/10.1016/j.semcdb.2011.11.003.
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DeMaere, Matthew Z., and Aaron E. Darling. "Deconvoluting simulated metagenomes: the performance of hard- and soft- clustering algorithms applied to metagenomic chromosome conformation capture (3C)." PeerJ 4 (November 8, 2016): e2676. http://dx.doi.org/10.7717/peerj.2676.
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BackgroundChromosome conformation capture, coupled with high throughput DNA sequencing in protocols like Hi-C and 3C-seq, has been proposed as a viable means of generating data to resolve the genomes of microorganisms living in naturally occuring environments. Metagenomic Hi-C and 3C-seq datasets have begun to emerge, but the feasibility of resolving genomes when closely related organisms (strain-level diversity) are present in the sample has not yet been systematically characterised.MethodsWe developed a computational simulation pipeline for metagenomic 3C and Hi-C sequencing to evaluate the accuracy of genomic reconstructions at, above, and below an operationally defined species boundary. We simulated datasets and measured accuracy over a wide range of parameters. Five clustering algorithms were evaluated (2 hard, 3 soft) using an adaptation of the extended B-cubed validation measure.ResultsWhen all genomes in a sample are below 95% sequence identity, all of the tested clustering algorithms performed well. When sequence data contains genomes above 95% identity (our operational definition of strain-level diversity), a naive soft-clustering extension of the Louvain method achieves the highest performance.DiscussionPreviously, only hard-clustering algorithms have been applied to metagenomic 3C and Hi-C data, yet none of these perform well when strain-level diversity exists in a metagenomic sample. Our simple extension of the Louvain method performed the best in these scenarios, however, accuracy remained well below the levels observed for samples without strain-level diversity. Strain resolution is also highly dependent on the amount of available 3C sequence data, suggesting that depth of sequencing must be carefully considered during experimental design. Finally, there appears to be great scope to improve the accuracy of strain resolution through further algorithm development.
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Chen, Yong, Yunfei Wang, Xin Liu, Jian Xu, and Michael Q. Zhang. "Model-based analysis of chromatin interactions from dCas9-Based CAPTURE-3C-seq." PLOS ONE 15, no. 7 (July 31, 2020): e0236666. http://dx.doi.org/10.1371/journal.pone.0236666.
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Magnitov, Mikhail D., Veronika S. Kuznetsova, Sergey V. Ulianov, Sergey V. Razin, and Alexander V. Tyakht. "Benchmark of software tools for prokaryotic chromosomal interaction domain identification." Bioinformatics 36, no. 17 (August 27, 2020): 4560–67. http://dx.doi.org/10.1093/bioinformatics/btaa555.
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Abstract Motivation The application of genome-wide chromosome conformation capture (3C) methods to prokaryotes provided insights into the spatial organization of their genomes and identified patterns conserved across the tree of life, such as chromatin compartments and contact domains. Prokaryotic genomes vary in GC content and the density of restriction sites along the chromosome, suggesting that these properties should be considered when planning experiments and choosing appropriate software for data processing. Diverse algorithms are available for the analysis of eukaryotic chromatin contact maps, but their potential application to prokaryotic data has not yet been evaluated. Results Here, we present a comparative analysis of domain calling algorithms using available single-microbe experimental data. We evaluated the algorithms’ intra-dataset reproducibility, concordance with other tools and sensitivity to coverage and resolution of contact maps. Using RNA-seq as an example, we showed how orthogonal biological data can be utilized to validate the reliability and significance of annotated domains. We also suggest that in silico simulations of contact maps can be used to choose optimal restriction enzymes and estimate theoretical map resolutions before the experiment. Our results provide guidelines for researchers investigating microbes and microbial communities using high-throughput 3C assays such as Hi-C and 3C-seq. Availability and implementation The code of the analysis is available at https://github.com/magnitov/prokaryotic_cids. Supplementary information Supplementary data are available at Bioinformatics online.
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Golov, Arkadiy K., Sergey V. Ulianov, Artem V. Luzhin, Ekaterina P. Kalabusheva, Omar L. Kantidze, Ilya M. Flyamer, Sergey V. Razin, and Alexey A. Gavrilov. "C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries." Methods 170 (January 2020): 48–60. http://dx.doi.org/10.1016/j.ymeth.2019.06.022.
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Hołówka, Joanna, and Małgorzata Płachetka. "Structure of bacterial chromosome: An analysis of DNA-protein interactions in vivo." Postępy Higieny i Medycyny Doświadczalnej 71 (December 8, 2017): 0. http://dx.doi.org/10.5604/01.3001.0010.6696.
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According to recent reports, bacterial chromosomes exhibit a hierarchical organization. The number of proteins that bind DNA are responsible for local and global organization of the DNA ensuring proper chromosome compaction. Advanced molecular biology techniques combined with high-throughput DNA sequencing methods allow a precise analysis of bacterial chromosome structures on a local and global scale. Methods such as in vivo footprinting and ChIP-seq allow to map binding sites of analyzed proteins in certain chromosomal regions or along the whole chromosome while analysis of the spatial interactions on global scale could be performed by 3C techniques. Additional insight into complex structures created by chromosome-organizing proteins is provided by high-resolution fluorescence microscopy techniques.
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Ferreira, Leonardo M. R., Torsten B. Meissner, Tarjei S. Mikkelsen, William Mallard, Charles W. O’Donnell, Tamara Tilburgs, Hannah A. B. Gomes, et al. "A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface." Proceedings of the National Academy of Sciences 113, no. 19 (April 13, 2016): 5364–69. http://dx.doi.org/10.1073/pnas.1602886113.
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HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal–fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation.
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Stadhouders, Ralph, Suleyman Aktuna, Supat Thongjuea, Ali Aghajanirefah, Farzin Pourfarzad, Wilfred van Ijcken, Boris Lenhard, et al. "HBS1L-MYB intergenic Variants Modulate Fetal Hemoglobin Via Long-Range MYB Enhancers." Blood 122, no. 21 (November 15, 2013): 43. http://dx.doi.org/10.1182/blood.v122.21.43.43.
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Abstract Three quantitative trait loci (QTLs) modifying fetal hemoglobin (HbF) levels have been identified, and these have been shown to have a predictive value of disease severity in β thalassemia and sickle cell disease in diverse ethnic groups. One of the HbF QTLs which consists of a set of common intergenic single nucleotide polymorphisms (SNPs) in the HBS1L-MYB intergenic region on chromosome 6q23, has also been consistently identified as highly associated with clinically important human erythroid traits. Despite extensive genetic evidence, a clear mechanistic basis for the association between the intergenic SNPs and erythroid biology has remained elusive, although the two flanking genes (HBS1L and MYB) are candidate target genes. Here, we set out to characterize the regulatory potential of the human HBS1L-MYB intergenic region in detail and to investigate its functional impact on the erythroid phenotype-associated variants. We profiled chromatin occupancy of the key erythroid LDB1 transcription factor (TF) complex in primary human erythroid progenitors (HEPs) using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq) and quantitative PCR (ChIP-qPCR). We detected an intergenic cluster containing 7 binding sites for the LDB1–complex, characterized by strong binding and co-occupancy of core complex proteins LDB1, GATA1, TAL1 and ETO2. One of these sites was co-occupied by the erythroid-specific TF KLF1, a protein which was also found to bind the murine intergenic region. Depletion of LDB1, TAL1 and KLF1 in K562 cells using RNA interference resulted in a specific downregulation of MYB expression while leaving HBS1L levels unaffected, demonstrating that the erythroid TFs occupying the intergenic enhancers are required for MYB expression. Using chromosome conformation capture (3C) coupled to high-throughput sequencing (3C-Seq), we profiled higher order chromatin structure within the locus, and detected several strong chromatin co-associations between the MYB promoter and intergenic sequences, almost all of which correlated with TF binding. The binding activities were distributed over a conserved core region identical to a 24-kb interval containing genetic variants in strong genetic association with erythroid traits in human populations. This block of SNPs is referred to as HBS1L-MYB intergenic polymorphisms block-2 (HMIP-2). The SNPs within HMIP-2 clustered in 2 regions positioned directly under the 2 LDB1-complex ChIP-Seq peaks, at -84 and -71 kb from the MYB transcription start site. Using allele-specific ChIP in K562 cells, we observed diminished (25-50%) binding of LDB1, GATA1, TAL1 and KLF1 to the minor rs66650371 allele, showing that rs66650371 affects local TF binding. An allele-specific 3C analysis also showed reduced interactions between the minor rs66650371 allele at -84 and MYB. We validated and expanded observations made in K562 cells using primary human cells: 1. HEPs from high HbF individuals homozygous for all minor alleles of the phenotype-associated -84kb and -71kb intergenic variants in the conserved core (‘SNP/SNP’) showed consistently lower MYB levels throughout phase II of the culture as compared to wildtype control cells (‘WT/WT’); 37% lower MYB on average; 2. ChIP experiments on SNP/SNP and WT/WT HEPs showed reduced binding of GATA1 and KLF1 to the -84 and -71 regulatory elements (containing the associated variants), the results were further confirmed by allele-specific ChIP assays in SNP/WT HEPs; 3. 3C-qPCR assays on cultured SNP/SNP and WT/WT cells demonstrated diminished looping between the -84 element and the MYB promoter in SNP/SNP individuals; 4. SNP/WT HEPs also showed allelic imbalance of MYB but not HBS1L transcripts when compared to controls (WT/WT and SNP/SNP HEPs). In conclusion, we show that the HBS1L-MYB gene-free interval contains distal enhancer elements that interact with MYB, a critical regulator of erythroid development and HbF levels. Key variants in the intergenic interval affect MYB expression by reducing TF binding to its regulatory elements and disrupting long-range enhancer-gene interaction. Our study identifies the first causal link between the 6q23 HbF QTL and MYB regulation, provides novel insights into the molecular control of clinically important haematological traits and adds another layer of complexity to the regulation of MYB, suggesting potential targets for therapeutic intervention. Disclosures: Thein: Sangart: Consultancy; Shire: Research Funding; Novartis: Speakers Bureau.
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Yu, Miao, Armen Abnousi, Yanxiao Zhang, Guoqiang Li, Lindsay Lee, Ziyin Chen, Rongxin Fang, et al. "SnapHiC: a computational pipeline to identify chromatin loops from single-cell Hi-C data." Nature Methods 18, no. 9 (August 26, 2021): 1056–59. http://dx.doi.org/10.1038/s41592-021-01231-2.
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AbstractSingle-cell Hi-C (scHi-C) analysis has been increasingly used to map chromatin architecture in diverse tissue contexts, but computational tools to define chromatin loops at high resolution from scHi-C data are still lacking. Here, we describe Single-Nucleus Analysis Pipeline for Hi-C (SnapHiC), a method that can identify chromatin loops at high resolution and accuracy from scHi-C data. Using scHi-C data from 742 mouse embryonic stem cells, we benchmark SnapHiC against a number of computational tools developed for mapping chromatin loops and interactions from bulk Hi-C. We further demonstrate its use by analyzing single-nucleus methyl-3C-seq data from 2,869 human prefrontal cortical cells, which uncovers cell type-specific chromatin loops and predicts putative target genes for noncoding sequence variants associated with neuropsychiatric disorders. Our results indicate that SnapHiC could facilitate the analysis of cell type-specific chromatin architecture and gene regulatory programs in complex tissues.
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Wang, Hongfang, Yumi Yashiro-Ohtani, Chongzhi Zang, Yinling Joey Wong, Will Bailis, Birgit Knoechel, Bradley Bernstein, et al. "Alternative Super-Enhancer States Determine MYC Sensitivity to Notch and Brd4 Inhibitors in T Lymphoblastic Leukemia/Lymphoma." Blood 124, no. 21 (December 6, 2014): 863. http://dx.doi.org/10.1182/blood.v124.21.863.863.
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Abstract Gain-of-function NOTCH1 mutations are oncogenic drivers in a high fraction of T-cell lymphoblastic leukemia/lymphoma (T-LL). These mutations variously cause increased production or stabilization of the free intracellular domain of NOTCH1, which regulates gene expression by forming a transcription complex with the DNA-binding factor RBPJ and coactivators of the MAML family. Using expression profiling and ChIP-seq, we have shown that NOTCH1/RBPJ complexes activate most target genes by binding to super-enhancers, large regulatory elements that switch on transcription through long-range interactions with gene promoters. MYC is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates MYC is unknown. To understand which regulatory element(s) regulate MYC expression, we used chromatin conformation capture (3C) assays to test the interaction between putative enhancer(s) and the MYC promoter in T-LL cell lines, and reporter gene assays to confirm enhancer function of candidate sites. We identified a distal site located >1 Mb 3’ of human and murine MYC termed the Notch-dependent MYC enhancer (NDME) that binds Notch transcription complexes and physically interacts with the MYC proximal promoter. An ~1 kb DNA fragment containing this site activates a luciferase reporter gene in a Notch-dependent fashion in T-LL cells but not in heterologous cell types. The Notch binding site lies within a large enhancer region (>600 kb in breadth) containing multiple discrete H3K27ac peaks. Remarkably, acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer region and the MYC promoter that correlate with NOTCH1/RBPJ complex binding and MYC expression. T-LL cells selected for resistance to gamma-secretase inhibitors (GSIs) exhibit epigenetic silencing of the NDME and loss of NDME looping interactions with the MYC promoter, yet maintain MYC expression. 3C analysis of GSI resistant cells shows preferential interaction between the MYC promoter and a more 3’ enhancer element recently described as a BRD4-dependent regulator of MYC expression in acute myeloid leukemia cells. In line with this observation, BRD4 antagonists are potent inhibitors of MYC expression in GSI resistant T-LL cells but not GSI-sensitive cells. We also studied a case of Notch-mutated early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL). ChIP-Seq analysis of the leukemic blasts revealed an “AML-like” MYC enhancer chromatin state, and as predicted from our analysis of cell lines, the blasts rapidly down-regulated MYC in response to BRD4 inhibitor but not in response to GSI. These findings suggest that specific MYC chromatin states predict responsiveness to Notch and BRD4 inhibitors, and provide a rationale for use of Notch and BRD4 inhibitor combinations in Notch-mutated leukemias. Disclosures No relevant conflicts of interest to declare.
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Kim, Ahran, Dahye Yoon, Yunjin Lim, Heyong Jin Roh, Suhkmann Kim, Chan-Il Park, Heui-Soo Kim, Hee-Jae Cha, Yung Hyun Choi, and Do-Hyung Kim. "Co-Expression Network Analysis of Spleen Transcriptome in Rock Bream (Oplegnathus fasciatus) Naturally Infected with Rock Bream Iridovirus (RBIV)." International Journal of Molecular Sciences 21, no. 5 (March 2, 2020): 1707. http://dx.doi.org/10.3390/ijms21051707.
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Rock bream iridovirus (RBIV) is a notorious agent that causes high mortality in aquaculture of rock bream (Oplegnathus fasciatus). Despite severity of this virus, no transcriptomic studies on RBIV-infected rock bream that can provide fundamental information on protective mechanism against the virus have been reported so far. This study aimed to investigate physiological mechanisms between host and RBIV through transcriptomic changes in the spleen based on RNA-seq. Depending on infection intensity and sampling time point, fish were divided into five groups: uninfected healthy fish at week 0 as control (0C), heavy infected fish at week 0 (0H), heavy mixed RBIV and bacterial infected fish at week 0 (0MH), uninfected healthy fish at week 3 (3C), and light infected fish at week 3 (3L). We explored clusters from 35,861 genes with Fragments Per Kilo-base of exon per Million mapped fragments (FPKM) values of 0.01 or more through signed co-expression network analysis using WGCNA package. Nine of 22 modules were highly correlated with viral infection (|gene significance (GS) vs. module membership (MM) |> 0.5, p-value < 0.05). Expression patterns in selected modules were divided into two: heavy infected (0H and 0MH) and control and light-infected groups (0C, 3C, and 3L). In functional analysis, genes in two positive modules (5448 unigenes) were enriched in cell cycle, DNA replication, transcription, and translation, and increased glycolysis activity. Seven negative modules (3517 unigenes) built in this study showed significant decreases in the expression of genes in lymphocyte-mediated immune system, antigen presentation, and platelet activation, whereas there was significant increased expression of endogenous apoptosis-related genes. These changes lead to RBIV proliferation and failure of host defense, and suggests the importance of blood cells such as thrombocytes and B cells in rock bream in RBIV infection. Interestingly, a hub gene, pre-mRNA processing factor 19 (PRPF19) showing high connectivity (kME), and expression of this gene using qRT-PCR was increased in rock bream blood cells shortly after RBIV was added. It might be a potential biomarker for diagnosis and vaccine studies in rock bream against RBIV. This transcriptome approach and our findings provide new insight into the understanding of global rock bream-RBIV interactions including immune and pathogenesis mechanisms.
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Houtman, M., X. Ge, A. Mcgovern, K. Klein, G. Orozco, M. Frank Bertoncelj, M. Marks, et al. "OP0016 IDENTIFICATION OF FUNCTIONAL VARIANTS IN THE RHEUMATOID ARTHRITIS ASSOCIATED JAZF1 LOCUS IN SYNOVIAL FIBROBLASTS." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 9.1–9. http://dx.doi.org/10.1136/annrheumdis-2021-eular.1400.
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Background:Over the past decade, genome wide association studies (GWAS) have identified the JAZF1 locus as a risk locus for several autoimmune diseases, including rheumatoid arthritis (RA)1. However, the exact causal variants in the JAZF1 locus and their underlying regulatory events contributing to RA are still not known. Here, we focus on the effect of these variants on gene expression in synovial fibroblasts (SF).Objectives:To characterize the functional consequences of RA-causal variants in the JAZF1 locus in SF.Methods:Genetic fine-mapping of RA loci was conducted by computing sets of credible variants driving GWAS signals. These credible variant sets were integrated with DNA architecture (ChIP-seq), 3D chromatin interactions (3C, HiC and capture HiC), DNA accessibility (ATAC-seq) and gene expression (RNA-seq and CAGE-seq) datasets to select putative RA-causal variants in SF. Selected variants in the JAZF1 locus were tested for regulatory function by luciferase reporter assays and electrophoretic mobility shift assays (EMSA) in the fibrosarcoma cell line HT1080. The JASPAR2020 database was used to identify putative transcription factors (TF) binding to the selected variants. The expression of HOTTIP was measured by quantitative PCR in hand SF (n=23). Genotyping was done by pyrosequencing.Results:Genetic fine mapping revealed 47 variants in the JAZF1 locus. Integration of these variants with the chromatin datasets prioritized rs2158624, rs57585717 and rs186735625 as the top candidates (posterior probability of causality >0.1) in the JAZF1 locus. We found that rs2158624 and rs186735625 are located in the vicinity of enhancer elements in SF as determined by ATAC-seq. In addition, the region of rs2158624 exhibited strong chromatin interactions with the genomic region of HOTTIP and HOXA13. Both these transcripts were previously shown to be specifically expressed in SF isolated from hands and feet2. Based on this, we selected rs2158624 as the most promising candidate in the JAZF1 locus. We found that the rs2158624-C allele (risk) is associated with lower expression of HOTTIP, but not HOXA13, in hand SF compared to the rs2158624-T allele (non-risk) (p=0.02). Luciferase assays in HT1080 cells demonstrated enhancer activity with both the rs2158624-C allele (p=0.006) and T allele (p=0.04), with no significant difference in enhancer activity between the rs2158624-C and T allele. EMSAs identified stronger specific binding of HT1080-cell nuclear extract for the rs2158624-T allele than for the C allele (risk). Based on the JASPAR2020 database, we identified NFAT5 as a potential TF that can bind to rs2158624 and may regulate the expression of HOTTIP.Conclusion:We were able to substantially narrow down the potential functional variants in the JAZF1 locus using our data integration approach and functional assays. We suggest that the risk allele of rs2158624 influences the binding of TFs controlling the expression of the long non-coding RNA HOTTIP in SF, which might confer specific risk to develop RA in hands.References:[1]Okada Y et al. Genetic of rheumatoid arthritis contributes to biology and drug discovery. Nature 2014;506:376.[2]Frank-Bertoncelj M et al. Epigenetically-driven anatomical diversity of synovial fibroblasts guides joint-specific fibroblast functions. Nat Commun 2017;8:14852.Disclosure of Interests:Miranda Houtman: None declared, Xiangyu Ge: None declared, Amanda McGovern: None declared, Kerstin Klein: None declared, Gisela Orozco: None declared, Mojca Frank Bertoncelj: None declared, Miriam Marks: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, iQone, Medscape, MSD, Novartis, Pfizer and Roche, Consultant of: Abbvie, Acceleron Pharma, Amgen, AnaMar, Arxx Therapeutics, Bayer, Baecon Discovery, Boehringer, CSL Behring, ChemomAb, Corbus Pharmaceuticals, Galapagos NV, GSK, Glenmark Pharmaceuticals, Horizon Pharmaceuticals, Inventiva, Italfarmaco, iQvia, Kymera, Lilly, Medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Pfizer, Roche, Roivant Sciences, Sanofi and UCB, Grant/research support from: Kymera Therapeutics and Mitsubishi Tanabe, Paul Martin: None declared, Stephen Eyre: None declared, Caroline Ospelt: None declared
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Vecellio, M., P. B. Wordsworth, and C. J. Cohen. "POS0362 INVESTIGATING THE ANKYLOSING SPONDYLITIS-ASSOCIATED REGULATORY SNPS AT THE RUNX3 LOCUS WITH A FUNCTIONAL GENOMICS APPROACH." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 411.2–411. http://dx.doi.org/10.1136/annrheumdis-2021-eular.4098.
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Background:Of the >100 genetic associations with ankylosing spondylitis (AS), RUNX3 transcription factor (TF) involved in diverse immunological processes, is robustly (10−15) associated.1 The biggest challenge is to understand the mechanism behind this association. We demonstrated the association between AS and the single nucleotide polymorphism (SNP) rs4648889 located in a 2kb regulatory locus upstream of the RUNX3 promoter can be explained by allele-specific effects on TF recruitment that alter gene expression, specifically in CD8+ T-cells.2 We recently dissected the full plethora of TFs affected by rs4648889 AS-associated allele showing the NuRD complex and IRF5 differentially bound and having a crucial role in CD8+ T-cells function3. Nevertheless, other post-GWAS approaches are needed to elucidate the function of RUNX3 AS-associated SNPs (i.e. the lead SNP rs6600247).Objectives:The purpose of this work is to define the functional effect of rs6622047 in CD8+ T-cells and monocytes. In specific, we expect to define the impact of AS-associated allele to TF binding and to evaluate chromosome looping between rs6600247 and the RUNX3 promoter.Methods:The epigenetic landscape of SNP rs6600247 was defined using Roadmap database. In vitro functional studies were performed to characterize the effects of this SNP on TFs binding. Chromosome conformation capture (3C) provided critical functional evidence for looping among AS-associated SNPs and the RUNX3 promoter.Results:(1) In silico data revealed a c-MYC ChIP-seq peak in GM12878 lymphoblastoid cells overlapping rs6600247; (2) Mobility shift assays (EMSAs) and WB-EMSAs showed reduced DNA/protein binding in the presence of the AS-risk allele in CD14+ monocytes. C-MYC binding-site is disrupted and binding abolished in the presence of the AS-risk allele; (3) 3C experiments indicate low interaction frequency between SNP rs6600247 and RUNX3 promoter.Conclusion:The enhancer upstream the RUNX3 gene has a plausible functional role in AS, probably by regulating gene transcription and DNA looping. These observations are critically important in defining dysregulated pathways and potential therapeutic drug targets.References:[1]IGAS et al. Nat Genet. 2013 Jul;45(7):730-8.[2]Vecellio M. et al, Ann Rheum Dis. 2016 Aug;75(8):1534-40.[3]Vecellio et al Arthritis Rheumatol 2020. doi: 10.1002/art.41628.Acknowledgements:We thank Professor Julian Knight for his constructive advices and comments to the present work.Disclosure of Interests:None declared
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van der Kouwe, Emiel, Gerwin Heller, Akos Czibere, Lucio H. Castilla, Ruud Delwel, Annalisa Di Ruscio, Alexander Ebralidze, et al. "Core Binding Factor Leukemias Utilize a Physiologic Sense/Antisense Promoter Switch Employed By T-Cells." Blood 136, Supplement 1 (November 5, 2020): 40–41. http://dx.doi.org/10.1182/blood-2020-140776.
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Introduction: Alterations of core binding factors (CBF), Runx1 and CBFβ are frequent mutational targets in acute myeloid leukemia (AML). Chromosomal translocations t(8;21)(q22;q22) and inv(16)(p13q22), creating the fusion proteins RUNX1-ETO and CBFβ-MYH11 respectively, account for 15% and thus the largest sub-group of AML called CBF-AML. CBF oncogenes induce global changes in chromatin structure and gene regulation, which lead to differentiation blockade. A critical leukemic event could be the inactivation of PU.1 transcription factor. Normal myeloid differentiation needs PU.1 levels to increase, failure to do so leads to a stop of differentiation and AML development. In contrast, T-cell differentiation requires PU.1 to be completely switched off. The exact mechanism of PU.1 suppression, physiological for T-lymphopoiesis or pathological for leukemia, remains elusive. Results: We assessed the activation of the PU.1 locus throughout human hematopoietic differentiation stages using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Interestingly, we observed high accessibility of a previously identified antisense promoter (AsPr) in intron 3 and antisense transcript (asRNA) expression during early lymphopoiesis which preceded locus shutdown in T cells. The ratio of AsPr/PrPr accessibility and of antisense/sense transcription clearly indicated cellular fate during hematopoiesis (Figure 1A). T-lymphoid differentiation was related to the timely expression of RUNX transcription factors. RUNX1, RUNX3, and the CBF fusions RUNX1-ETO and CBFβ-MYH11 were capable to transactivate PU.1 AsPr. In CBF-AML patient samples we strikingly found elevated asRNA/mRNA ratios compared to normal karyotype AML or healthy CD34+ cells (Figure 1B) and increased AsPr/PrPr ratios unsing DNaseI-seq data in RUNX1-ETO AML patients. Functionally we found that PU.1 asRNA depletion in t(8;21) xenografted immune-deficient (NOD/SCID) mice restored a normal survival (Figure 1C) demonstrating that PU.1 antisense transcripts are required for CBF leukemia outgrow in vivo. To further dissect the mechanism of how CBFs could drive PU.1 antisense transcription we applied active RNA polymerase mapping (PRO-seq) and chromatin accessibility (ATAC-seq) and found a shift from PU.1 antisense to sense transcription after RUNX1-ETO depletion. Using chromosomal conformation capture sequencing (3C, Hi-C and CHiC) in T-lymphoid, myeloid and RUNX1-ETO cells combined with transcript quantification we observed that competitive interaction of an upstream enhancer with the proximal or the antisense promoter are at the heart of differential PU.1 expression during myeloid and T-cell development (Figure 2A). Leukemic CBF fusions thus utilize a physiologic mechanism employed by T-cells to decrease sense PU.1 transcription (Figure 2B). Conclusion: The data suggest that silencing transcription factor PU.1 is an active process that requires a specific chromosome formation that is induced by CBF fusions. Sense/antisense promoter competition represents a crucial functional switch for gene expression perturbation by oncogenes and provide a potential strategy for future precise therapeutic targeting of oncogene-induced chromatin remodeling. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Staber:Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; msd: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria.
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Schwartzman, Omer, Zohar Mukamel, Shai Izraeli, and Amos Tanay. "Umi-4C: A Quantitative, Robust and Multiplexed Method to Study the Regulatory Three Dimensional Chromatin Organization - Application for the Mgakaryocytic-Eythroid Lineage." Blood 126, no. 23 (December 3, 2015): 1183. http://dx.doi.org/10.1182/blood.v126.23.1183.1183.
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Abstract Background: The role of the spatial three dimensional (3D) chromatin organization in regulation of gene expression is at the forefront of epigenetic research. Chromatin Conformation Capture (3C) technologies are increasingly being used to map physical proximity between distal regulatory elements. The underlying principal is similar in all these assays and involves chromatin cross-linking, digestion, and ligation. The proximity ligation junctions are then analyzed as a proxy to physical proximity. These methods vary in terms of scope and resolution, from Hi-C, which allows whole-genome coverage but requires massive sequencing burden, to traditional 3C which is simpler but allows only pairwise contact mapping. Of particular recent interest are methods allowing targeted sequencing of ligation products such as 4C-seq. However, 4C is heavily dependent on PCR amplification and requires elaborate statistical models to account for biases introduced. Consequently, a major drawback of all current methodologies is the lack of precise quantitation. To control for these drawbacks we developed a new simple and directly quantitative 4C methodology applying the concept of Unique Molecular Identifiers (UMI). Methods: We have developed a modified 4C-seq protocol (see figure). After the standard fixation, digestion and ligation, the chromatin DNA is sonicated, resulting in random breakpoints that are exploited as bona-fida UMIs. To target specific loci we utilize a version of ligation mediated (LM)-PCR, ligating a universal adapter to one end of the insert and a target-specific primer, to focus on the region of interest, to the other end. In addition, we developed a novel computational framework to process the data and filter potential artifacts and non-specific priming events. We applied this highly quantitative method to study the chromatin spatial landscape of important megakaryocytic and eryhtroid genes - GATA1, ANK1 and the HBB region. We generated high-complexity contact profiles of these regions in six cell lines - four Megaerythroid cell lines (CMK, CMY, K562 and CHRF), that express these genes at variable levels, and a T-ALL cell line (DND41) and primary human fibroblasts where these loci are silenced. Results: We are able to recover on average 5,000-20,000 ligation events per 1μg of starting 4C template. Estimating the sequencing requirement by inference and subsampling, we find that 500,000 reads are enough to recover more than 90% of the ligation events. By applying our assay to GATA1 locus we were able to detect and precisely quantify hotspots of differential contact intensity, likely to reflect differences in the contacting probabilities between erythroid and megakaryocytic cells. These regions coincided with active histone marks in either of the cell types. Next, we interrogated ANK1 promoter region and detected differential contact intensity of the promoter with enhancer elements -15kb, and -27kb upstream and +15kb downstream of the transcription start site (TSS). The differences were also correlated with the expression pattern of ANK1 in these cells. Finally we utilized our assay to multiplex different regions in the HBB locus and generated very high complexity contact profiles of the region revealing activity-associated hierarchical looping structure that was previously not described. Conclusions: We have developed a powerful sensitive methodology to study the chromatin structure of specific targets in a multiplexed, cost-effective and simple manner. We applied it to a variety of regions and cells and were able to precisely detect and quantify minute differences in contact intensities between cells belonging to related but different lineages. We suggest UMI-4C as a precise and practical tool to study 3D epigenetic regulation of gene expression. Figure 1. A scheme of the UMI-4C methodology and a snapshot of the GATA1 locus in CMK megakaryocytic-eryhthroid and K562 erythroid leukemia cells. Figure 1. A scheme of the UMI-4C methodology and a snapshot of the GATA1 locus in CMK megakaryocytic-eryhthroid and K562 erythroid leukemia cells. Disclosures No relevant conflicts of interest to declare.
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Hellmuth, Johannes C., Chi-Shuen Chu, Rajat Singh, Lucy A. Skrabanek, Ashley Doane, Olivier Elemento, Robert G. Roeder, and Ari Melnick. "An OCT2 / OCA-B / MEF2B Ternary Complex Controls the Activity and Architecture of an Essential Locus Control Region for Normal and Malignant Germinal Center B-Cells." Blood 134, Supplement_1 (November 13, 2019): 24. http://dx.doi.org/10.1182/blood-2019-130692.
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The transcriptional repressor BCL6 is considered the master regulator of the germinal center (GC) reaction and is a key proto-oncogene in GC-derived lymphoma pathogenesis. At GC initiation, chromatin architecture is dramatically remodeled around a BCL6-associated locus control region (LCR) with presumed enhancer function. The BCL6 LCR is located 150kb upstream of the BCL6 gene and is fundamentally required for GC formation. Herein, we functionally dissect the BCL6 LCR to uncover the crucial genetic elements and transcription factor binding events that drive BCL6 LCR function. We generated a custom gRNA library densely tiling the BCL6 LCR and surrounding regulatory elements. In total, the library covered a genomic region of 316.8kb with 25,698 gRNAs. We used a GC derived diffuse large B-cell lymphoma line, OCI-LY7, stably expressing dCas9-KRAB (CRISPRi) to screen for depletion of gRNAs. Surprisingly, BCL6 LCR function relied on only 4 of 21 constituent enhancers. These 4 essential enhancers were critically required for cell growth and BCL6 expression. In contrast, all other constituent enhancers were completely dispensable for LCR function. These results indicate that the BCL6 LCR is governed by a strong internal hierarchy and pinpoint the crucial genetic elements that drive LCR function. To understand what distinguishes essential enhancers from non-essential enhancers, we interrogated ChIP-seq profiles of key GC transcription factors (TF) and transcriptional co-activators. While most TFs and co-activators bound constituent enhancers indiscriminately, MEF2B specifically bound only to essential enhancers (p=0.01). Interestingly, essential enhancers did not contain MEF2B binding motifs. Furthermore, MEF2B did not bind to essential enhancer DNA in electrophoretic mobility shift (EMSA) and immobilized template assays. De novo motif analysis of MEF2B ChIP-seq data prominently featured the canonical octamer transcription factor motif (p=10-79) indicating a role for OCT2 in the recruitment of MEF2B to essential enhancers. Indeed, MEF2B binding to essential enhancer DNA in EMSA and immobilized template assays was dependent upon the presence of OCT2 and its co-activator OCA-B. We assayed occupancy of OCT2, OCA-B and MEF2B at essential enhancers in OCI-LY7 cells by qChIP and found that each factor required the other two for full binding activity. Furthermore, all three factors were required for BCL6 expression (p<0.001 for each factor). These results indicate that OCT2, OCA-B and MEF2B cooperatively bind to essential enhancer elements and act as an intimately linked ternary complex to drive BCL6 expression through the BCL6 LCR. To elucidate how the OCT2 / OCA-B / MEF2B complex promotes target gene transcription, we performed IP of OCA-B followed by mass spectrometry and found highly significant interactions with the majority of Mediator proteins (p<10-8). We furthermore showed that OCA-B directly and specifically interacts with MED1, through which it recruits the remainder of the Mediator complex and that OCA-B and OCT2 are required to recruit Mediator to essential enhancer elements. The Mediator complex is thought to serve as a drawbridge across enhancers and promoters to facilitate enhancer-promoter looping. Using 3C assays, we found that OCA-B is required for chromatin contacts between the BCL6 promoter and the LCR highlighting its importance in recruiting Mediator. Similarly, essential enhancer elements were crucially required for intact chromatin conformation at the BCL6 locus as determined by 3C. In summary, BCL6 LCR function completely relies on very few but highly essential enhancer elements. OCT2, OCA-B and MEF2B cooperatively bind these essential enhancers forming an intimately linked trimeric complex. By recruiting Mediator, OCA-B provides a direct link to the basal transcriptional machinery. Finally, essential enhancers as well as the OCT2 / OCA-B / MEF2B complex are required for BCL6 expression and intact chromatin conformation at the BCL6 locus - key determinants of the GC B cell state. Disclosures Melnick: Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding.
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Biancon, Giulia, Poorval Joshi, Torben Hunck, Josh Zimmer, Yimeng Gao, Martin Machyna, Valentina Botti, et al. "High-Resolution Binding Atlas of U2AF1 Mutants Uncovers New Complexity in Splicing Alterations and Kinetics in Myeloid Malignancies." Blood 136, Supplement 1 (November 5, 2020): 3–4. http://dx.doi.org/10.1182/blood-2020-142854.
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Spliceosomal gene mutations function as drivers of hematologic malignancies and other cancers with an occurrence of more than 50% in myelodysplastic syndromes and secondary acute myeloid leukemia. Hotspot mutations S34F and Q157R in the two zinc finger domains of the splicing factor U2AF1, forming with U2AF2 the U2AF complex that recognizes 3' splice site (3'SS) of U2 introns, alter exon usage in a sequence-specific manner. However, how pathological U2AF1 mutations disrupt ordered splicing, from binding to recruitment of cooperating RNA binding proteins and ultimately splicing kinetics, is still not known at the molecular level. To obtain unique insights into in vivo RNA binding mechanisms, we performed fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA sequencing (freCLIP-seq) on human erythroleukemia (HEL) cells expressing wild-type (WT) or mutant (S34F, Q157R) U2AF1. Transcriptome-wide analysis of binding at single nucleotide resolution in light and heavy fractions, corresponding respectively to U2AF1 only and U2AF complex, allowed to: i) deconvolute U2AF1 signal peaking over the AG dinucleotide at the intronic end of the 3'SS region, and U2AF2 signal sitting on the adjacent polypyrimidine tract (PPT); ii) identify conformational changes in mutant U2AF1 binding with a novel peak in position -3 of the 3'SS region for S34F and in position +1 for Q157R. Alternative splicing analysis on newly collected RNA-seq data showed that less included exons present higher probability of U in position -3 for S34F and A in position +1 for Q157R, pinpointing a match with nucleotide positions affected by aberrant binding in freCLIP-seq. In both U2AF1 mutants, aberrant binding and splicing mechanisms affected genes involved in mRNA processing and transport (P-value<0.01) highlighting the involvement of U2AF1 mutations in the dysregulation of these key biological processes. We then performed a combined analysis of differential binding and aberrant splicing in U2AF1 mutants vs WT considering 4 categories: ">inclusion/>binding", "<inclusion/<binding", "<inclusion/>binding", ">inclusion/<binding". The first 2 categories correspond to the loss-of-function binding model suggested in literature to explain the splicing outcome of U2AF1 mutations: U2AF1 mutants bind certain splicing junctions with less affinity, leading to their exclusion. The last 2 categories represent a non-canonical gain-of-function model where increased mutant U2AF1 binding results in the impairment of the splicing machinery. Surprisingly, while Q157R mainly exhibited a loss-of-function mechanism where ineffective splicing is related to absence of binding ("<inclusion/<binding", 51.1%), S34F mostly follows a gain-of-function mechanism affecting splicing progression by an increased, yet skewed, binding. The most represented category was, indeed, "<inclusion/>binding" with 123 events out of 309 (Figure 1A). Moreover, differential binding was not dependent on specific nucleotides in position -3: events characterized by increased S34F binding (Figure 1B, top), as well as events characterized by decreased S34F binding (Figure 1B, bottom), showed -3U in less included exons or -3C in more included exons. The binding analysis across the 4 categories showed that increased S34F binding was associated with reduced U2AF2 binding (Figure 1C, top) particularly in less included exons, while decreased S34F binding was associated with increased U2AF2 binding (Figure 1C, bottom) especially in more included exons. Finally, analysis of branch point and splice junction features revealed that PPT strength influences the splicing outcome with "<inclusion/>binding" category characterized by a weak PPT that impairs U2AF2 binding in the presence of skewed U2AF1 S34F binding (Figure 1D). Additionally, transcriptome-wide RNA kinetics analysis by TimeLapse-seq demonstrated that U2AF1 S34F and Q157R, compared to WT, globally decrease synthesis of aberrantly spliced and bound 3'SS regions. Of note, this shutdown effect was particularly evident in the downstream exons pointing towards a role of U2AF1 mutations in a widespread alteration of RNA synthesis and splicing dynamics. Collectively, these results disclose novel molecular mechanisms of pathogenic U2AF1 mutations in the context of myeloid malignancies and provide the basis for the development of effective U2AF1 directed therapeutic strategies. Disclosures Hunck: Boehringer Ingelheim Fonds: Other: MD Fellowship.
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Zhang, Ya, Xinting Hu, Yang Han, Xiangxiang Zhou, Huimin Zhang, Xiaoya Yun, and Xin Wang. "Targeting Inhibition of N6-Methyladenosine Demethylase Fto Displays Potent Anti-Tumor Activities in Chronic Lymphocytic Leukemia." Blood 136, Supplement 1 (November 5, 2020): 35–36. http://dx.doi.org/10.1182/blood-2020-139526.
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Introduction Accumulating evidence indicates that Fat mass and obesity-associated protein (FTO), a N6-methyladenosine (m6A) RNA demethylase exerts crucial roles in oncogenesis. FB23-2, a novel inhibitor selectively targeting FTO m6A demethylase activity displayed promising potency in acute myeloid leukemia. Yet, no literature has been reported regarding the effects of FTO and FB23-2 in the tumorigenesis and development of chronic lymphocytic leukemia (CLL). Hence, the aim of this study was to investigate the clinical significance and mechanisms of FTO regulation in CLL. Methods Peripheral blood samples from 55 de novo CLL patients (36 males and 19 females; age range 32-82 years, median 62 years) were collected with informed consents in Shandong Provincial Hospital. CD19+ B cells were isolated with informed consents from healthy donors. Expression levels of FTO mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-seq) were conduct to profile RNA m6A methylation and expression of CLL cells. Lentiviral vectors were constructed to stably silence and overexpress FTO in CLL cells. Besides, cell viability, apoptosis and cell cycle were assessed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. Results Aberrantly increased expression of FTO was observed in CLL patients and CLL cell lines at mRNA and protein level compared with normal B cells from healthy donors (Figure 1A-B). Clinical correlation analyses suggested FTO high expression was significantly associated with 11q23 deletion (p=0.012; Figure 1C). Furthermore, Keplan-Meier plot indicated that elevated FTO expression predicted adverse outcome in CLL patients (HR=1.758, p=0.019; Figure 1D). ROC curve confirmed the prognostic value of FTO in survival of CLL patients (AUC=0.600, p=0.018; Figure 1E). To explore the potential role of FTO in CLL tumorigenesis, CLL cells were transfected with lentiviral vectors to stably silence and overexpress FTO. CLL primary cells and MEC1 cells with silence of FTO exhibited attenuated cell proliferation, increased fast-onset apoptosis (Figure 2A-D). Western blotting assay suggested significant down-regulated Bcl-2, enhanced cleaved-PARP and BAX expression in FTO-deficient CLL cells. Whereas, gain-of-function assay showed promoted cell survival in FTO-overexpressed CLL cells (Figure 2F-G). Additionally, serial dilution of FTO inhibitor FB23-2 decreased viability of MEC1 and primary CLL cells in time-dependent manner, and displayed rare cytotoxicity in normal B cells (Figure 3A-B). Besides, annexin V-PE/7AAD and western blotting assay indicated obvious apoptosis was induced with treatment of FB23-2 in CLL primary cells from 9 de novo CLL patients (Figure 3C-D). Importantly, obvious G2/M phase arrest and enhanced sensitivity to Venetoclax were also detected in FTO-reduced CLL cells. Furthermore, interactive MERIP-seq and RNA-seq of CLL cells with control and deleted FTO expression were performed to investigate the m6A Methylation-Mediated mechanism of FTO regulation of CLL pathogenesis. A total of 573 significantly changed peaks, of which 301 were significantly up-regulated and 272 peaks were significantly down-regulated (Figure 4A). Differentiated peaks were located in 3'UTR (42.58%) and 5'UTR (24.43%). Annotations of bioinformatics analyses indicated that FTO was functionally enriched in cell apoptosis in CLL progression (Figure 4B). Western blotting assay suggested significant down-regulated p-CHK2, c-myc, p-p53, cyclinD1 and enhanced p-H2AX expression in FTO-deficient CLL cells, indicating FTO accelerated CLL cell survival via DNA damage pathway (Figure 4C). Conclusion Taken together, our investigations identified for the first time the oncogenic role of FTO in CLL tumorigenesis and regulatory mechanism of FTO inhibitor FB23-2 in CLL cells by MERIP sequencing and ex vivo evaluation. Expression of FTO was upregulated and associated with inferior prognosis of CLL patients. FB23-2 exerted potent therapeutic potential in abrogating cell survival and inducing cell cycle arrest via m6A methylation. This study provides a rationale on evaluation of FTO-targeted intervention formulating a novel treatment paradigm in progressed CLL that warrants clinical investigation. Disclosures No relevant conflicts of interest to declare.
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Xiong, Qian, Zhaojun Zhang, Hongzhu Qu, Xiuyan Ruan, Hai Wang, Qian Zhang, Heyuan Qi, et al. "Deciphering the Cis- and Trans-regulatory Roles of KLF6 in Primitive Hematopoiesis." Blood 120, no. 21 (November 16, 2012): 4730. http://dx.doi.org/10.1182/blood.v120.21.4730.4730.
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Abstract Abstract 4730 Krüppel-like factors (KLFs) are a conserved family of Cys2His2 zinc finger proteins which are important components of eukaryotic cellular transcriptional machinery that controls many biological processes including erythroid differentiation and development. As a transcriptional activator and a tumor suppressor, KLF6 was also involved in hematopoiesis. Klf6−/− mice is embryonic lethal by embryonic day 12.5 and associated with markedly reduced hematopoiesis as well as poorly organized yolk sac vascularization. Moreover, the expression of erythroid differentiation markers including Klf1, Gata1 and Scl are delayed and hematopoietic differentiation is impaired in klf6−/− ES cells. However, the detailed mechanism that KLF6 regulates hematopoiesis is not fully understood. To characterize the role of KLF6 in hematopoiesis, we firstly detected the dynamic expression pattern of KLF6 during erythroid differentiation by mRNA-seq in undifferentiated human embryonic stem cells (hESC), three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The transcriptome analysis showed that KLF6 expressed at significantly higher level in ESER cells compared with that in other cells. Meanwhile, chromatin immunoprecipitation (ChIP) studies in human K562 cells demonstrated the enrichment of KLF6 on the promoter region of embryonic epsilon-globin gene. These results probably indicate that KLF6 play an important role in primitive hematopoiesis. To clarify whether the erythroid-specific enhancers in the genomic region of KLF6 participate in the regulation of primitive hematopoiesis, we extensively screened the erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF6 locus, from 70 kb upstream of the transcription start site to 20 kb downstream of the poly(A) site, from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of these erythroid-specific DHSs was comprehensively characterized by dual-luciferase reporter assay in K562 cells as well as non-erythroid HeLa and HEK293 cells. Three erythroid-specific enhancers located 18–24 kb upstream of human KLF6 were finally characterized, which not only helps to understand the higher expression of KLF6 in ESER, but also hints that KLF6 could participate in primitive hematopoiesis through erythroid-specific enhancers. In conclusion, we depicted the dynamic expression pattern of KLF6 during erythroid differentiation, characterized three erythroid-specific enhancers in KLF6 gene locus, and disclosed the potential role of KLF6 in primitive hematopoiesis. Next, the overexpression and depletion of KLF6 in K562 cells will be executed to further explore whether the abnormal KLF6 will affect the expression and functions of globin genes as well as erythroid-specific transcription factors. Chromosome conformation capture (3C) analysis will be performed to evaluate the interactions between the erythroid-specific enhancers and the cis-regulatory elements of hematopoiesis related genes. Moreover, we will establish morpholino-based klf6 knockdown zebrafish model and study the target genes, interacting networks and pathways in which KLF6 involved. Collectively, these results will address the detailed cis- and trans- regulatory functions and molecular mechanism of KLF6 in regulating hematopoiesis. Disclosures: No relevant conflicts of interest to declare.
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Gillinder, Kevin R., Jim R. Hughes, Michael R. Tallack, Graham Magor, Melissa Ilsley, James Davies, Douglas Higgs, and Andrew C. Perkins. "Dynamics and Mechanics Of KLF1 Regulation In Erythropoiesis." Blood 122, no. 21 (November 15, 2013): 2176. http://dx.doi.org/10.1182/blood.v122.21.2176.2176.
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Abstract Krûppel-like factor-1 (KLF1) is a C2H2 zinc finger transcription factor which is essential for broad erythroid gene expression and erythropoiesis in vivo. A number of studies have shown ∼700 genes are poorly expressed when KLF1 is absent [1-8]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice [9,10], and partly responsible for congenital anemia in humans and mice with dominant mutations in KLF1 [11,12]. To determine whether KLF1-dependent genes are direct or indirect targets of KLF1, we have previously performed global ChIP-seq experiments identifying 945-1350 regions of KLF1 occupancy in the mouse genome [7]. About 15% of these regions fall within the promoters of KLF1 target genes but surprisingly, most are thousands of kilobases distant from any known gene. Many of these distant sites exhibit co-occupancy with other transcriptional regulators involved in erythropoiesis, including GATA1. Approximately half of the KLF1 occupied sites are found within regions of mono-methylation of lysine 4 on histone 3 (H3K4me1). These regions are devoid of histones tri-methylated at the same residue (H3K4me3). This methylation signature is commonly associated with regions of the genome that act as transcriptional enhancers [13,14] and many are also bound by the co-activator, p300. The nature and function of these distant sites, particularly those without enhancer marks, is interesting as they may shed light on novel mechanisms of action of KLF1 and associated transcription factors. The transcriptional machinery of the cell, including many transcription factors is found in large sub-nuclear compartments called transcriptional factories [15]. KLF1 has been found localized to a subset of these in erythroid cells. KLF1 is also required for long-range looping of the β-globin gene into these transcription factories [16]. Other erythroid genes involved in the production of a functional haemoglobin molecule such as α-globin and haem synthesis enzymes are often found in the same transcription factory. This strongly suggests KLF1 can employ this sub-nuclear machine to co-ordinate the transcriptional output from many genes and thereby direct erythroid cell differentiation. To explore the function of KLF1-bound loci, we have performed multiplexed chromosome conformation capture (3C) coupled with sequencing (Capture-seq) using a tamoxifen responsive, KLF1 inducible cell line to investigate the role of KLF1 in chromosomal looping. In addition, we have analysed primary transcriptional output of KLF1 target genes by nascent RNA-seq. As expected β-globin and a-globin transcription is rapidly induced, becoming detectable within 5 minutes. However, the transcriptional response of dematin and a set direct KLF1 target genes is much slower. Thus, the mechanism of KLF1 transcriptional activation differs between target gene loci. We find a dynamic role of KLF1-dependent chromosomal looping and transcriptional co-factor recruitment required to effect gene transcription during erythropoiesis. We will discuss models of differentiation transcription regulation by KLF1. References: 1. Drissen R, et al. (2005). Molecular and Cellular Biology 25: 5205–5214. 2. Funnell APW, et al. (2007). Molecular and Cellular Biology 27: 2777–2790. 3. Hodge D, et al. (2006). Blood 107: 3359–3370. 4. Pilon AM, et al. (2008). Molecular and Cellular Biology 28: 7394–7401. 5. Siatecka M, et al. (2010). PNAS 107: 15151–15156. 6. Siatecka M, Bieker JJ (2011). Blood 118: 2044–2054. 7. Tallack MR, et al. (2010). Genome Res 20: 1052–1063. 8. Tallack MR, Perkins AC (2010). IUBMB Life 62: 886–890. 9. Perkins AC, Sharpe AH, Orkin SH (1995). Nature 375: 318–322. 10. Nuez B, et al. (1995). Nature 375: 316–318. 11. Arnaud L, S et al. (2010). Am J Hum Genet 87: 721–727. 12. Borg J, et al. (2011). Haematologica 96: 635–638. 13. Zentner GE, et al. (2011). Genome Res 21: 1273–1283. 14. Pekowska A, et al. (2011). EMBO J 30: 4198–4210. 15. Osborne CS, et al. (2004). Nat Genet 36: 1065–1071. 16. Schoenfelder S, et al. (2010). Nat Genet 42: 53–61. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.
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Wang, Anqi, Rene Welch, Bo Zhao, Tram Ta, Sündüz Keleş, and Eric Johannsen. "Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites." Journal of Virology 90, no. 6 (December 30, 2015): 2906–19. http://dx.doi.org/10.1128/jvi.02737-15.
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ABSTRACTLatent infection of B lymphocytes by Epstein-Barr virus (EBV)in vitroresults in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A- or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A- and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors.IMPORTANCEEpstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell linesin vitro. EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for lymphocyte growth and survival. An important unresolved question is how four different EBNAs (EBNA2, -3A, -3B, and -3C) exert unique effects via a single transcription factor, RBPJ. Here, we report that each EBNA binds to distinct but partially overlapping sets of genomic sites. EBNA3A and EBNA3C specifically regulate EBNA2's access to different RBPJ sites, providing a mechanism by which each EBNA can regulate distinct cell genes. We show that IRF4, an essential regulator of B cell differentiation, is critical for EBNA3C binding specificity; EBNA3A and EBNA3B specificities are likely due to interactions with other cell transcription factors. EBNA3 titration of EBNA2 transcriptional function at distinct sites likely limits cell defenses that would be triggered by unchecked EBNA2 prooncogenic activity.
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Yang, Juan, Xiangxiang Zhou, Ying Li, Ya Zhang, Xiaosheng Fang, Na Chen, and Xin Wang. "Targeting Sirt6 with OSS_128167 Displays Anti-Tumor Activities in Diffuse Large B-Cell Lymphoma through Down-Regulation of PI3K Signaling." Blood 134, Supplement_1 (November 13, 2019): 5065. http://dx.doi.org/10.1182/blood-2019-127926.
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Introduction Sirt6, a member of the mammalian sirtuins family, functions as a highly conserved ADP-ribosylase and NAD+ dependent deacylase. Evidence from recent years indicates that Sirt6 is closely related to the occurrence and development of various solid tumors and multiple myeloma (MM). OSS_128167 is a novel small molecular inhibitor specifically targeting Sirt6, and induced chemosensitization in MM cells. Nevertheless, the role of Sirt6 in lymphoid malignancies is still unclear. Herein, we investigated the function of Sirt6 and identified the anti-tumor effects of OSS_128167 in diffuse large B-cell lymphoma (DLBCL). Methods Immunohistochemistry (IHC) was conducted to assess the expression of Sirt6 on paraffin-embedded tissues from 70 de novo DLBCL patients and 35 reactive hyperplasia patients with informed contents. Microarray dataset GSE32918 was obtained from Gene Expression Omnibus and survival analysis was performed. Lentivirus vectors either encoding shSirt6, lvSirt6 or empty lentiviral vector were stably transfected into DLBCL cells. shSirt6 transfected or shControl transfected LY1 cell samples were performed RNA sequencing (RNA-seq) analysis, functional enrichment analyses of gene ontology (GO) and gene set enrichment analysis (GSEA). DLBCL cells were subcutaneously injected to SCID-Beige mice to establish xenograft models. All animal experiments were performed in accordance with the principles of the Institutional Animal Care. A p value of less than 0.05 was interpreted as having statistical significance. Results Aberrantly overexpression of Sirt6 was confirmed in DLBCL tissues compared with reactive hyperplasia by IHC staining (Fig 1a). The expression level of Sirt6 was revealed in significant positive correlation with advanced age (p=0.009), Ann Arbor stage (p=0.036) and IPI score (p=0.045, Fig. 1b). Sirt6 high expression was turned up to be correlated with reduced overall survival of enrolled patients according to GSE32918 (n=249, Fig. 1c). Stable shSirt6 transfected DLBCL cells exhibited growth suppression compared to those transfected with empty vector in vitro (Fig. 2a) and in vivo (Fig. 2b-c). Lower expression level of Ki-67 was confirmed in the xenograft tumor tissues derived from shSirt6 cells by IHC staining (Fig. 2d). Sirt6 knockdown caused enforced apoptosis rates, induced G2/M phase arrest (Fig. 2e-f) and enhanced sensitivity to Doxorubicin (ADR) and bendamustine (Fig. 2g-h). These results were further confirmed in GO analysis according to RNA-seq (Fig. 2i) and WB (Fig. 2j). The exciting finding was, OSS_128167 obviously decreased cell proliferation (Fig. 3a), induced cell apoptosis (Fig. 3b) and blocked cell cycle (Fig. 3c) in DLBCL cell lines. Addition to ADR or bendamustine with 100µM OSS_128167 exhibited enhanced cytotoxicity in DLBCL cells (p< 0.05; Fig. 3d-e). Xenograft DLBCL mice model were treated with intraperitoneal injection of either OSS_128167 or a control vehicle every two days for 2 weeks. Consistent with our in vitro results, obviously decreased tumor growth were observed in the OSS_128167 group (Fig. 3f). Also, lowly expression level of c-Myc was found in OSS_128167 treated mice (Fig. 3g). We then investigated the underlying mechanism driving the tumor genesis potential of Sirt6. In accordance with the GSEA analysis, we observed that the phosphorylation of PI3K p110α and its downstream targets, including Akt (Ser473) and mTOR protein, were dramatically decreased in Sirt6 knockdown cells than in the control cells (Fig. 4a-b). In vivo western blotting analysis further confirmed it (Fig. 4c). The Sirt6 deletion-induced reductive of cell viability was only fewer restored by IGF-1 (Fig. 4d). Conclusion At present, the function of Sirt6 in tumorigenesis is still controversial. Our findings demonstrated for the first time the oncogenic role of Sirt6 in the pathogenesis of DLBCL. High expression of Sirt6 may serve as a prognostic marker for inferior outcome in DLBCL patients. OSS_128167, a novel targeted inhibitor of Sirt6, exerted excellent anti-lymphoma effects via activating PI3K/Akt/mTOR signaling. In addition, blockade of Sirt6 expression enhanced the sensitivity of DLBCL cells to chemotherapeutic agents. These findings provide mechanistic insights into the oncogenic activity of Sirt6 and highlighted the potency of OSS_128167 for novel therapeutic strategies in DLBCL. Disclosures No relevant conflicts of interest to declare.
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Kang, Yuanyuan, Bhavita Patel, Kairong Cui, Keji Zhao, Yi Qiu, and Suming Huang. "A T-Cell Specific Element Activates the TAL1 Oncogene Via an Interchromosomal Interaction During Leukemogenesis." Blood 120, no. 21 (November 16, 2012): 3507. http://dx.doi.org/10.1182/blood.v120.21.3507.3507.
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Abstract Abstract 3507 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant disease of thymocytes that mainly affects children and has very poor prognosis with high rates of relapse. A prominent feature observed in 60% of T-ALL childhood patients is the ectopic expression of a key hematopoietic transcription factor TAL1/SCL. Although several enhancers has been identified to play an important role in normal hematopoietic differentiation, the histone modification patterns and chromatin organization over the whole TAL1 locus reveled that none of them is active in T-ALL cell lines such as Jurkat and Rex cells. It remains currently unknown how TAL1 is activated in the majority of T-ALL patients lacking the TAL1 locus rearrangements. To understand the molecular mechanism underlying regulation of the TAL1 oncogene in leukemic T-cells, we employed circularized chromosome conformation capture (4C) methodology to identify new regulatory elements that activate TAL1 specifically in T-ALL leukemia. Using the TAL1 promoter 1a as the bait, we discovered that the TAL1 promoter 1a interacts with the TIL16 element (TAL1 interacting locus in chromosome 16) that is located at ∼15 Kb downstream of T-cell specific CD2BP2 gene in T-ALL cell line Jurkat, but not in erythroid progenitor K562 cells. The CD2BP2 protein is a cellular adapter protein that was originally identified as a binding partner of the T cell adhesion protein CD2 in the context of T cell signaling. The TIL16 element contains the bind sites for several transcription factors that are important for hematopoiesis such as C-Maf, Pax5, HoxA7 and USF2. The inter-chromosomal interaction between the TIL16 and the TAL1 promoter 1a was further confirmed by chromosome conformation capture (3C) assay in three TAL1 over-expressing T-ALL cell lines, Jurkat, REX and Molt4, but not in K562 cells. Recent genome wide study has correlates H3K4 mono- or dimethyl marks with distal enhancers while trimethyl H3K4 is enriched in promoters of active genes. To further test if the TIL16 acts as T-cell specific enhancer for TAL1 activation in T-ALL cells, we carried out ChIP-seq and ChIP analysis in CD4 T cells, Jurkat, and K562 cells. We found that the TIL16 element is specifically marked by H3K4me1 in Jurkat and CD4+ T-cells but not in K562 cells. The enrichment of H3K4me1 is correlated with the binding of c-Maf, a T-cell specific transcription factor. To further test whether the TIL16 element contributes to transcription activity, a DNA fragments containing the TIL16 element were cloneed into SV40 minimal promoter driven luciferase reporter and introduced into K562 and several T-ALL cell lines. Compared to the pGL3-SV40 vector that showed only minimal luciferase activity, the 1 Kb TIL element specifically activated transcription of the luciferase reporter in T-ALL cells, but not in erythroid progenitor K562 cells suggesting that the TIL16 element functions as a T-cell specific TAL1 enhancer. Thus, our data revealed a novel epigenetic mechanism by which the TAL1 oncogene is ectopically activated in T-cell leukemia. Disclosures: No relevant conflicts of interest to declare.
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Zhang, Ya, Xinting Hu, Hongzhi Xu, Ying Li, Lingyan Zhang, Lili Feng, Xiangxiang Zhou, et al. "Comprehensive Profiling of the Epitranscriptomic N6-Methyladenosine RNA Methylation in Chronic Lymphocytic Leukemia." Blood 136, Supplement 1 (November 5, 2020): 17–18. http://dx.doi.org/10.1182/blood-2020-141758.
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Introduction N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA. Accumulating evidence suggests that RNA m6A methylation exerts crucial roles in oncogenesis. However, the mRNA m6A methylation pattern in chronic lymphocytic leukemia (CLL) has not been investigated. Hence, the aim of this study is to perform a comprehensive profiling to identify distinct m6A methylation signatures in CLL patients. Methods Peripheral blood samples from de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong First Medical University. CD19+ B cells were isolated with informed consents from healthy donors. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was conduct to profile mRNA m6A methylation of CLL-B cells and normal CD19+ B cells at Novogene (Beijing, China). The library preparations were sequenced on an Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with 3 independent biological replicates. After mapping reads to the reference genome, exomePeak R package was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER. Differential peak calling was performed using exomePeak R package with parameters of p-value < 0.05 and fold_change > 1. Functional enrichment analyses of gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) of differentiated peaks associated genes were performed. All investigators comply with the guiding principles for experimental procedures found in the Declaration of Helsinki of the World Medical Association. Results By MeRIP sequencing, significant distribution of methylation peaks were detected in 5'UTR, 3'UTR and CDS regions of CLL primary cells (Figure 1A) and normal B cells (Figure 1B). Figure 1C-D illustrated the percentage of methylation peaks in the five regions, suggesting distinct m6A patterns in CLL cells. Moreover, Figure 1E-F revealed the positions of m6A methylation peaks in chromosomes of CLL and normal B cells. Furthermore, the compared distributions of m6A methylation peaks in CLL and normal B cells were presented in Figure 2A. Besides, the bean plot visibly displayed the obvious differentiation of methylation peaks in CLL group and normal B cells in read density (Figure 2B). Importantly, a total of 1836 significantly changed peaks, of which 1519 were significantly up-regulated and 317 peaks were significantly down-regulated (p<0.05, |log2Foldchange|>1; Figure 2C). These m6A peaks were located across 1850 genes. Functional enrichment analyses identified that differentiated peaks associated genes were potentially regulate RNA metabolic process via oncogenic pathways in CLL pathogenesis (Figure 3A-B). In addition, HOMER analysis identified 38 significant de novo m6A peak motifs, top 10 most significant peak motifs of which were presented (Figure 3C), illuminating potential detailed mechanism of m6A RNA methylation in the tumorigenesis and progression of CLL. Conclusion Taken together, our investigations explored for the first time the m6A methylation pattern of mRNA in CLL. m6A modifications play crucial roles in the progression and survival of CLL patients,highlighting m6A modifications-targeted intervention formulating a novel treatment paradigm in progressed CLL that warrants clinical investigation. Disclosures No relevant conflicts of interest to declare.
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Pospisil, Vitek, Pavle Krsmanovic, Jan Valecka, Kamila Chramostová, Vojtech Kulvait, Jiri Zavadil, Martin Vokurka, Peter Laslo, and Tomas Stopka. "Graded PU.1 Levels Regulate Granulocyte Vs. Macrophage Genes Via Multiple Enhancer Elements." Blood 128, no. 22 (December 2, 2016): 403. http://dx.doi.org/10.1182/blood.v128.22.403.403.
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Abstract PU.1 is a transcription factor absolutely required for normal hematopoiesis. Cumulating evidence indicates that precise levels of PU.1 expression are critical for differentiation to distinct blood lineages, and if perturbed, even modest decreases in PU.1 can lead to leukemogenesis. In contrast to extensive knowledge of regulation of PU.1 gene itself, the mechanism of how target genes senses different PU.1 levels remain largely unknown. To address this, we used PU.1-/- mouse myeloid progenitors encoding inducible PU.1 transgene (PU.1ER, PUER, Walsh 2002) that allows tight control of PU.1 activity. Interestingly, intermediate PU.1 activity induced differentiation of PUER progenitors into granulocyte like cells, while high PU.1 produced macrophages, supporting the model that different PU.1 expression is not a consequence but a driver of cell fate choice. Global expression analysis using 4 different levels of PU.1 at 8 time points (2-96 hrs) revealed that granulocyte specific genes were activated exclusively by intermediate PU.1 levels in 3 distinct modes: 1. not expressed in progenitors while strongly induced at intermediate PU.1 (e.g. Gelatinase B (Mmp9) and Neutrophil collagenase (NC) 2. moderately expressed in progenitors while strongly activated at intermediate PU.1 and repressed at high PU.1 (e.g. Myeloperaxidase (Mpo) 3. highly expressed in unstimulated progenitors with expression maintained at intermediate PU.1 but strongly repressed at high PU.1 (e.g. Neutrophil elastase (NE), Proteinase 3 (primary granule proteins), Cebpe and Gfi1 (Growth factor independent1) Majority of macrophage genes (incl. CD14, Csf1R, Egr2) were regulated as early PU.1 target genes; being gradually activated by high PU.1 activity within 8hrs. However, most granulocyte genes (NE, Mmp9, Mpo, NC but not Cebpe and GFI1) were late activated PU.1 targets (48 and 96hrs) indicating that these genes are coregulated by additional factor(s), likely an early PU.1 target. Next we analyzed the regulatory sequences (+-50kb) of two genes activated exclusively by intermediate PU.1, Mpo and Mmp9, using own and public ChIP(seq) data of transcription factors (TFs) (PU.1, GFI.1), DNAseI hypersensitive sites, histone modifications (H3K4Me, H3K27Ac, H3K9Ac) and expression of enhancer specific bidirectional ncRNAs (eRNA) (CAGE). 14 Mpo and 16 Mmp9 putative enhancers, selected by above mentioned criteria, were cloned into luciferase vector containing their proximal promoter (PP) and were tested for functional activity in response to PU.1 levels. Interestingly, the PU.1 binding motifs within these regions have a low to intermediate affinity (log of score, Jaspar) and are often present in multiples and/or enriched for binding sites of other lineage determining transcription factors. Although PU.1 bound to all of these DNA regions resembling superenhancer, just a small fraction of PU.1 binding was functionally responsive. Specifically, we identified novel enhancer elements at -3.4 kb and -15kb of MPO which were activated by intermediate (but not high) PU.1 levels. Interestingly, activity of -3.4 kb enhancer required presence of PP, while the -15kb element required presence of both PP and the -3.4kb element. Similar phenomenon was observed at -5kb and +4.6kb (intronic) MMP9 enhancers. Collectively, these observations suggest that a cooperative assembly of several cell type-specific enhancers is required for optimal Mpo and Mmp9 activation. This model is supported by our Chromosome conformation capture (3C) data identifying 3D interaction of these enhancer elements at intermediate PU.1 levels suggesting that PU.1 binding mediates DNA looping that allows enhancer cooperation. In addition, activity of these enhancers at intermediate PU.1 levels was associated with expression of bidirectional noncoding enhancer RNAs, confirming functionality of these elements. In conclusion, our data support the model that PU.1 at intermediate concentration binds to low and intermediate affinity binding sites in several enhancers of granulocyte genes, causing their successive looping and interaction with proximal promoter that leads to transcription activation. The role of cooperating TFs, mechanisms of how granulocyte genes are switched off at high PU.1 concentration and deregulation of these mechanisms in AML are being further studied. Grants 16-05649S P305/12/1033 16-31586A 16-27790A 16-31586A UNCE 204021 PRVOUK P24 Disclosures No relevant conflicts of interest to declare.
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Roccaro, Aldo M., Antonio Sacco, Dongdong Ma, Jiantao Shi, Yuji Mishima, Michele Moschetta, Robert I. Handin, and Irene M. Ghobrial. "A New Model for Studying the Dissemination of Myeloma Cells throughout the Bone Marrow Using Embryonic Zebrafish." Blood 126, no. 23 (December 3, 2015): 915. http://dx.doi.org/10.1182/blood.v126.23.915.915.
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Abstract Multiple myeloma develops from a pre-malignant clonal proliferation of plasma cells. The dissemination of myeloma cells throughout the bone marrow (BM) is an important early step in myeloma pathogenesis. Studies of myeloma cell homing in mouse models are not quantitative and cannot be used for functional genomics or drug screening. To overcome these limitations, we have developed a novel in vivo model to assess multiple myeloma (MM) cell homing, that takes advantage of the optical clarity of zebrafish (Danio rerio) embryos. We performed intra-cardiac (i.c.) injection of GFP+MM.1S cells into zebrafish embryos 48 hpf, and assessed the ability of the injected cells to enter the peripheral circulation and then traced their homing to the trunk region of zebrafish embryos, an area referred as the caudal hematopoietic tissue (CHT), by using intra-vital confocal microscopy. We next injected primary CD138+ cells derived from MM patient BM that had been stained with either DiO or DiD and demonstrated that they also homed to CHT. As a control, we injected DiO-labeled CD41-GFPlow zebrafish hematopoietic stem cells (HSCs) that are known to home to the CHT hematopoietic niche; and observed that zebrafish-derived CD41-HSCs homed to the same area as MM cells. We next analyzed changes in the transcriptome of those MM cells that homed to the CHT-niche. We dissected the zebrafish embryos to separate the CHT from other tissue containing non-adherent MM cells and performed whole human exome enrichment prior to sequencing of total RNA. We had an alignment rate of 10-15%, with a high intragenic rate an exonic rate (> 95%) and a low mismatch rate (~0.5%). RNAseq revealed that the MM cells that homed to the CHT were enriched in transcripts important for cytokine/chemokine mediated signaling, the IL-6 signalling pathway, cell-cell adhesion and angiogenesis (FDR<0.25; P<0.05). Overall, these findings indicate that the changes observed in MM cells that have homed to the CHT mirror those that are seen in MM cells that are resident in the human BM. In order to investigate the functional relevance of the zebrafish model, we established CXCR4-, VLA-4- and FAK-silenced MM cells and compared their ability to home to CHT to that of control scrambled shRNA-transfected cells. DiO-labeled-CXCR4-silenced and DiO-labeled-scrambled-probe control MM cells were mixed in equal numbers and subsequently injected into recipient zebrafish. We found a significant reduction in the number of CXCR4-silenced MM cells homing to the CHT, compared to the control cells (P<0.00). We then examined VLA-4- and FAK-knock-down MM cells and observed that the homing of MM cells to CHT was impaired when either VLA-4 or FAK were silenced (P<0.001; Fig. 3C-D). Having demonstrated the role of CXCF4, FAK and VLA4 in MM cell homing to the CHT niche, we next performed qRT-PCR for those transcripts and confirmed that MM cells harvested from the CHT areas expressed higher levels of CXCR4, FAK and VLA4, compared to MM cells harvested from non-CHT areas. (P<0.05). To ascertain whether homing to zebrafish embryo CHT is occurs in other hematologic malignancies that are known to home to the human and murine BM, we used a cultured cell lined derived from a patient with Waldenstrom's Macroglobulinemia (WM). We injected either CXCR4-overexpressing or CXCR4-silenced WM cells and found that increased CXCR4 expression in WM cells led to enhanced CHT-homing of WM cells (P<0.001), while the homing of CXCR4-silenced WM cells to the CHT was reduced compared to scrambled control (P<0.001). These findings demonstrate that zebrafish can be used to study the homing of human myeloma cells to a hematopoietic niche. The rapidity of homing to CHT, which occurs within seconds of cell injection, suggests that a fraction of the CD138+ harvested from patient bone marrow already express those RNA transcripts and proteins needed for stable adhesion and residence in CHT. This hypothesis is confirmed by the RNA seq and qRT-PCR studies which directly demonstrate increased expression of relevant transcripts in adherent cells. This zebrafish model may provide new insights into the pathogenesis of MM and may be useful as a means to screen for agents which can disrupt homing and dissemination of MM cells. Disclosures No relevant conflicts of interest to declare.
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Jiang, Hua, Abdulraouf Ramadan, Becquet Laurine, Tu Szu-Wei, Hong Liu, Courtney Rowan, Xiaowen Liu, Huanmei Wu, Jun Wan, and Sophie Paczesny. "IL-33 Therapy Prevents Acute Lung Injury after Transplantation Via IL-9-Producing Type 2 Innate Lymphoid Cells Induction." Blood 134, Supplement_1 (November 13, 2019): 583. http://dx.doi.org/10.1182/blood-2019-123821.
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Idiopathic pneumonia syndrome (IPS) is a noninfectious acute lung injury, often fatal, following allogeneic hematopoietic cell transplantation (HCT). Similar to graft-versus-host disease (GVHD), IPS is mediated by type 1 cytopathic T cells accompanied with high levels of proinflammatory cytokines. We previously showed that elevated plasma soluble Stimulation-2 (sST2), which acts as a decoy receptor for IL-33, is a risk factor of death by GVHD (N. Engl. J. Med, 2013) or by IPS (Biol Blood Marrow Transplant, 2018). ST2 blockade of the excess of sST2 with a neutralizing antibody or small molecules released plasmatic IL-33, increasing its availability to cytoprotective T cells expressing the transmembrane molecule form of ST2, such as regulatory T cells (Tregs) reducing the type 1 proinflammatory T cell-response (Sci Transl Med, 2015; JCI Insight, 2019). The membrane-ST2 is also expressed on type 2 innate lymphoid cells (ILC2s), mostly present in lungs. Herein, we first confirmed in a cohort of 673 HCT patients that plasma sST2 measured 14 days following HCT is increased 10 fold in IPS patients (n=22) as compared to controls with no IPS/GVHD (n=271), and is 6 fold higher as compared to GVHD patients (n=380) (Figure 1A). Patients with IPS and high sST2 levels above the median of 200 ng/ml, were significantly more likely to die than patients with lower sST2 levels (Figure 1B). In a therapeutic translational purpose, we then inquired if local administration of IL-33 via intranasal route at a dose of 500 ng/mouse daily (5 doses from day -1 to +3) will ameliorate the recipients' pulmonary function tests in a major-mismatched B6 → Balb/c HCT murine model. Allogeneic recipients that received IL-33 improved their lung compliance (C), lung resistance (R), and elastance (E) as compared to vehicle treated mice (Figure 2A). Based on our patients' data, we further explored the sST2/IL-33 ratio. Although the treatment was local, plasma IL-33 increased at day +7 post-HCT and therefore the sST2/IL-33 ratio was significantly decreased in IL-33 treated mice (Figure 2B). Parraleling this decrease, both systemic IFNγ and TNFα at day +7 post-HCT were significantly lower in mice treated with IL-33 compared to vehicle treated mice (Figure 2B). Findings in the plasma were also correlated with a local decrease of IFNγ secretion in the bronchoalveolar lavage of IL-33 treated mice (not shown). The frequencies and numbers of donor CD45.1+ IFNg+CD4+ and IFNg+CD8+ donor T cells in the lungs of IL-33 treated mice were also significantly decreased as compared to vehicle treated mice (Figure 2C). We next sought to determine if IL-33 had an impact on recipient ILC2s (CD45.2+Lin-CD90.2+GATA3+ST2+). As shown in Figure 2D, recipient mice treated with IL-33 had significant higher frequencies of lung ILC2s at day +7 post-HCT compared to mice treated with vehicle. RNA-seq analysis of sorted ILC2s from the lungs of naïve GATA3 reporter mice treated with IL-33 showed increased Il9 and PU.1 transcripts in lung ILC2s, validated at the protein level in allogeneic mice treated with IL-33 as compared to allogeneic vehicle treated mice in which ILC2s were undetectebale (Figure 2D). As antibody (Ab) injection is more clinically relevant than local cytokine instillation, and since we have shown that anti-ST2 Ab results in IL-33 increase, we tested this in a minor-mismatched B6 → C3H.SW HCT murine model, and respectively treated mice with anti-ST2 Ab 100μg/dose every other day (6 doses total) or anti-ST2 Ab 200μg/dose for 2 doses at days -1 and +1 or isotype 100μg/dose for 6 doses. Prophylactic administration of anti-ST2 Ab with 6 doses and 2 doses significantly decreases mortality as compared to isotype with six doses allowing a better survival than the peritransplant administration (Figure 3A). Plasma IL-33 increased in both anti-ST2 treated groups vs. isotype (Figure 3B). Consistently, both plasma IFNγ and TNFα were significantly decreased in anti-ST2 Ab treated groups (Figures 3C, 3D). Percentages of cytopathic lung donor CD4+IFNγ+ and CD8+IFNγ+ T cells were decreased (Figure 3E) while cytoprotective lung recipient total, IL-9+, and PU.1+ ILC2s were increased in anti-ST2 Ab treated groups vs. isotype (Figures 3F, 3G). Tregs in both anti-ST2 Ab treated groups were concomitantly increased (Figure 3H). We concluded that not only is sST2 a prognostic biomarker for IPS but it is also a promising therapeutic target that may prevent IPS via IL-33 induced IL-9 secreting ILC2s. Disclosures Paczesny: Viracor Eurofins Clinical Diagnostic: Patents & Royalties.
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Guin, Krishnendu, Yao Chen, Radha Mishra, Siti Rawaidah BM Muzaki, Bhagya C. Thimmappa, Caoimhe E. O'Brien, Geraldine Butler, Amartya Sanyal, and Kaustuv Sanyal. "Spatial inter-centromeric interactions facilitated the emergence of evolutionary new centromeres." eLife 9 (May 29, 2020). http://dx.doi.org/10.7554/elife.58556.
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Centromeres of Candida albicans form on unique and different DNA sequences but a closely related species, Candida tropicalis, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of centromere type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of C. tropicalis by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in C. tropicalis. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of C. albicans and C. tropicalis. Identification of putative centromeres in closely related Candida sojae, Candida viswanathii and Candida parapsilosis indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in C. albicans. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.
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Statkiewicz, Malgorzata, Natalia Maryan, Maria Kulecka, Urszula Kuklinska, Jerzy Ostrowski, and Michal Mikula. "Functional analyses of a low-penetrance risk variant rs6702619/1p21.2 associating with colorectal cancer in Polish population." Acta Biochimica Polonica, September 17, 2019. http://dx.doi.org/10.18388/abp.2019_2775.
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Several studies employed the genome-wide association (GWA) analysis of single-nucleotide polymorphisms (SNPs) to identify susceptibility regions in colorectal cancer (CRC). However, the functional studies exploring the role of associating SNPs with cancer biology are limited. Herein, using chromatin immunoprecipitation assay (ChIP), reporter assay and chromosome conformation capture sequencing (3C-Seq) augmented with publically available genomic and epigenomic databases we aimed to define the function of rs6702619/1p21.2 region associated with CRC in the Polish population. Using ChIP we confirmed that rs6702619 region is occupied by a CTCF, a master regulator of long-range genomic interactions, and is decorated with enhancer-like histone modifications. The enhancer blocking assay revealed that rs6702619 region acts as an insulator with activity dependent on the SNP genotype. Finally, a 3C-Seq survey indicated more than a hundred loci in the rs6702619 locus interactome, including GNAS gene that is frequently amplified in CRC. Taken together, we showed that the CRC-associated rs6702619 region has in vitro and in vivo properties of an insulator that demonstrates long-range physical interactions with CRC-relevant loci.
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Zheng, Jun-Yi, Chen-Yu Wang, Chuan Gao, Qiong Xiao, Cheng-Wei Huang, Min Wu, and Lian-Yun Li. "MLL3 suppresses tumorigenesis through regulating TNS3 enhancer activity." Cell Death & Disease 12, no. 4 (April 2021). http://dx.doi.org/10.1038/s41419-021-03647-2.
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AbstractMLL3 is a histone H3K4 methyltransferase that is frequently mutated in cancer, but the underlying molecular mechanisms remain elusive. Here, we found that MLL3 depletion by CRISPR/sgRNA significantly enhanced cell migration, but did not elevate the proliferation rate of cancer cells. Through RNA-Seq and ChIP-Seq approaches, we identified TNS3 as the potential target gene for MLL3. MLL3 depletion caused downregulation of H3K4me1 and H3K27ac on an enhancer ~ 7 kb ahead of TNS3. 3C assay indicated the identified enhancer interacts with TNS3 promoter and repression of enhancer activity by dCas9-KRAB system impaired TNS3 expression. Exogenous expression of TNS3 in MLL3 deficient cells completely blocked the enhanced cell migration phenotype. Taken together, our study revealed a novel mechanism for MLL3 in suppressing cancer, which may provide novel targets for diagnosis or drug development.
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van der Weide, Robin H., Teun van den Brand, Judith H. I. Haarhuis, Hans Teunissen, Benjamin D. Rowland, and Elzo de Wit. "Hi-C analyses with GENOVA: a case study with cohesin variants." NAR Genomics and Bioinformatics 3, no. 2 (April 9, 2021). http://dx.doi.org/10.1093/nargab/lqab040.
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Abstract Conformation capture-approaches like Hi-C can elucidate chromosome structure at a genome-wide scale. Hi-C datasets are large and require specialised software. Here, we present GENOVA: a user-friendly software package to analyse and visualise chromosome conformation capture (3C) data. GENOVA is an R-package that includes the most common Hi-C analyses, such as compartment and insulation score analysis. It can create annotated heatmaps to visualise the contact frequency at a specific locus and aggregate Hi-C signal over user-specified genomic regions such as ChIP-seq data. Finally, our package supports output from the major mapping-pipelines. We demonstrate the capabilities of GENOVA by analysing Hi-C data from HAP1 cell lines in which the cohesin-subunits SA1 and SA2 were knocked out. We find that ΔSA1 cells gain intra-TAD interactions and increase compartmentalisation. ΔSA2 cells have longer loops and a less compartmentalised genome. These results suggest that cohesinSA1 forms longer loops, while cohesinSA2 plays a role in forming and maintaining intra-TAD interactions. Our data supports the model that the genome is provided structure in 3D by the counter-balancing of loop formation on one hand, and compartmentalization on the other hand. By differentially controlling loops, cohesinSA1 and cohesinSA2 therefore also affect nuclear compartmentalization. We show that GENOVA is an easy to use R-package, that allows researchers to explore Hi-C data in great detail.
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Khalil, Ahmed Ibrahim Samir, Siti Rawaidah Binte Mohammad Muzaki, Anupam Chattopadhyay, and Amartya Sanyal. "Identification and utilization of copy number information for correcting Hi-C contact map of cancer cell lines." BMC Bioinformatics 21, no. 1 (November 7, 2020). http://dx.doi.org/10.1186/s12859-020-03832-8.
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Abstract Background Hi-C and its variant techniques have been developed to capture the spatial organization of chromatin. Normalization of Hi-C contact map is essential for accurate modeling and interpretation of high-throughput chromatin conformation capture (3C) experiments. Hi-C correction tools were originally developed to normalize systematic biases of karyotypically normal cell lines. However, a vast majority of available Hi-C datasets are derived from cancer cell lines that carry multi-level DNA copy number variations (CNVs). CNV regions display over- or under-representation of interaction frequencies compared to CN-neutral regions. Therefore, it is necessary to remove CNV-driven bias from chromatin interaction data of cancer cell lines to generate a euploid-equivalent contact map. Results We developed the HiCNAtra framework to compute high-resolution CNV profiles from Hi-C or 3C-seq data of cancer cell lines and to correct chromatin contact maps from systematic biases including CNV-associated bias. First, we introduce a novel ‘entire-fragment’ counting method for better estimation of the read depth (RD) signal from Hi-C reads that recapitulates the whole-genome sequencing (WGS)-derived coverage signal. Second, HiCNAtra employs a multimodal-based hierarchical CNV calling approach, which outperformed OneD and HiNT tools, to accurately identify CNVs of cancer cell lines. Third, incorporating CNV information with other systematic biases, HiCNAtra simultaneously estimates the contribution of each bias and explicitly corrects the interaction matrix using Poisson regression. HiCNAtra normalization abolishes CNV-induced artifacts from the contact map generating a heatmap with homogeneous signal. When benchmarked against OneD, CAIC, and ICE methods using MCF7 cancer cell line, HiCNAtra-corrected heatmap achieves the least 1D signal variation without deforming the inherent chromatin interaction signal. Additionally, HiCNAtra-corrected contact frequencies have minimum correlations with each of the systematic bias sources compared to OneD’s explicit method. Visual inspection of CNV profiles and contact maps of cancer cell lines reveals that HiCNAtra is the most robust Hi-C correction tool for ameliorating CNV-induced bias. Conclusions HiCNAtra is a Hi-C-based computational tool that provides an analytical and visualization framework for DNA copy number profiling and chromatin contact map correction of karyotypically abnormal cell lines. HiCNAtra is an open-source software implemented in MATLAB and is available at https://github.com/AISKhalil/HiCNAtra.
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Jonsson Boezelman, M., A. Dashi, W. Tan, B. Pan, A. M. Ilmari, Z. Tiang, R. J. G. Hartman, et al. "VENTHEART is required for cardiomyocyte specification and function." European Heart Journal 41, Supplement_2 (November 1, 2020). http://dx.doi.org/10.1093/ehjci/ehaa946.3575.
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Abstract Background Long noncoding RNAs (lncRNAs) control early stages of cardiac differentiation, however their role in later specification and maturation is still not well explored. Methods and results We performed single cell RNA-seq for 2, 6 and 12 week-old hESC-CM. Weighted correlation network analysis (WGCNA) identified core genes significantly upregulated, along with a subset of lncRNAs. Importantly, these lncRNAs are highly abundant and unique to human heart. Through independent integrative analysis of genome-wide association studies (GWAS) and expression quantitative trait locus (eQTL) data using human hearts, we also identified a long intergenic noncoding RNA (we call VENTHEART, VHRT) as co-regulated with core cardiac contractile genes, and strongly associated with heart failure. VHRT was highly expressed in MYL2+ hESC-CMs in our single cell dataset, and its locus is antisense and downstream of MYL2. VHRT knockdown (KD) in 6-weeks old hESC-CMs downregulated MYL2 and other key cardiac genes. Patch clamp recordings with VHRT KD cells showed a loss of the ventricular-like action potential. Concordantly, CRISPR-mediated excision of the VHRT locus led to impaired CM sarcomere formation, and loss of CM specification gene programs. VHRT transcript replacement in VHRT-KO cells was however insufficient to rescue the phenotype. Instead, we established by 3C assay, that the VHRT locus loops and interacts with the MYL2 promoter, bearing histone marks characteristic of a super-enhancer. Conclusion Thus, we conclude that both the VHRT lncRNA transcript and its genomic locus are required for proper CM specification and function, and may play a role in heart failure progression. Funding Acknowledgement Type of funding source: Other. Main funding source(s): EMBO, Singapore National Research Council
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Wang, Fang, Song Chen, Deyang Liang, Guan-Zheng Qu, Su Chen, and Xiyang Zhao. "Transcriptomic analyses of Pinus koraiensis under different cold stresses." BMC Genomics 21, no. 1 (January 3, 2020). http://dx.doi.org/10.1186/s12864-019-6401-y.
Full textAbstract:
Abstract Background Pinus koraiensis is an evergreen tree species with strong cold resistance. However, the transcriptomic patterns in response to cold stress are poorly understood for P. koraiensis. In this study, global transcriptome profiles were generated for P. koraiensis under cold stress (− 20 °C) over time by high-throughput sequencing. Results More than 763 million clean reads were produced, which assembled into a nonredundant data set of 123,445 unigenes. Among them, 38,905 unigenes had homology with known genes, 18,239 were assigned to 54 gene ontology (GO) categories and 18,909 were assigned to 25 clusters of orthologous groups (COG) categories. Comparison of transcriptomes of P. koraiensis seedlings grown at room temperature (20 °C) and low temperature (− 20 °C) revealed 9842 differential expressed genes (DEGs) in the 6 h sample, 9250 in the 24 h sample, and 9697 in the 48 h sample. The number of DEGs in the pairwise comparisons of 6 h, 24 h and 48 h was relatively small. The accuracy of the RNA-seq was validated by analyzing the expression patterns of 12 DEGs by quantitative real-time PCR (qRT-PCR). In this study, 34 DEGs (22 upregulated and 12 downregulated) were involved in the perception and transmission of cold signals, 96 DEGs (41 upregulated and 55 downregulated) encoding 8 transcription factors that regulated cold-related genes expression, and 27 DEGs (17 upregulated and 10 downregulated) were involved in antioxidant mechanisms in response to cold stress. Among them, the expression levels of c63631_g1 (annexin D1), c65620_g1 (alpha-amylase isozyme 3C), c61970_g1 (calcium-binding protein KIC), c51736_g1 (ABA), c58408_g1 (DREB3), c66599_g1 (DREB3), c67548_g2 (SOD), c55044_g1 (CAT), c71938_g2 (CAT) and c11358_g1 (GPX) first increased significantly and then decreased significantly with the extension of stress time. Conclusions A large number of DEGs were identified in P. koraiensis under cold stress, especially the DEGs involved in the perception and transmission of cold signals, the DEGs encoding TFs related to cold regulation and the DEGs removing ROS in antioxidation mechanisms. The transcriptome and digital expression profiling of P. koraiensis could facilitate the understanding of the molecular control mechanism related to cold responses and provide the basis for the molecular breeding of conifers.