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1
Takeda, Kotaro, Hector L. Aguila, Nehal S. Parikh, et al. "Regulation of adult erythropoiesis by prolyl hydroxylase domain proteins." Blood 111, no. 6 (2008): 3229–35. http://dx.doi.org/10.1182/blood-2007-09-114561.
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Abstract Polycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)–containing proteins, which tag hypoxia-inducible factor (HIF) α subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-α stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1−/− or Phd3−/− mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2α, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1α accumulated in both the kidney and liver. Elevated HIF-1α levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2α/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway.
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Takeda, Kotaro, Vivienne C. Ho, Hiromi Takeda, Li-Juan Duan, Andras Nagy та Guo-Hua Fong. "Placental but Not Heart Defects Are Associated with Elevated Hypoxia-Inducible Factor α Levels in Mice Lacking Prolyl Hydroxylase Domain Protein 2". Molecular and Cellular Biology 26, № 22 (2006): 8336–46. http://dx.doi.org/10.1128/mcb.00425-06.
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ABSTRACT PHD1, PHD2, and PHD3 are prolyl hydroxylase domain proteins that regulate the stability of hypoxia-inducible factor α subunits (HIF-α). To determine the roles of individual PHDs during mouse development, we disrupted all three Phd genes and found that Phd2 − / − embryos died between embryonic days 12.5 and 14.5 whereas Phd1 −/− or Phd3 −/− mice were apparently normal. In Phd2 − / − mice, severe placental and heart defects preceded embryonic death. Placental defects included significantly reduced labyrinthine branching morphogenesis, widespread penetration of the labyrinth by spongiotrophoblasts, and abnormal distribution of trophoblast giant cells. The expression of several trophoblast markers was also altered, including an increase in the spongiotrophoblast marker Mash2 and decreases in the labyrinthine markers Tfeb and Gcm1. In the heart, trabeculae were poorly developed, the myocardium was remarkably thinner, and interventricular septum was incompletely formed. Surprisingly, while there were significant global increases in HIF-α protein levels in the placenta and the embryo proper, there was no specific HIF-α increase in the heart. Taken together, these data indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.
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Pappalardi, Melissa B., Dean E. McNulty, John D. Martin, et al. "Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites." Biochemical Journal 436, no. 2 (2011): 363–69. http://dx.doi.org/10.1042/bj20101201.
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The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1–3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography–tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.
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Barth, Sandra, Jutta Nesper, Philippe A. Hasgall, et al. "The Peptidyl Prolyl cis/trans Isomerase FKBP38 Determines Hypoxia-Inducible Transcription Factor Prolyl-4-Hydroxylase PHD2 Protein Stability." Molecular and Cellular Biology 27, no. 10 (2007): 3758–68. http://dx.doi.org/10.1128/mcb.01324-06.
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ABSTRACT The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-α subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-α for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.
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Wu, Qi, and Chuan Tang Wang. "Genome Wide Analysis of PHD Finger Family in Soybean (Glycine max)." Advanced Materials Research 864-867 (December 2013): 2503–8. http://dx.doi.org/10.4028/www.scientific.net/amr.864-867.2503.
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The PHD finger is a highly conserved structural domain in roles with regulating transcription and modification of chromatin structure. Forty-five PHD finger genes encoding PHD finger protein were identified from soybean (Glycine max) database. And sixty - four unique typical PHD finger domains were retrieved. NJ phylogenetic tree of all 64 PHD finger domains consisted of ten main clades (A-J). Subcellular localization analysis shows that Glyma06g33590.1, Glyma10g05080.1 and Glyma11g11720.1 may localize in Golgi body, chloroplast thylakoid membrane and mitochondrial inner membrane, respectively. The function of domain is loyal to the cause of protein situated in particular site of cell. Eight unique domains have been found concomitant with PHD domain in a certain protein. The cooperative relationship between diverse domains may important for particular biological event.
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Kampantais, Spyridon, Ilias Kounatidis, Vasiliki Kotoula, Ioannis Vakalopoulos, Konstantinos Gkagkalidis, and Georgios Dimitriadis. "Decreased prolyl hydroxylase 3 mRNA expression in oncocytomas compared with clear cell renal cell carcinoma." International Journal of Biological Markers 35, no. 4 (2020): 80–86. http://dx.doi.org/10.1177/1724600820960478.
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Introduction: Hypoxia inducible factors (HIF) and prolyl hydroxylase domain (PHD) enzymes play a central role in tumor progression in clear cell renal cell carcinoma (ccRCC). However, there are currently no data regarding the behavior of this pathway (HIF/PHD) in a large number of benign renal tumors, the oncocytomas. The aim of the present study was to compare the expression levels of these factors between ccRCC and oncocytoma tumors. Material and methods: A total of 56 fresh frozen specimens from patients with ccRCC and 14 oncocytoma specimens were analyzed via reverse transcription-quantitative polymerase chain reaction in order to assess the expression levels of HIF-1α, HIF-2α, PHD1, PHD2, and PHD3. The analysis involved both fresh frozen tumor samples as well as adjacent normal kidney tissues. Results: In ccRCC, HIF-1α and HIF-2α levels were upregulated in 65.5% and 71.4% of cases, respectively. PHD3 was downregulated only in 15.4% of the ccRCC cases, in contrast with oncocytoma cases, which exhibited low expression levels in the majority. The upregulation of PHD3 messenger RNA (mRNA) levels in ccRCC when compared with oncocytoma was statistically significant ( P<0.001). No other comparisons (HIF-1α, HIF-2α, PHD1, and PHD2) were significantly different. HIF-2α and PHD3 mRNA expression levels were negatively correlated with Fuhrman Grade ( P=0.029 and P=0.026, respectively) in ccRCC. Conclusion: To the best of our knowledge, this is the first time that the HIF/PHD pathway was compared between ccRCC and a common benign tumor, identifying the upregulation of PHD3 as the possible underlying factor guiding the difference in the behavior of ccRCC.
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Köditz, Jens, Jutta Nesper, Marieke Wottawa, et al. "Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor." Blood 110, no. 10 (2007): 3610–17. http://dx.doi.org/10.1182/blood-2007-06-094441.
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Abstract The activating transcription factor-4 (ATF-4) is translationally induced under anoxic conditions, mediates part of the unfolded protein response following endoplasmic reticulum (ER) stress, and is a critical regulator of cell fate. Here, we identified the zipper II domain of ATF-4 to interact with the oxygen sensor prolyl-4-hydroxylase domain 3 (PHD3). The PHD inhibitors dimethyloxalylglycine (DMOG) and hypoxia, or proteasomal inhibition, all induced ATF-4 protein levels. Hypoxic induction of ATF-4 was due to increased protein stability, but was independent of the ubiquitin ligase von Hippel–Lindau protein (pVHL). A novel oxygen-dependent degradation (ODD) domain was identified adjacent to the zipper II domain. Mutations of 5 prolyl residues within this ODD domain or siRNA-mediated down-regulation of PHD3, but not of PHD2, was sufficient to stabilize ATF-4 under normoxic conditions. These data demonstrate that PHD-dependent oxygen-sensing recruits both the hypoxia-inducible factor (HIF) and ATF-4 systems, and hence not only confers adaptive responses but also cell fate decisions.
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Machado, Luciana E. F., Yulia Pustovalova, Andrew C. Kile, et al. "PHD domain from human SHPRH." Journal of Biomolecular NMR 56, no. 4 (2013): 393–99. http://dx.doi.org/10.1007/s10858-013-9758-2.
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Zheng, Shuangping, Yucong Bi, Haining Chen, Bo Gong, Shunji Jia, and Haitao Li. "Molecular basis for bipartite recognition of histone H3 by the PZP domain of PHF14." Nucleic Acids Research 49, no. 15 (2021): 8961–73. http://dx.doi.org/10.1093/nar/gkab670.
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Abstract Histone recognition constitutes a key epigenetic mechanism in gene regulation and cell fate decision. PHF14 is a conserved multi-PHD finger protein that has been implicated in organ development, tissue homeostasis, and tumorigenesis. Here we show that PHF14 reads unmodified histone H3(1–34) through an integrated PHD1-ZnK-PHD2 cassette (PHF14PZP). Our binding, structural and HDX-MS analyses revealed a feature of bipartite recognition, in which PHF14PZP utilizes two distinct surfaces for concurrent yet separable engagement of segments H3-Nter (e.g. 1–15) and H3-middle (e.g. 14–34) of H3(1–34). Structural studies revealed a novel histone H3 binding mode by PHD1 of PHF14PZP, in which a PHF14-unique insertion loop but not the core β-strands of a PHD finger dominates H3K4 readout. Binding studies showed that H3-PHF14PZP engagement is sensitive to modifications occurring to H3 R2, T3, K4, R8 and K23 but not K9 and K27, suggesting multiple layers of modification switch. Collectively, our work calls attention to PHF14 as a ‘ground’ state (unmodified) H3(1–34) reader that can be negatively regulated by active marks, thus providing molecular insights into a repressive function of PHF14 and its derepression.
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Li, Ningjun, Fan Yi, Christina M. Sundy, et al. "Expression and actions of HIF prolyl-4-hydroxylase in the rat kidneys." American Journal of Physiology-Renal Physiology 292, no. 1 (2007): F207—F216. http://dx.doi.org/10.1152/ajprenal.00457.2005.
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Hypoxia inducible factor (HIF) prolyl-4-hydroxylase domain-containing proteins (PHDs) promote the degradation of HIF-1α. Because HIF-1α is highly expressed in the renal medulla and HIF-1α-targeted genes such as nitric oxide synthase, cyclooxygenase, and heme oxygenase are important in the regulation of renal medullary function, we hypothesized that PHD regulates HIF-1α levels in the renal medulla and, thereby, participates in the control of renal Na+ excretion. Using real-time RT-PCR, Western blot, and immunohistochemical analyses, we have demonstrated that all three isoforms of PHD, PHD1, PHD2, and PHD3, are expressed in the kidneys and that PHD2 is the most abundant isoform. Regionally, all PHDs exhibited much higher levels in renal medulla than cortex. A furosemide-induced increase in renal medullary tissue Po2 significantly decreased PHD levels in renal medulla, whereas hypoxia significantly increased mRNA levels of PHDs in cultured renal medullary interstitial cells, indicating that O2 regulates PHDs. Functionally, the PHD inhibitor l-mimosine (l-Mim, 50 mg·kg−1·day−1 ip for 2 wk) substantially upregulated HIF-1α expression in the kidneys, especially in the renal medulla, and remarkably enhanced (by >80%) the natriuretic response to renal perfusion pressure in Sprague-Dawley rats. Inhibition of HIF transcriptional activity by renal medullary transfection of HIF-1α decoy oligodeoxynucleotides attenuated l-Mim-induced enhancement of pressure natriuresis, which confirmed that HIF-1α mediated the effect of l-Mim. These results indicate that highly expressed PHDs in the renal medulla make an important contribution to the control of renal Na+ excretion through regulation of HIF-1α and its targeted genes.
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Mazurek, Sylwia, Urszula Oleksiewicz, Patrycja Czerwińska, Joanna Wróblewska, Marta Klimczak, and Maciej Wiznerowicz. "Disruption of RING and PHD Domains of TRIM28 Evokes Differentiation in Human iPSCs." Cells 10, no. 8 (2021): 1933. http://dx.doi.org/10.3390/cells10081933.
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TRIM28, a multi-domain protein, is crucial in the development of mouse embryos and the maintenance of embryonic stem cells’ (ESC) self-renewal potential. As the epigenetic factor modulating chromatin structure, TRIM28 regulates the expression of numerous genes and is associated with progression and poor prognosis in many types of cancer. Because of many similarities between highly dedifferentiated cancer cells and normal pluripotent stem cells, we applied human induced pluripotent stem cells (hiPSC) as a model for stemness studies. For the first time in hiPSC, we analyzed the function of individual TRIM28 domains. Here we demonstrate the essential role of a really interesting new gene (RING) domain and plant homeodomain (PHD) in regulating pluripotency maintenance and self-renewal capacity of hiPSC. Our data indicate that mutation within the RING or PHD domain leads to the loss of stem cell phenotypes and downregulation of the FGF signaling. Moreover, impairment of RING or PHD domain results in decreased proliferation and impedes embryoid body formation. In opposition to previous data indicating the impact of phosphorylation on TRIM28 function, our data suggest that TRIM28 phosphorylation does not significantly affect the pluripotency and self-renewal maintenance of hiPSC. Of note, iPSC with disrupted RING and PHD functions display downregulation of genes associated with tumor metastasis, which are considered important targets in cancer treatment. Our data suggest the potential use of RING and PHD domains of TRIM28 as targets in cancer therapy.
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Soshnikova, N. V., A. A. Sheynov, Eu V. Tatarskiy, and S. G. Georgieva. "The DPF Domain As a Unique Structural Unit Participating in Transcriptional Activation, Cell Differentiation, and Malignant Transformation." Acta Naturae 12, no. 4 (2020): 57–65. http://dx.doi.org/10.32607/actanaturae.11092.
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The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modifications interacting with the DPF domain have already been identified. They include acetylation of H3K14 and H3K9, as well as crotonylation of H3K14. These modifications are found predominantly in transcriptionally active chromatin. Proteins containing DPF belong to two classes of protein complexes, which are the transcriptional coactivators involved in the regulation of the chromatin structure. These are the histone acetyltransferase complex belonging to the MYST family and the SWI/SNF chromatin-remodeling complex. The DPF domain is responsible for the specificity of the interactions between these complexes and chromatin. Proteins containing DPF play a crucial role in the activation of the transcription of a number of genes expressed during the development of an organism. These genes are important in the differentiation and malignant transformation of mammalian cells.
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Park, Sangho, Robyn L. Stanfield, Maria A. Martinez-Yamout, H. Jane Dyson, Ian A. Wilson, and Peter E. Wright. "Role of the CBP catalytic core in intramolecular SUMOylation and control of histone H3 acetylation." Proceedings of the National Academy of Sciences 114, no. 27 (2017): E5335—E5342. http://dx.doi.org/10.1073/pnas.1703105114.
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The histone acetyl transferases CREB-binding protein (CBP) and its paralog p300 play a critical role in numerous cellular processes. Dysregulation of their catalytic activity is associated with several human diseases. Previous work has elucidated the regulatory mechanisms of p300 acetyltransferase activity, but it is not known whether CBP activity is controlled similarly. Here, we present the crystal structure of the CBP catalytic core encompassing the bromodomain (BRD), CH2 (comprising PHD and RING), HAT, and ZZ domains at 2.4-Å resolution. The BRD, PHD, and HAT domains form an integral structural unit to which the RING and ZZ domains are flexibly attached. The structure of the apo-CBP HAT domain is similar to that of acyl-CoA–bound p300 HAT complexes and shows that the acetyl-CoA binding site is stably formed in the absence of cofactor. The BRD, PHD, and ZZ domains interact with small ubiquitin-like modifier 1 (SUMO-1) and Ubc9, and function as an intramolecular E3 ligase for SUMOylation of the cell cycle regulatory domain 1 (CRD1) of CBP, which is located adjacent to the BRD. In vitro HAT assays suggest that the RING domain, the autoregulatory loop (AL) within the HAT domain, and the ZZ domain do not directly influence catalytic activity, whereas the BRD is essential for histone H3 acetylation in nucleosomal substrates. Several lysine residues in the intrinsically disordered AL are autoacetylated by the HAT domain. Upon autoacetylation, acetyl-K1596 (Ac-K1596) binds intramolecularly to the BRD, competing with histones for binding to the BRD and acting as a negative regulator that inhibits histone H3 acetylation.
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Ozurumba, Ezinne, Omana Mathew, Katsuri Ranganna, Myung Choi та Adebayo Oyekan. "Regulation of hypoxia inducible factor/prolyl hydroxylase binding domain proteins 1 by PPARα and high salt diet". Journal of Basic and Clinical Physiology and Pharmacology 29, № 2 (2018): 165–73. http://dx.doi.org/10.1515/jbcpp-2017-0074.
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Abstract Background: Hypoxia inducible factor (HIF)/prolyl hydroxylase domain (PHD)-containing proteins are involved in renal adaptive response to high salt (HS). Peroxisome proliferator activated receptor alpha (PPARα), a transcription factor involved in fatty acid oxidation is implicated in the regulation of renal function. As both HIF-1α/PHD and PPARα contribute to the adaptive changes to altered oxygen tension, this study tested the hypothesis that PHD-induced renal adaptive response to HS is PPARα-dependent. Methods: PPARα wild type (WT) and knock out (KO) mice were fed a low salt (LS) (0.03% NaCl) or a HS (8% NaCl) diet for 8 days and treated with hydralazine. PPARα and heme oxygenase (HO)-1 expression were evaluated in the kidney cortex and medulla. A 24-h urinary volume (UV), sodium excretion (UNaV), and nitrite excretion (UNOx V) were also determined. Results: PHD1 expression was greater in the medulla as compared to the cortex of PPARα WT mice (p<0.05) fed with a LS (0.03% NaCl) diet. The HS diet (8% NaCl) downregulated PHD1 expression in the medulla (p<0.05) but not the cortex of WT mice whereas expression was downregulated in the cortex (p<0.05) and medulla (p<0.05) of KO mice. These changes were accompanied by HS-induced diuresis (p<0.05) and natriuresis (p<0.05) that were greater in WT mice (p<0.05). Similarly, UNOx V, index of renal nitric oxide synthase (NOS) activity or availability and heme oxygenase (HO)-1 expression was greater in WT (p<0.05) but unchanged in KO mice on HS diet. Hydralazine, a PHD inhibitor, did not affect diuresis or natriuresis in LS diet-fed WT or KO mice but both were increased (p<0.05) in HS diet-fed WT mice. Hydralazine also increased UNOx V (p<0.05) with no change in diuresis, natriuresis, or HO-1 expression in KO mice on HS diet. Conclusions: These data suggest that HS-induced PPARα-mediated downregulation of PHD1 is a novel pathway for PHD/HIF-1α transcriptional regulation for adaptive responses to promote renal function via downstream signaling involving NOS and HO.
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Panne, Daniel. "Chromatin recognition and regulation of the acetyltransferase CBP/p300." Acta Crystallographica Section A Foundations and Advances 70, a1 (2014): C1586. http://dx.doi.org/10.1107/s2053273314084137.
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Gene regulation in higher eukaryotes requires recruitment of the transcriptional co-activators CBP/p300 that associate with transcriptional regulators and integrate a large number of signal transduction pathways. Recruitment of CBP/p300 results in acetylation and remodeling of inhibitory chromatin. Recently we have determined the 2.8Å crystal structure of the catalytic core of p300 containing its Bromodomain, the CH2 region and HAT domain in complex with the bi-substrate inhibitor, Lys-CoA. Unexpectedly the structure reveals that the CH2 region contains a discontinuous PHD domain which is interrupted by a RING domain. The Bromodomain, PHD, RING and HAT domains adopt an assembled configuration in which the RING domain is positioned over the HAT substrate binding pocket. Disease mutations that disrupt RING attachment lead to upregulation of HAT activity, revealing an auto-inhibitory role for this domain. Detailed investigation of chromatin substrate recognition showed that the Bromodomain preferentially interacts with histones containing combinations of acetylations rather than singly modified sequences, whereas the p300 PHD domain did not interact with canonical substrates. Our results demonstrate that the Bromodomain substrate specificity is compatible with HAT substrate acetylation patterns suggesting that positive feedback is likely an important component in establishment of active chromatin states. We here present progress in our understanding of the regulation of p300 activity, chromatin modification, readout and how disease-related mutations result in dysregulation of these activities.
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Bienz, Mariann. "The PHD finger, a nuclear protein-interaction domain." Trends in Biochemical Sciences 31, no. 1 (2006): 35–40. http://dx.doi.org/10.1016/j.tibs.2005.11.001.
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Kalkhoven, Eric, Hans Teunissen, Ada Houweling, C. Peter Verrijzer, and Alt Zantema. "The PHD Type Zinc Finger Is an Integral Part of the CBP Acetyltransferase Domain." Molecular and Cellular Biology 22, no. 7 (2002): 1961–70. http://dx.doi.org/10.1128/mcb.22.7.1961-1970.2002.
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ABSTRACT Histone acetyltransferases (HATs) such as CBP and p300 are regarded as key regulators of RNA polymerase II-mediated transcription, but the critical structural features of their HAT modules remain ill defined. The HAT domains of CBP and p300 are characterized by the presence of a highly conserved putative plant homeodomain (PHD) (C4HC3) type zinc finger, which is part of the functionally uncharacterized cysteine-histidine-rich region 2 (CH2). Here we show that this region conforms to the PHD type zinc finger consensus and that it is essential for in vitro acetylation of core histones and the basal transcription factor TFIIE34 as well as for CBP autoacetylation. PHD finger mutations also reduced the transcriptional activity of the full-length CBP protein when tested on transfected reporter genes. Importantly, similar results were obtained on integrated reporters, which reflect a more natural chromatinized state. Taken together, our results indicate that the PHD finger forms an integral part of the enzymatic core of the HAT domain of CBP.
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Chen, Jianji, John Horton, Cari Sagum, Jujun Zhou, Xiaodong Cheng, and Mark T. Bedford. "Histone H3 N-terminal mimicry drives a novel network of methyl-effector interactions." Biochemical Journal 478, no. 10 (2021): 1943–58. http://dx.doi.org/10.1042/bcj20210203.
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The reader ability of PHD fingers is largely limited to the recognition of the histone H3 N-terminal tail. Distinct subsets of PHDs bind either H3K4me3 (a transcriptional activator mark) or H3K4me0 (a transcriptional repressor state). Structural studies have identified common features among the different H3K4me3 effector PHDs, including (1) removal of the initiator methionine residue of H3 to prevent steric interference, (2) a groove where arginine-2 binds, and (3) an aromatic cage that engages methylated lysine-4. We hypothesize that some PHDs might have the ability to engage with non-histone ligands, as long as they adhere to these three rules. A search of the human proteome revealed an enrichment of chromatin-binding proteins that met these criteria, which we termed H3 N-terminal mimicry proteins (H3TMs). Seven H3TMs were selected, and used to screen a protein domain microarray for potential effector domains, and they all had the ability to bind H3K4me3-interacting effector domains. Furthermore, the binding affinity between the VRK1 peptide and the PHD domain of PHF2 is ∼3-fold stronger than that of PHF2 and H3K4me3 interaction. The crystal structure of PHF2 PHD finger bound with VRK1 K4me3 peptide provides a molecular basis for stronger binding of VRK1 peptide. In addition, a number of the H3TMs peptides, in their unmethylated form, interact with NuRD transcriptional repressor complex. Our findings provide in vitro evidence that methylation of H3TMs can promote interactions with PHD and Tudor domain-containing proteins and potentially block interactions with the NuRD complex. We propose that these interactions can occur in vivo as well.
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Perrin, Laurent, Sébastien Bloyer, Conchita Ferraz, Namita Agrawal, Pradip Sinha, and Jean Maurice Dura. "The Leucine Zipper Motif of the Drosophila AF10 Homologue Can Inhibit PRE-Mediated Repression: Implications for Leukemogenic Activity of Human MLL-AF10 Fusions." Molecular and Cellular Biology 23, no. 1 (2003): 119–30. http://dx.doi.org/10.1128/mcb.23.1.119-130.2003.
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ABSTRACT In a screen for Drosophila genes that interfere with transcriptional repression mediated by the Polycomb group of genes, we identified a dominant mutation affecting the Alhambra (Alh) gene, the fly homologue of the human AF10 gene. AF10 has been identified as a fusion partner of both MLL and CALM in infant leukemias. Both fusion proteins retain the leucine zipper domain of AF10 but not its PHD domain. We show here that, while the full-length ALH protein has no activity on Polycomb group-responsive elements (PREs), overexpression of the isolated ALH leucine zipper domain activates several PREs. Within the ALH full-length protein, the PHD domain inhibits the PRE deregulation mediated by the leucine zipper domain. This deregulation is conserved in the human AF10 leucine zipper domain, which confers the same activity on an oncogenic MLL-AF10 fusion protein expressed in Drosophila melanogaster. These data reveal new properties for the leucine zipper domain and thus might provide new clues to understanding the mechanisms by which AF10 fusion proteins in which the PHD domain is lost might trigger leukemias in humans.
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Muntean, Andrew G., and Jay L. Hess. "The MLL PHD Fingers of MLL Block MLL Fusion Protein Mediated Transformation." Blood 110, no. 11 (2007): 976. http://dx.doi.org/10.1182/blood.v110.11.976.976.
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Abstract Mixed Lineage Leukemia (MLL) is a histone H3K4 methyltransferase that is rearranged in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). MLL is required for the maintenance of Hox gene expression. Deregulation of Hox genes by MLL fusion proteins, which fuse MLL in frame to one of over 50 different translocation partners, is critical for transformation. In these translocations, the DNMT homology (CXXC) domain is always included, but the set of adjacent plant homeodomains (PHD), which includes four PHD fingers and a bromodomain, is invariably excluded. PHD fingers have recently been described to bind tri-methylated histone H3K4 and others report PHD domains binding transcriptional co-repressors, such as Mi-2a of the NuRD complex. However, the role of the PHD fingers in MLL is not well understood. To determine the function of the PHD fingers in MLL, we performed bone marrow transduction and colony assays with the MLL fusion protein MLL-AF9, engineered to contain the PHD domain region (MLL-PHD-AF9). These experiments showed that inclusion of the PHD fingers inhibited immortalization as shown by the absence of compact colonies in methylcellulose replating assays and inhibition of proliferation in liquid cultures. Initial experiments with PHD finger deletions to map the inhibiting activity suggest inclusion of any PHD fingers beyond the first PHD finger, results in inhibition of transformation. To monitor the transcriptional activity of the retrovirally infected bone marrow cells, total RNA was isolated from cells harvested after the second replating, when significant differences were seen in colony morphology and size. Consistent with the transformation inhibition, Hoxa9 gene expression was found to be significantly repressed with respect to expression detected in transformed MLL-AF9 cells as determined by qPCR. To confirm this effect is directly due to the MLL fusion proteins, we performed luciferase assays with an MLL responsive myc E-box luciferase construct in MLL −/− MEFs. We found a specific and robust activation of the reporter in the presence of MLL-AF9, which was severely compromised by the inclusion of the PHD fingers. Together, these results suggest the PHD fingers act as transcriptional repressors that inhibit transformation. Our results provide an explanation for the finding that translocations including the coding region for C terminal PHD fingers do not occur in human leukemias and suggest that this region is also involved in the regulation of wild type MLL. We are currently studying the mechanisms of transcriptional repression mediated by the PHD fingers by isolating interacting proteins and assessing their effect on transcription and transformation.
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Capili, A. D. "Solution structure of the PHD domain from the KAP-1 corepressor: structural determinants for PHD, RING and LIM zinc-binding domains." EMBO Journal 20, no. 1 (2001): 165–77. http://dx.doi.org/10.1093/emboj/20.1.165.
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Zieseniss, Anke, Amke R. Hesse, Aline Jatho, et al. "Cardiomyocyte-Specific Transgenic Expression of Prolyl-4-Hydroxylase Domain 3 Impairs the Myocardial Response to Ischemia." Cellular Physiology and Biochemistry 36, no. 3 (2015): 843–51. http://dx.doi.org/10.1159/000430260.
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Aims: The prolyl-4-hydroxylase domain (PHD) enzymes are representing novel therapeutic targets for ischemic tissue protection. Whereas the consequences of a knock out of the PHDs have been analyzed in the context of cardioprotection, the implications of PHD overexpression is unknown so far. Methods and Results: We generated cardiomyocyte-specific PHD3transgenic mice (cPhd3tg). Resting cPhd3tg mice did not show constitutive accumulation of HIF-1α or HIF-2α or changes in HIF target gene expression in the heart. Cardiac function was followed up for 14 months in these mice and found to be unchanged. After challenging the cPhd3tg mice with ligation of the left anterior descending artery, HIF-1α/-2α accumulation in the left ventricles was blunted. This was associated with a significantly increased infarct size of the cPhd3tg compared to wild type mice. Conclusion: Whereas overexpression of PHD3 in the resting state does not significantly influence cardiac function, it is crucial for the cardiac response to ischemia by affecting HIFα accumulation in the ischemic tissue.
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Gough, Sheryl M., Fan Lee, Robert L. Walker, et al. "Leukemia Driven By a NUP98-Phd Domain Fusion Is Highly Sensitive To Disruption Of H3K4me3-Phd Domain Binding By a Small Molecule Inhibitor." Blood 122, no. 21 (2013): 3759. http://dx.doi.org/10.1182/blood.v122.21.3759.3759.
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Abstract NUP98 gene fusions, created by non-random chromosomal translocations, are associated with a wide spectrum of high risk hematologic malignancies, and typically lead to overexpression of abdominal-b HOXA genes, a common theme shared by ∼50% of AML patients. We have generated a transgenic mouse model of the NUP98-PHF23 (NP23) gene fusion, initially identified in patients with AML, which develop AML, erythroleukemia, pre-T lymphoblastic leukemia (pre-T LBL), and a novel pre-B1 B cell acute lymphoblastic leukemia (pre-B1 ALL). A common theme in the leukemias and the premalignant hematopoietic tissues, is the overexpression of a Hoxa/b +Meis1 stem cell-like gene expression signature. GSEA analysis reveals this signature to be enriched in both human AML and ALL malignancies, and in human HSPC profiles. In addition, we found Bahcc1, a gene not previously associated with malignancy, to consistently segregate with the Hoxa/b+Meis1 signature in the NP23 leukemias and the premalignant tissues, independent of hematopoietic cell lineage. Furthermore, data-mining revealed BAHCC1 to be markedly overexpressed in AML patients with HOXA9/MEIS1 overexpression, and in a subset of MLL rearranged pre-B-ALL patients, suggesting BAHCC1 may be a previously unsuspected marker of leukemic transformation. NUP98-PHF23 belongs to a subset of fusion oncoproteins (including some MLL- and NUP98-fusions) that are potently tumorigenic and act by abrogating the normal reading, writing and erasure of histone methylation. Wild type PHF23 binds H3K4me3 residues via a PHD domain, therefore we used ChIP-seq to characterize global chromatin H3K4me3 and NP23 enrichment in NP23 leukemia derived cell lines. The vast majority (88%) of NP23 binding sites were enriched for H3K4me3 binding. Conversely, the NP23 protein co-localized at only 1.6% of all H3K4me3 enriched sites (including Hoxa, Hoxb and Meis1 loci) identifying these sites as direct targets of the NP23 fusion protein. Given that the NP23 fusion appears to function, at least in part, via binding to H3K4me3 sites at specific loci, we hypothesized that NP23 cells would be sensitive to disruption of the H3K4me4 binding by the NP23 PHD domain. Treatment of leukemic NP23 cells with Tetraethylthiuram disulfide (Disulfiram), a small molecule shown to inhibit PHD domain binding of H3K4me3 marks in vitro, rapidly and selectively killed NP23 myeloblasts but not control myeloblast cell lines (188G3, 189E6 and 32D) at 2 µM. Cell death was rapid, being 100% complete within 24 hours. Cell death was preceded by decreased levels of NP23 protein bound at target loci and decreased expression of these loci (e.g., Hoxa7/9/10, Hoxb5 and Meis1). We conclude that inhibitors of H3K4me3 PHD domain readers are promising therapeutic compounds that can kill leukemic cells driven by proteins that aberrantly read or write the histone code. The NP23 model provides a robust platform on which to identify and improve such compounds. Disclosures: Denu: Sirtris-GSK: Consultancy.
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Remich, Robin, Michelle E. Naffziger-Hirsch, J. Lynn Gazley, and Richard McGee. "Scientific Growth and Identity Development during a Postbaccalaureate Program: Results from a Multisite Qualitative Study." CBE—Life Sciences Education 15, no. 3 (2016): ar25. http://dx.doi.org/10.1187/cbe.16-01-0035.
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This report builds upon our previous study, which described five patterns of why college graduates join National Institutes of Health (NIH)-funded diversity-focused Postbaccalaureate Research Education Programs (PREP). A 2015 report from the NIH showed that a high fraction of PREP participants matriculate into PhD and MD/PhD programs. This current study reveals how participants change during PREP, the program elements that facilitate change, and how identity as a graduate student and future scientist develops. Data come from in-depth interviews done at the beginning and end of PREP with 48 individuals from seven PREP programs. Results reveal three domains of development: academics, research, and presentation of oneself; each domain contains a developmental continuum. Key attributes of PREP enabling development include opportunities to attend graduate-level classes and seminars; time to practice reading literature; extended lab time with one’s own project; high and explicit expectations from mentors; and multiple opportunities to talk about science and improve communication skills. PREP enabled participants to develop their identities as graduate students and to anticipate being seen by others as highly prepared for PhD training. After PREP, 85% (n = 41) started the PhD or MD/PhD, making PREP an intervention approach with great potential to broaden participation in biomedical PhD programs.
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Zhu, Xuemei, and Cheryl M. Craft. "Modulation of CRX Transactivation Activity by Phosducin Isoforms." Molecular and Cellular Biology 20, no. 14 (2000): 5216–26. http://dx.doi.org/10.1128/mcb.20.14.5216-5226.2000.
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ABSTRACT Phosducin (Phd) and Phd-like proteins (PhLPs) selectively bind guanine nucleotide protein (G protein) βγ subunits (Gβγ), while Phd-like orphan proteins (PhLOPs) lack the major functional domain for the binding of Gβγ. A retina- and pineal gland-specific transcription factor, cone-rod homeobox (CRX), was identified by a yeast two-hybrid screen using PhLOP1 as the bait. Direct protein-protein interactions between Phd or PhLOP1 and CRX were demonstrated using a β-galactosidase quantitative assay in the yeast two-hybrid system and were confirmed by an in vitro binding assay and a glutathione S-transferase (GST) pull-down assay. To determine if the interaction with Phd or PhLOP1 affected CRX transactivation, a 120-bp interphotoreceptor retinoid binding protein (IRBP) promoter-luciferase reporter construct containing a CRX consensus element (GATTAA) was cotransfected into either COS-7 or retinoblastoma Weri-Rb-1 cells with expression constructs for CRX and either Phd or PhLOP1. Phd and PhLOP1 inhibited the transcriptional activation activity of CRX by 50% during transient cotransfection in COS-7 cells and by 70% in Weri-Rb-1 cells and COS-7 cells stably transfected with CRX. Phd inhibited CRX transactivation in a dose-dependent manner. Whereas Phd is a cytoplasmic phosphoprotein, coexpression of Phd with CRX results in Phd being localized both in the cytoplasm and nucleus. By contrast, PhLOP1 is found in the nucleus even without CRX coexpression. To address the physiological relevance of these potential protein interacting partners, we identified immunoreactive proteins for Phd and CRX in retinal cytosolic and nuclear fractions. Immunohistochemical analysis of bovine retinas reveals colocalization of Phd isoforms with CRX predominantly in the inner segment of cone cells, with additional costaining in the outer nuclear layer and the synaptic region. Our findings demonstrate that both Phd and PhLOP1 interact directly with CRX and that each diminishes the transactivation activity of CRX on the IRBP promoter. A domain that interacts with CRX is found in the carboxyl terminus of the Phd isoforms. Phd antibody-immunoreactive peptides are seen in light-adapted mouse retinal cytosolic and nuclear extracts. Neither Phd nor PhLOP1 affected CRX binding to its consensus DNA element in electrophoretic mobility shift assays. A model that illustrates separate functional roles for interactions between Phd and either SUG1 or CRX is proposed. The model suggests further a mechanism by which Phd isoforms could inhibit CRX transcriptional activation.
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Pastor, Oscar. "Design Science for PhD Research in the Software Engineering Domain." ACM SIGSOFT Software Engineering Notes 44, no. 3 (2019): 22. http://dx.doi.org/10.1145/3356773.3372314.
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Dar, Srishti, and Thomas J. Pucadyil. "The pleckstrin-homology domain of dynamin is dispensable for membrane constriction and fission." Molecular Biology of the Cell 28, no. 1 (2017): 152–60. http://dx.doi.org/10.1091/mbc.e16-09-0640.
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Classical dynamins bind the plasma membrane–localized phosphatidylinositol-4,5-bisphosphate using the pleckstrin-homology domain (PHD) and engage in rapid membrane fission during synaptic vesicle recycling. This domain is conspicuously absent among extant bacterial and mitochondrial dynamins, however, where loop regions manage membrane recruitment. Inspired by the core design of bacterial and mitochondrial dynamins, we reengineered the classical dynamin by replacing its PHD with a polyhistidine or polylysine linker. Remarkably, when recruited via chelator or anionic lipids, respectively, the reengineered dynamin displayed the capacity to constrict and sever membrane tubes. However, when analyzed at single-event resolution, the tube-severing process displayed long-lived, highly constricted prefission intermediates that contributed to 10-fold reduction in bulk rates of membrane fission. Our results indicate that the PHD acts as a catalyst in dynamin-induced membrane fission and rationalize its adoption to meet the physiologic requirement of a fast-acting membrane fission apparatus.
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He, Chao, Ning Liu, Dongya Xie, Yanhong Liu, Yazhong Xiao, and Fudong Li. "Structural basis for histone H3K4me3 recognition by the N-terminal domain of the PHD finger protein Spp1." Biochemical Journal 476, no. 13 (2019): 1957–73. http://dx.doi.org/10.1042/bcj20190091.
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Abstract Saccharomyces cerevisiae Spp1, a plant homeodomain (PHD) finger containing protein, is a critical subunit of the histone H3K4 methyltransferase complex of proteins associated with Set1 (COMPASS). The chromatin binding affinity of the PHD finger of Spp1 has been proposed to modulate COMPASS activity. During meiosis, Spp1 plays another role in promoting programmed double-strand break (DSB) formation by binding H3K4me3 via its PHD finger and interacting with a DSB protein, Mer2. However, how the Spp1 PHD finger performs site-specific readout of H3K4me3 is still not fully understood. In the present study, we determined the crystal structure of the highly conserved Spp1 N-terminal domain (Sc_Spp1NTD) in complex with the H3K4me3 peptide. The structure shows that Sc_Spp1NTD comprises a PHD finger responsible for methylated H3K4 recognition and a C3H-type zinc finger necessary to ensure the overall structural stability. Our isothermal titration calorimetry results show that binding of H3K4me3 to Sc_Spp1NTD is mildly inhibited by H3R2 methylation, weakened by H3T6 phosphorylation, and abrogated by H3T3 phosphorylation. This histone modification cross-talk, which is conserved in the Saccharomyces pombe and mammalian orthologs of Sc_Spp1 in vitro, can be rationalized structurally and might contribute to the roles of Spp1 in COMPASS activity regulation and meiotic recombination.
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Fortschegger, Klaus, Petra de Graaf, Nikolay S. Outchkourov, Frederik M. A. van Schaik, H. T. Marc Timmers, and Ramin Shiekhattar. "PHF8 Targets Histone Methylation and RNA Polymerase II To Activate Transcription." Molecular and Cellular Biology 30, no. 13 (2010): 3286–98. http://dx.doi.org/10.1128/mcb.01520-09.
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ABSTRACT Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. PHF8 contains a plant homeodomain (PHD) in its N terminus and is a member of a family of JmjC domain-containing proteins. While PHDs can act as methyl lysine recognition motifs, JmjC domains can catalyze lysine demethylation. Here, we show that PHF8 is a histone demethylase that removes repressive histone H3 dimethyl lysine 9 marks. Our biochemical analysis revealed specific association of the PHF8 PHD with histone H3 trimethylated at lysine 4 (H3K4me3). Chromatin immunoprecipitation followed by high-throughput sequencing indicated that PHF8 is enriched at the transcription start sites of many active or poised genes, mirroring the presence of RNA polymerase II (RNAPII) and of H3K4me3-bearing nucleosomes. We show that PHF8 can act as a transcriptional coactivator and that its activation function largely depends on binding of the PHD to H3K4me3. Furthermore, we present evidence for direct interaction of PHF8 with the C-terminal domain of RNAPII. Importantly, a PHF8 disease mutant was defective in demethylation and in coactivation. This is the first demonstration of a chromatin-modifying enzyme that is globally recruited to promoters through its association with H3K4me3 and RNAPII.
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Boulay, Gaylor, Claire Rosnoblet, Cateline Guérardel, Pierre-Olivier Angrand, and Dominique Leprince. "Functional characterization of human Polycomb-like 3 isoforms identifies them as components of distinct EZH2 protein complexes." Biochemical Journal 434, no. 2 (2011): 333–42. http://dx.doi.org/10.1042/bj20100944.
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PcG (Polycomb group) proteins are conserved transcriptional repressors essential to regulate cell fate and to maintain epigenetic cellular memory. They work in concert through two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2–4. In Drosophila, PRC2 contains the H3K27 histone methyltransferase E(Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (Polycomb-like). In the present study, we have examined hPCL3, one of its three human paralogues. Through alternative splicing, hPCL3 encodes a long isoform, hPCL3L, containing an N-terminal TUDOR domain and two PHDs (plant homeodomains) and a smaller isoform, hPCL3S, lacking the second PHD finger (PHD2). By quantitative reverse transcription–PCR analyses, we showed that both isoforms are widely co-expressed at high levels in medulloblastoma. By co-immunoprecipitation analyses, we demonstrated that both isoforms interact with EZH2 through their common TUDOR domain. However, the hPCL3L-specific PHD2 domain, which is better conserved than PHD1 in the PCL family, is also involved in this interaction and implicated in the self-association of hPCL3L. Finally, we have demonstrated that both hPCL3 isoforms are physically associated with EZH2, but in different complexes. Our results provide the first evidence that the two hPCL3 isoforms belong to different complexes and raise important questions about their relative functions, particularly in tumorigenesis.
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Bettridge, John, Chan Hyun Na, Akhilesh Pandey, and Stephen Desiderio. "H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase." Proceedings of the National Academy of Sciences 114, no. 8 (2017): 1904–9. http://dx.doi.org/10.1073/pnas.1615727114.
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V(D)J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin.
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Metzen, Eric. "Enzyme substrate recognition in oxygen sensing: how the HIF trap snaps." Biochemical Journal 408, no. 2 (2007): e5-e6. http://dx.doi.org/10.1042/bj20071306.
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The transcriptional activator HIF (hypoxia-inducible factor) is a focal point of biomedical research because many situations in physiology and in pathology coincide with hypoxia. The effects of HIF activation may be a facet of normal growth, as in embryonic development, they may counterbalance a disease, as seen in the stimulation of erythropoiesis in anaemia, and they may be part of the pathological processes, as exemplified by tumour angiogenesis. The oxygen-sensitive α-subunits of HIF are primarily regulated by the enzymatic hydroxylation that induces rapid proteasomal degradation. The HIFα hydroxylases belong to a superfamily of dioxygenases that require the co-substrates oxygen and 2-oxoglutarate as well as the cofactors Fe2+ and ascorbate. The regulation of enzyme turnover by the concentration of the cosubstrate oxygen constitutes the interface between tissue oxygen level and the activity of HIF. The HIFα prolyl hydroxylases, termed PHDs/EGLNs (prolyl hydroxylase domain proteins/EGL nine homologues), bind to a conserved Leu-Xaa-Xaa-Leu-Ala-Pro motif present in all substrates identified so far. This recognition motif is present twice in HIF1α, which gives rise to a NODD [N-terminal ODD (oxygen-dependent degradation domain)] containing Pro402 of HIF1α and a CODD (C-terminal ODD) where Pro564 is hydroxylated. PHD1/EGLN2 and PHD2/EGLN1 hydroxylate both ODDs with higher activity towards CODD, whereas PHD3/EGLN3 is specific for CODD. The reason for this behaviour has been unclear. In this issue of the Biochemical Journal, Villar and colleagues demonstrate that distinct PHD/EGLN domains, that are remote from the catalytic site, function in substrate discrimination. This elegant study improves our understanding of the interaction of the oxygen-sensing PHDs/EGLNs with their substrates, which include, but are not limited to, the HIFα proteins.
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Arita, Kyohei, Mariko Ariyoshi, Kazuya Sugita, Hidehito Tochio, and Masahiro Shirakawa. "Structure study of UHRF1, recoginition of epigenetic marks." Acta Crystallographica Section A Foundations and Advances 70, a1 (2014): C1590. http://dx.doi.org/10.1107/s2053273314084095.
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Two major epigenetic traits, histone modifications and DNA methylation, regulate various chromatin-template processes in mammals. The pattern of these epigenetic traits is cooperatively established in early embryogenesis and cell development, and inherited during the cell cycle. UHRF1 (also known as Np95 or ICBP90) is believed to play an important role in linking the two major epigenetic traits. UHRF1 has five functional domains, UBL, Tandem Tudor (TTD), pland homeo domain (PHD), SET and RING-associated doain (SRA) and RING finger. To maintain DNA methylation pattern, UHRF1 recognizes hemi-methylated DNA generated during DNA replication through interactions with its SRA domain, and recruit maintenance of DNA methyltransferase Dnmt1 to the site [1], [2]. UHRF1 also recognizes histone H3 containing tri-methylated Lys9 (H3K9me3) via its TTD-PHD moiety. [3]. To obtain the structural basis for recognition of epigenetic marks by UHRF1, we determined the crystal structure of the SRA domain in complex with hemi-methylated DNA. The structure showed that the DNA binding caused a loop and an N-terminal tail of the SRA domain. Interestingly, the methyl-cytosine base at the hemi-methylation site was flipped out from the DNA helix, which has not observed in other DNA binding proteins. These results suggest that the Base flip out mechanism is important event for maintenance of DNA methylation. We also determined the crystal structure of TTD-PHD region of UHRF1 in complex with H3K9me3 peptide. To our surprise, the linker region between the reader modules, which is predicted as an intrinsically disorder, was formed a stable structure with binding to the groove of TTD and plays an essential role in the formation of histone H3 binding hole between the reader modules. The structure revealed how multiple histone modifications were simultaneously decoded by the linked histone reader modules of UHRF1.
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Xing, Weirong, Sheila Pourteymoor, and Subburaman Mohan. "Ascorbic acid regulates osterix expression in osteoblasts by activation of prolyl hydroxylase and ubiquitination-mediated proteosomal degradation pathway." Physiological Genomics 43, no. 12 (2011): 749–57. http://dx.doi.org/10.1152/physiolgenomics.00229.2010.
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Mouse genetic studies reveal that ascorbic acid (AA) is essential for osteoblast (OB) differentiation and that osterix (Osx) was a key downstream target of AA action in OBs. To determine the molecular pathways for AA regulation of Osx expression, we evaluated if AA regulates Osx expression by regulating production and/or actions of local growth factors and extracellular matrix (ECM) proteins. Inhibition of actions of IGFs by inhibitory IGFBP-4, BMPs by noggin, and ECM-mediated integrin signaling by RGD did not block AA effects on Osx expression in OBs. Furthermore, blockade of components of MAPK signaling pathway had no effect on AA-induced Osx expression. Because AA is required for prolyl hydroxylase domain (PHD) activity and because PHD-induced prolyl-hydroxylation targets proteins to proteosomal degradation, we next tested if AA effect on Osx expression involves activation of PHD to hydroxylate and induce ubiquitin-proteosome-mediated degradation of transcriptional repressor(s) of Osx gene. Treatment of OBs with dimethyloxallyl glycine and ethyl 3, 4-dihydroxybenzoate, known inhibitors of PHD, completely blocked AA effect on Osx expression and OB differentiation. Knockdown of PHD2 expression by Lentivirus-mediated shRNA abolished AA-induced Osx induction and alkaline phosphatase activity. Furthermore, treatment of OBs with MG115, inhibitor of proteosomal degradation, completely blocked AA effects on Osx expression. Based on these data, we conclude that AA effect on Osx expression is mediated via a novel mechanism that involves PHD2 and proteosomal degradation of a yet to be identified transcriptional repressor that is independent of BMP, IGF-I, or integrin-mediated signaling in mouse OBs.
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Papait, Roberto, Christian Pistore, Ursula Grazini, et al. "The PHD Domain of Np95 (mUHRF1) Is Involved in Large-Scale Reorganization of Pericentromeric Heterochromatin." Molecular Biology of the Cell 19, no. 8 (2008): 3554–63. http://dx.doi.org/10.1091/mbc.e07-10-1059.
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Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH.
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Kwon, Hyuk Sung, Yien Kyoung Choi, Jeong Won Kim, Yong Keun Park, Eun Gyeong Yang, and Dae-Ro Ahn. "Inhibition of a prolyl hydroxylase domain (PHD) by substrate analog peptides." Bioorganic & Medicinal Chemistry Letters 21, no. 14 (2011): 4325–28. http://dx.doi.org/10.1016/j.bmcl.2011.05.050.
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Liu, Bing, Chunlian Jin, Nico De Storme, et al. "A Hypomorphic Mutant of PHD Domain Protein Male Meiocytes Death 1." Genes 12, no. 4 (2021): 516. http://dx.doi.org/10.3390/genes12040516.
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Meiosis drives reciprocal genetic exchanges and produces gametes with halved chromosome number, which is important for the genetic diversity, plant viability, and ploidy consistency of flowering plants. Alterations in chromosome dynamics and/or cytokinesis during meiosis may lead to meiotic restitution and the formation of unreduced microspores. In this study, we isolated an Arabidopsis mutant male meiotic restitution 1 (mmr1), which produces a small subpopulation of diploid or polyploid pollen grains. Cytological analysis revealed that mmr1 produces dyads, triads, and monads indicative of male meiotic restitution. Both homologous chromosomes and sister chromatids in mmr1 are separated normally, but chromosome condensation at metaphase I is slightly affected. The mmr1 mutant displayed incomplete meiotic cytokinesis. Supportively, immunostaining of the microtubular cytoskeleton showed that the spindle organization at anaphase II and mini-phragmoplast formation at telophase II are aberrant. The causative mutation in mmr1 was mapped to chromosome 1 at the chromatin regulator Male Meiocyte Death 1 (MMD1/DUET) locus. mmr1 contains a C-to-T transition at the third exon of MMD1/DUET at the genomic position 2168 bp from the start codon, which causes an amino acid change G618D that locates in the conserved PHD-finger domain of histone binding proteins. The F1 progenies of mmr1 crossing with knockout mmd1/duet mutant exhibited same meiotic defects and similar meiotic restitution rate as mmr1. Taken together, we here report a hypomorphic mmd1/duet allele that typically shows defects in microtubule organization and cytokinesis.
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Sethumadhavan, Devadathan Valiyamangalath, Gayathri Govindaraju, C. A. Jabeena, and Arumugam Rajavelu. "Plasmodium falciparum SET2 domain is allosterically regulated by its PHD-like domain to methylate at H3K36." Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1864, no. 10 (2021): 194744. http://dx.doi.org/10.1016/j.bbagrm.2021.194744.
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Shi, Yan, Xiushan Wu, Shuoji Zhu, et al. "Structure and function of Pygo in organ development dependent and independent Wnt signalling." Biochemical Society Transactions 48, no. 4 (2020): 1781–94. http://dx.doi.org/10.1042/bst20200393.
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Pygo is a nuclear protein containing two conserved domains, NHD and PHD, which play important roles in embryonic development and carcinogenesis. Pygo was first identified as a core component of the Wnt/β-catenin signalling pathway. However, it has also been reported that the function of Pygo is not always Wnt/β-catenin signalling dependent. In this review, we summarise the functions of both domains of Pygo and show that their functions are synergetic. The PHD domain mainly combines with transcription co-factors, including histone 3 and Bcl9/9l. The NHD domain mainly recruits histone methyltransferase/acetyltransferase (HMT/HAT) to modify lysine 4 of the histone 3 tail (H3K4) and interacts with Chip/LIM-domain DNA-binding proteins (ChiLS) to form enhanceosomes to regulate transcriptional activity. Furthermore, we summarised chromatin modification differences of Pygo in Drosophila (dPygo) and vertebrates, and found that Pygo displayes a chromatin silencing function in Drosophila, while in vertebates, Pygo has a chromatin-activating function due to the two substitution of two amino acid residues. Next, we confirmed the relationship between Pygo and Bcl9/9l and found that Pygo–Bcl/9l are specifically partnered both in the nucleus and in the cytoplasm. Finally, we discuss whether transcriptional activity of Pygo is Wnt/β-catenin dependent during embryonic development. Available information indications that the transcriptional activity of Pygo in embryonic development is either Wnt/β-catenin dependent or independent in both tissue-specific and cell-specific-modes.
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Zhu, Huihui, Tao Wei, Yong Cai, and Jingji Jin. "Small Molecules Targeting the Specific Domains of Histone-Mark Readers in Cancer Therapy." Molecules 25, no. 3 (2020): 578. http://dx.doi.org/10.3390/molecules25030578.
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Epigenetic modifications (or epigenetic tags) on DNA and histones not only alter the chromatin structure, but also provide a recognition platform for subsequent protein recruitment and enable them to acquire executive instructions to carry out specific intracellular biological processes. In cells, different epigenetic-tags on DNA and histones are often recognized by the specific domains in proteins (readers), such as bromodomain (BRD), chromodomain (CHD), plant homeodomain (PHD), Tudor domain, Pro-Trp-Trp-Pro (PWWP) domain and malignant brain tumor (MBT) domain. Recent accumulating data reveal that abnormal intracellular histone modifications (histone marks) caused by tumors can be modulated by small molecule-mediated changes in the activity of the above domains, suggesting that small molecules targeting histone-mark reader domains may be the trend of new anticancer drug development. Here, we summarize the protein domains involved in histone-mark recognition, and introduce recent research findings about small molecules targeting histone-mark readers in cancer therapy.
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Oliver, Antony W., Sarah A. Jones, Stephen Mark Roe, Steve Matthews, Graham H. Goodwin, and Laurence H. Pearl. "Crystal structure of the proximal BAH domain of the polybromo protein." Biochemical Journal 389, no. 3 (2005): 657–64. http://dx.doi.org/10.1042/bj20050310.
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The BAH domain (bromo-associated homology domain) was first identified from a repeated motif found in the nuclear protein polybromo – a large (187 kDa) modular protein comprising six bromodomains, two BAH domains and an HMG box. To date, the BAH domain has no ascribed function, although it is found in a wide range of proteins that contain additional domains involved in either transcriptional regulation (e.g. SET, PHD and bromodomain) and/or DNA binding (HMG box and AT hook). The molecular function of polybromo itself also remains unclear, but it has been identified as a key component of an SWI/SNF (switching/sucrose non-fermenting)-related, ATP-dependent chromatin-remodelling complex PBAF (polybromo, BRG1-associated factors; also known as SWI/SNF-B or SWI/SNFβ). We present in this paper the crystal structure of the proximal BAH domain from chicken polybromo (BAH1), at a resolution of 1.6 Å (1 Å=0.1 nm). Structure-based sequence analysis reveals several features that may be involved in mediating protein–protein interactions.
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Bera, Rabindranath, Der-Cherng Liang, Ming-Chun Chiu, Ying-Jung Huang, Sung-Tzu Liang, and Lee-Yung Shih. "PHD Domain Deletion Mutations of ASXL1 Promote Myeloid Leukemia Transformation Through Epigenetic Dysregulation and Inhibit Megakaryocytic Differentiation Through the Inactivation of FOSB in K562 Cells." Blood 120, no. 21 (2012): 2393. http://dx.doi.org/10.1182/blood.v120.21.2393.2393.
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Abstract Abstract 2393 Somatic mutations of ASXL1 gene have been described in patients with myeloid malignancies and were associated with inferior outcomes. ASXL1 mutations have also been detected in myeloid blast crisis of chronic myeloid leukemia (CML) patients. The mechanisms of acute myeloid leukemia (AML) transformation and functional role of ASXL1 mutations in the leukemogenesis remain to be determined. Recently, we identified PHD domain deletion mutations (R693X and L885X) in patients with CML in myeloid blast crisis and/or AML with minimal differentiation (M0). In the present study, we aimed to investigate the role of PHD domain deletion mutations in the pathogenesis of AML transformation. The K562 cells carrying Philadelphia chromosome, serves as a model to study the molecular mechanisms associated with leukemogenesis. Our result showed that R693X/L885X mutations inhibited PMA-treated megakaryocytic differentiation with the change of physiological characteristic features and suppressed the induction of CD61, a specific cell surface marker of megakaryocytes. We also found that FOSB, a member of Fos family of AP-1 transcription factors was down-regulated in K562 cells expressing R693X and L885X compared to wild-type ASXL1 during PMA-mediated megakaryocytic differentiation. Examination of intracellular signaling pathways showed that the mutant ASXL1 protein prevented PMA-induced megakaryocytic differentiation through the inactivation of ERK, AKT and STAT5 which are required for differentiation. Further, ASXL1 depletion by shRNA in K562 cells led to enhanced cell proliferation, increased colony formation and impaired PMA-mediated differentiation. Previous studies in Drosophila had revealed that Asxl forms the protein complexes of both Trithorax and Polycomb groups that are required for maintaining chromatin in both activated and repressed transcriptional states. By using Western blot analysis, we demonstrated that PHD domain deletion mutations of ASXL1 significantly suppressed the transcriptionally repressive mark H3K27 trimethylation, however no effect on methylated H3K4 (H3K4me2 and H3K4me3), an active histone mark in K562 cells. Co-immunoprecipitation analysis revealed that wild-type, but not PHD domain deletion mutations of ASXL1 interact with EZH2, a member of the polycomb repressive complex 2 (PRC2). Importantly, PHD deletion mutations or downregulation of ASXL1 resulted in the suppression of EZH2 in K562 cells. Our study demonstrated that PHD deletion mutations of ASXL1 resulted in a loss-of-function which exhibited direct effects on the proliferation and differentiation and also proposed a specific role for ASXL1 in epigenetic regulation of gene expression in K562 cells. Disclosures: No relevant conflicts of interest to declare.
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Licht, Jonathan D. "Aberrant Histone Methylation in Myeloma: What Are the Rules?" Blood 120, no. 21 (2012): SCI—5—SCI—5. http://dx.doi.org/10.1182/blood.v120.21.sci-5.sci-5.
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Abstract Abstract SCI-5 Aberrant regulation of histone methylation is a recurrent theme in multiple myeloma, lymphoma, and other B-cell malignancies. MMSET Multiple myeloma SET domain (MMSET) is a histone methyltransferase (HMT) overexpressed as a result of the translocation t(4;14) and is present in about 15 percent of multiple myeloma patients. MMSET is a nuclear protein with multiple domains critical for gene regulation, including the SET domain, which encodes histone methyltransferase activity, and protein and DNA interaction domains, including PHD and PWWP domains. Overexpression of MMSET induces a global increase in H3K36 methylation with concomitant loss of global H3K27 methylation. Kinetic studies using isotopic labeling and mass spectroscopy demonstrate that this change in methylation is due to both an increase in the rate of methylation of H3K36 and an increase in the demethylation of H3K27. These changes cause physical loosening of the chromatin structure, demonstrated by an increase in micrococcal nuclease accessibility, changes in DNA damage response, and aberrant gene expression. The HMT activity of MMSET is essential for growth stimulation by MMSET, as shown by the fact that reexpression of MMSET in a t(4;14) myeloma cell line, in which the rearranged MMSET allele was disrupted by homologous recombination (KMS11-TKO), rescued growth only when the HMT activity of the protein was intact. The complete H3K36/H3K27 switch mediated by MMSET requires all PHD finger domains of the protein, the second PWWP domain, and the functional SET domain. For example, a single point mutation in one PHD domain abrogated chromatin binding, histone methylation, and growth stimulation by the protein. Furthermore, deletion of the PHD domain 4 was able to increase H3K36 methylation but unable to reduce H3K37 methylation, leading to only partial growth stimulation. Despite the global change in histone methylation in response to MMSET, microarray and RNA-Seq analysis showed that only ∼1000 genes are appreciably changed in response to MMSET. The basis of the specificity of differential gene expression is under investigation. For example, many genes activated by MMSET display a peak of H3K27me3 near the transcription start site in MMSET-low cells, which is absent in MMSET-overexpressing cells, displaced by a broad pattern of H3K36me2 modification. We also found a subset of genes repressed in response to MMSET overexpression. While H3K27 methylation is decreased on a genome-wide basis in MMSET-overexpressing cells, H3K27me3 levels at repressed genes were increased in association with increased occupancy by EZH2. These regions did not show an increase in H3K36 methylation and are enriched with GC-rich elements, representing putative polycomb complex recruitment sites. We hypothesize that the global increase in H3K36me2 and drop of H3K27me3 levels on many genes leads to the displacement of the PRC2 complex from lower-affinity sites to such higher-affinity loci. These modes of action likely considerably diverge from the normal role of MMSET and EZH2 in gene regulation. Similarly, EZH2 point mutations in lymphoma lead to global chromatin dysfunction and aberrant regulation of specific sets of genes, only some of which represent previously identified EZH2 targets. Collectively, oncogenic lesions in histone-modifying enzymes in myeloma and other lymphoid neoplasms need to be understood on their own terms, as the lessons learned from the normal function of these enzymes may not predict their activity in malignancy. Disclosures: Licht: Epizyme, Inc: Research Funding.
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Pavlakis, Dimitrios, Spyridon Kampantais, Konstantinos Gkagkalidis, et al. "Hypoxia-Inducible Factor 2a Expression Is Positively Correlated With Gleason Score in Prostate Cancer." Technology in Cancer Research & Treatment 20 (January 1, 2021): 153303382199001. http://dx.doi.org/10.1177/1533033821990010.
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Background: One of the main factors in response to hypoxia in the tumor microenvironment is the hypoxia-inducible factor (HIF) pathway. Although its role in other solid tumors, particularly renal cell carcinoma, has been sufficiently elucidated, it remains elusive in prostate cancer. The aim of the present study was to investigate the expression of main proteins involved in this pathway and determine the correlation of the results with clinicopathological outcomes of patients with prostate cancer. Methods: The immunohistochemical expression of HIF-1a, HIF-2a and their regulators, prolyl hydroxylase domain (PHD)1, PHD2 and PHD3 and factor inhibiting HIF (FIH), was assessed on a tissue microarray. This was constructed from radical prostatectomy specimens, involving both tumor and corresponding adjacent non-tumoral prostate tissues from 50 patients with localized or locally advanced prostate cancer. Results: In comparison with non-tumoral adjacent tissue, HIF-1a exhibited an equal or lower expression in 86% of the specimens (P = 0.017), while HIF-2a was overexpressed in 52% (P = 0.032) of the cases. HIF-1a protein expression was correlated with HIF-2a (P < 0.001), FIH (P = 0.004), PHD1 (P < 0.001), PHD2 (P < 0.001) and PHD3 (P = 0.035). HIF-2a expression was positively correlated with Gleason score (P = 0.017) and International Society of Urological Pathologists (ISUP) grade group (P = 0.022). Conclusions: The findings of the present study suggest a key role for HIF-2a in prostate cancer, as HIF-2a expression was found to be correlated with Gleason score and ISUP grade of the patients. However, further studies are required to validate these results and investigate the potential value of HIF-2a as a therapeutic target in prostate cancer.
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Slama, Patrick. "Two-domain analysis of JmjN-JmjC and PHD-JmjC lysine demethylases: Detecting an inter-domain evolutionary stress." Proteins: Structure, Function, and Bioinformatics 86, no. 1 (2017): 3–12. http://dx.doi.org/10.1002/prot.25394.
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Wei, George Z., Sujata Saraswat Ohri, Nicolas K. Khattar, et al. "Hypoxia-inducible factor prolyl hydroxylase domain (PHD) inhibition after contusive spinal cord injury does not improve locomotor recovery." PLOS ONE 16, no. 4 (2021): e0249591. http://dx.doi.org/10.1371/journal.pone.0249591.
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Traumatic spinal cord injury (SCI) is a devastating neurological condition that involves both primary and secondary tissue loss. Various cytotoxic events including hypoxia, hemorrhage and blood lysis, bioenergetic failure, oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation contribute to secondary injury. The HIF prolyl hydroxylase domain (PHD/EGLN) family of proteins are iron-dependent, oxygen-sensing enzymes that regulate the stability of hypoxia inducible factor-1α (HIF-1α) and also mediate oxidative stress caused by free iron liberated from the lysis of blood. PHD inhibition improves outcome after experimental intracerebral hemorrhage (ICH) by reducing activating transcription factor 4 (ATF4)-driven neuronal death. As the ATF4-CHOP (CCAAT-enhancer-binding protein homologous protein) pathway plays a role in the pathogenesis of contusive SCI, we examined the effects of PHD inhibition in a mouse model of moderate T9 contusive SCI in which white matter damage is the primary driver of locomotor dysfunction. Pharmacological inhibition of PHDs using adaptaquin (AQ) moderately lowers acute induction of Atf4 and Chop mRNAs and prevents the acute decline of oligodendrocyte (OL) lineage mRNAs, but does not improve long-term recovery of hindlimb locomotion or increase chronic white matter sparing. Conditional genetic ablation of all three PHD isoenzymes in OLs did not affect Atf4, Chop or OL mRNAs expression levels, locomotor recovery, and white matter sparing after SCI. Hence, PHDs may not be suitable targets to improve outcomes in traumatic CNS pathologies that involve acute white matter injury.
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May, Meiling R., John T. Bettridge, and Stephen Desiderio. "Binding and allosteric transmission of histone H3 Lys-4 trimethylation to the recombinase RAG-1 are separable functions of the RAG-2 plant homeodomain finger." Journal of Biological Chemistry 295, no. 27 (2020): 9052–60. http://dx.doi.org/10.1074/jbc.ra120.014382.
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V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.
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Nytko, Katarzyna J., Patrick Spielmann, Gieri Camenisch, Roland H. Wenger, and Daniel P. Stiehl. "Regulated Function of the Prolyl-4-Hydroxylase Domain (PHD) Oxygen Sensor Proteins." Antioxidants & Redox Signaling 9, no. 9 (2007): 1329–38. http://dx.doi.org/10.1089/ars.2007.1683.
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Guo, Xue, Youwei Xu, Ping Wang, Ze Li, Yanhui Xu, and Huirong Yang. "Crystallization and preliminary crystallographic analysis of a PHD domain of human JARID1B." Acta Crystallographica Section F Structural Biology and Crystallization Communications 67, no. 8 (2011): 907–10. http://dx.doi.org/10.1107/s1744309111021981.
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Schillinger, Mark. "Safe and dynamic design of experiments." at - Automatisierungstechnik 68, no. 5 (2020): 387–88. http://dx.doi.org/10.1515/auto-2020-0011.
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AbstractThis PhD thesis covers multiple approaches for dynamic design of experiments considering safety constraints. Special emphasis is put on the evaluation of these methods at real-world systems in the combustion engine domain.