Journal articles: 'Pou5f1'

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Hasegawa, Shun, Isseki Nakao, Yuki Ootani, Ami Ogawa, Miku Takano, and Tsutomu Kinoshita. "Identification and characterization of POU class V family genes in Japanese red bellied newt, Cynops pyrrhogaster." Zygote 27, no. 05 (August 15, 2019): 329–36. http://dx.doi.org/10.1017/s0967199419000339.

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SummaryMammalian Pou5f1 encodes the POU family class V (POU-V) transcription factor which is essential for the pluripotency of embryonic cells and germ cells. In vertebrates, various POU-V family genes have been identified and classified into the POU5F1 family or its paralogous POU5F3 family. In this study, we cloned two cDNAs named CpPou5f1 and CpPou5f3, which encode POU-V family proteins of the Japanese red bellied newt Cynops pyrrhogaster. In the predicted amino acid sequence encoded by CpPou5f1, the typical MAGH sequence at the N-terminus and deletion of arginine at the fifth position of POU-homeodomain were recognized, but not in the sequence encoded by CpPou5f3. Phylogenetic analysis using Clustal Omega software indicated that CpPou5f1 and CpPou5f3 are classified into the clade of the POU5F1 and POU5F3 families, respectively. In a real-time polymerase chain reaction (RT-PCR) analysis, the marked gene expression of CpPou5f1 was observed during oogenesis and early development up to the tail-bud stage, whereas weak gene expression of CpPou5f3 was detected only in the early stages of oogenesis and gastrula. In adult organs, CpPou5f1 was expressed only in the ovary, while gene expression of CpPou5f3 was recognized in various organs. A regeneration experiment using larval forelimb revealed that transient gene expression of CpPou5f1 occurred at the time of wound healing, followed by gene activation of CpPou5f3 during the period of blastema formation. These results suggest that CpPou5f1 and CpPou5f3 might play different roles in embryogenesis and limb regeneration.

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Zheng, Yu, LeAnna J. Phillips, Rachel Hartman, Junhui An, and Christina T. Dann. "Ectopic POU5F1 in the male germ lineage disrupts differentiation and spermatogenesis in mice." Reproduction 152, no. 4 (October 2016): 363–77. http://dx.doi.org/10.1530/rep-16-0140.

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Expression levels of the pluripotency determinant, POU5F1, are tightly regulated to ensure appropriate differentiation during early embryogenesis. POU5F1 is also present in the spermatogonial stem cell/progenitor cell population in mice and it is downregulated as spermatogenesis progresses. To test if POU5F1 downregulation is required for SSCs to differentiate, we produced transgenic mice that ubiquitously express POU5F1 in Cre-expressing lineages. Using a Vasa-Cre driver to produce ectopic POU5F1 in all postnatal germ cells, we found that POU5F1 downregulation was necessary for spermatogonial expansion during the first wave of spermatogenesis and for the production of differentiated spermatogonia capable of undergoing meiosis. In contrast, undifferentiated spermatogonia were maintained throughout adulthood, consistent with a normal presence of POU5F1 in these cells. The results suggest that POU5F1 downregulation in differentiating spermatogonia is a necessary step for the progression of spermatogenesis. Further, the creation of a transgenic mouse model for conditional ectopic expression of POU5F1 may be a useful resource for studies of POU5F1 in other cell lineages, during tumorogenesis and cell fate reprogramming.

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Choi, Y. H., H. D. Harding, D. L. Hartman, A. D. Obermiller, S. Kurosaka, K. J. McLaughlin, and K. Hinrichs. "The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts." REPRODUCTION 138, no. 3 (September 2009): 589–99. http://dx.doi.org/10.1530/rep-08-0394.

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The reported patterns of trophectodermal expression of POU5F1 protein in blastocysts vary among species, and are possibly related to the differences in placental growth and function. This study investigated the pattern of embryonic POU5F1 expression in the horse, a species with delayed placental formation. Immature equine oocytes expressed POU5F1 protein in the cytoplasm and nucleus. Staining for POU5F1 protein inin vitro-produced (IVP) embryos decreased to day 5 of culture, then the nuclear staining increased to day 7. IVP day-7 to -11 blastocysts showed POU5F1 staining in nuclei throughout the blastocysts. In contrast,in vivo-produced day-7 to -10 blastocysts showed greatly reduced trophoectodermal POU5F1 protein expression. To determine whether the uterine environment modulates POU5F1 expression, IVP blastocysts were transferred to the uteri of mares, then recovered 2–3 days later (IVP-ET embryos). These embryos showed similar POU5F1 expression as thein vivo-produced embryos. Levels ofPOU5F1,SOX2, andNANOGmRNA in IVP-ET blastocysts were significantly higher in the inner cell mass than in trophectoderm (TE) cells. These data suggest that the differentiation of equine TE, as indicated by loss of POU5F1 expression, is impaired duringin vitroculture, but proceeds normally when the embryos are exposed to the uterine environment. Previously reported differences in trophectodermal expression of POU5F1 among species may thus be in part artifactual, i.e. related toin vitroculture. Failure for correction of such changes by the uterine environment is a potential factor in the placental abnormalities seen after transfer of cultured embryos in some species.

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Gupta, Rangan, Toshihiko Ezashi, and R. Michael Roberts. "Squelching of ETS2 Transactivation by POU5F1 Silences the Human Chorionic Gonadotropin CGA Subunit Gene in Human Choriocarcinoma and Embryonic Stem Cells." Molecular Endocrinology 26, no. 5 (May 1, 2012): 859–72. http://dx.doi.org/10.1210/me.2011-1146.

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Abstract The subunit genes encoding human chorionic gonadotropin, CGA, and CGB, are up-regulated in human trophoblast. However, they are effectively silenced in choriocarcinoma cells by ectopically expressed POU domain class 5 transcription factor 1 (POU5F1). Here we show that POU5F1 represses activity of the CGA promoter through its interactions with ETS2, a transcription factor required for both placental development and human chorionic gonadotropin subunit gene expression, by forming a complex that precludes ETS2 from interacting with the CGA promoter. Mutation of a POU5F1 binding site proximal to the ETS2 binding site does not alter the ability of POU5F1 to act as a repressor but causes a drop in basal promoter activity due to overlap with the binding site for DLX3. DLX3 has only a modest ability to raise basal CGA promoter activity, but its coexpression with ETS2 can up-regulate it 100-fold or more. The two factors form a complex, and both must bind to the promoter for the combination to be transcriptionally effective, a synergy compromised by POU5F1. Similarly, in human embryonic stem cells, which express ETS2 but not CGA, ETS2 does not occupy its binding site on the CGA promoter but is found instead as a soluble complex with POU5F1. When human embryonic stem cells differentiate in response to bone morphogenetic protein-4 and concentrations of POU5F1 fall and hCG and DLX3 rise, ETS2 then occupies its binding site on the CGA promoter. Hence, a squelching mechanism underpins the transcriptional silencing of CGA by POU5F1 and could have general relevance to how pluripotency is maintained and how the trophoblast lineage emerges from pluripotent precursor cells.

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Castillo-Martín, Miriam, Marc Yeste, Eva Pericuesta, Roser Morató, Alfonso Gutiérrez-Adán, and Sergi Bonet. "Effects of vitrification on the expression of pluripotency, apoptotic and stress genes in in vitro-produced porcine blastocysts." Reproduction, Fertility and Development 27, no. 7 (2015): 1072. http://dx.doi.org/10.1071/rd13405.

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The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX : BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.9 ± 0.4% vs 11.9 ± 2.0%) and peroxide levels (80.4 ± 2.6 vs 97.2 ± 3.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition, after blastocyst vitrification, relative transcript abundance was downregulated for POU5F1 and upregulated for HSPA1A. Finally, there was a significant correlation of POU5F1 and HSPA1A with DNA fragmentation (POU5F1, r = –0.561; HSPA1A, r = 0.604) and peroxide levels (POU5F1, r = –0.590; HSPA1A, r = 0.621). In conclusion, under the conditions of the present study, vitrification and warming of IVP porcine blastocysts resulted in altered expression of POU5F1 and HSPA1A, but had no effect on BAX, BCL2L1, SOD1 and SOD2 expression.

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Stamatiadis, P., A. Boel, G. Cosemans, M. Popovic, B. Bekaert, R. Guggilla, M. Tang, et al. "Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences." Human Reproduction 36, no. 5 (February 20, 2021): 1242–52. http://dx.doi.org/10.1093/humrep/deab027.

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Abstract STUDY QUESTION What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? SUMMARY ANSWER POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. WHAT IS KNOWN ALREADY Clustered regularly interspaced short palindromic repeats—CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. STUDY DESIGN, SIZE, DURATION The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain—B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. LIMITATIONS, REASONS FOR CAUTION One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

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Gil-Kulik, Paulina, Piotr Chomik, Arkadiusz Krzyżanowski, Elżbieta Radzikowska-Büchner, Ryszard Maciejewski, Anna Kwaśniewska, Mansur Rahnama, and Janusz Kocki. "Influence of the Type of Delivery, Use of Oxytocin, and Maternal Age on POU5F1 Gene Expression in Stem Cells Derived from Wharton’s Jelly within the Umbilical Cord." Oxidative Medicine and Cellular Longevity 2019 (December 14, 2019): 1–8. http://dx.doi.org/10.1155/2019/1027106.

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The paper presents an evaluation of the POU5F1 gene expression in mesenchymal stem cells derived from Wharton’s jelly within the umbilical cord, collected from 36 patients during labor. The study is the first one to show that the expression of POU5F1 in mesenchymal stem cells has been dependent on maternal age, birth order, route of delivery, and use of oxytocin. Our research proves that the POU5F1 gene expression in mesenchymal stem cells decreases with each subsequent pregnancy and delivery. Wharton’s jelly stem cells obtained from younger women and during their first delivery, as well as patients treated with oxytocin, show higher POU5F1 gene expression when compared with the subsequent deliveries. This leads to a conclusion that they are characterized by a lower level of differentiation, which in turn results in their greater plasticity and greater proliferative potential. Probably, they are also clinically more useful.

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Liao, Ji, Yikun He, and Piroska E. Szabó. "The Pou5f1 distal enhancer is sufficient to drive Pou5f1 promoter-EGFP expression in embryonic stem cells." International Journal of Developmental Biology 57, no. 9-10 (2013): 725–29. http://dx.doi.org/10.1387/ijdb.120186ps.

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Yamamoto, Yu, Osamu Miura, and Takashi Ohyama. "Cruciform Formable Sequences within Pou5f1 Enhancer Are Indispensable for Mouse ES Cell Integrity." International Journal of Molecular Sciences 22, no. 7 (March 26, 2021): 3399. http://dx.doi.org/10.3390/ijms22073399.

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DNA can adopt various structures besides the B-form. Among them, cruciform structures are formed on inverted repeat (IR) sequences. While cruciform formable IRs (CFIRs) are sometimes found in regulatory regions of transcription, their function in transcription remains elusive, especially in eukaryotes. We found a cluster of CFIRs within the mouse Pou5f1 enhancer. Here, we demonstrate that this cluster or some member(s) plays an active role in the transcriptional regulation of not only Pou5f1, but also Sox2, Nanog, Klf4 and Esrrb. To clarify in vivo function of the cluster, we performed genome editing using mouse ES cells, in which each of the CFIRs was altered to the corresponding mirror repeat sequence. The alterations reduced the level of the Pou5f1 transcript in the genome-edited cell lines, and elevated those of Sox2, Nanog, Klf4 and Esrrb. Furthermore, transcription of non-coding RNAs (ncRNAs) within the enhancer was also upregulated in the genome-edited cell lines, in a similar manner to Sox2, Nanog, Klf4 and Esrrb. These ncRNAs are hypothesized to control the expression of these four pluripotency genes. The CFIRs present in the Pou5f1 enhancer seem to be important to maintain the integrity of ES cells.

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Desmarais, Joëlle A., Simon-Pierre Demers, Joao Suzuki, Simon Laflamme, Patrick Vincent, Sheila Laverty, and Lawrence C. Smith. "Trophoblast stem cell marker gene expression in inner cell mass-derived cells from parthenogenetic equine embryos." REPRODUCTION 141, no. 3 (March 2011): 321–32. http://dx.doi.org/10.1530/rep-09-0536.

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Although putative horse embryonic stem (ES)-like cell lines have been obtained recently from in vivo-derived embryos, it is currently not known whether it is possible to obtain ES cell (ESC) lines from somatic cell nuclear transfer (SCNT) and parthenogenetic (PA) embryos. Our aim is to establish culture conditions for the derivation of autologous ESC lines for cell therapy studies in an equine model. Our results indicate that both the use of early-stage blastocysts with a clearly visible inner cell mass (ICM) and the use of pronase to dissect the ICM allow the derivation of a higher proportion of primary ICM outgrowths from PA and SCNT embryos. Primary ICM outgrowths express the molecular markers of pluripotency POU class 5 homeobox 1 (POU5F1) and (sex determining region-Y)-box2 (SOX2), and in some cases, NANOG. Cells obtained after the passages of PA primary ICM outgrowths display alkaline phosphatase (AP) activity and POU5F1, SOX2, caudal-related homeobox-2 (CDX2) and eomesodermin (EOMES) expression, but may lose NANOG. Cystic embryoid body-like structures expressing POU5F1, CDX2 and EOMES were produced from these cells. Immunohistochemical analysis of equine embryos reveals the presence of POU5F1 in trophectoderm, primitive endoderm and ICM. These results suggest that cells obtained after passages of primary ICM outgrowths are positive for trophoblast stem cell markers while expressing POU5F1 and displaying AP activity. Therefore, these cells most likely represent trophoblast cells rather than true ESCs. This study represents an important first step towards the production of autologous equine ESCs for pre-clinical cell therapy studies on large animal models.

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Frankenberg, S., A. J. Pask, and M. B. Renfree. "259. Pluripotency genes in a marsupial, the tammar wallaby." Reproduction, Fertility and Development 20, no. 9 (2008): 59. http://dx.doi.org/10.1071/srb08abs259.

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Markers of pluripotency and early differentiation in the early embryo have been extensively characterised in eutherian species, most notably the mouse. By comparison, mechanisms controlling pluripotency and early lineage specification have received surprisingly little attention in marsupials, which represent the second major infraclass of mammals. Early marsupial embryogenesis exhibits overt morphological differences to that of eutherians, however the underlying developmental mechanisms may be conserved. In order to characterise early marsupial development at the molecular level, we have identified, cloned and analysed expression of orthologueues of several eutherian genes encoding transcription factors and signalling molecules involved in regulating pluripotency and early lineage specification. These genes include POU5F1 (OCT4), SOX2, NANOG, FGF4, FGFR2, CDX2, EOMES, TEAD4, GATA6 and KITL and are all expressed at early stages of development in the tammar. In addition, we have identified and cloned tammar POU2, which has orthologueues in non-mammalian vertebrates. POU2 is a paralogue of POU5F1 – a master regulator of pluripotency in eutherians. Genomic analysis indicates that POU5F1 arose via gene duplication of POU2 before the monotreme-therian divergence. Both genes have persisted in marsupials and monotremes, while POU2 was lost early during eutherian evolution. Similar expression profiles of tammar POU5F1 and POU2 in early embryos and gonadal tissues suggest possible overlapping roles in the maintenance of pluripotency.

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Chew, Joon-Lin, Yuin-Han Loh, Wensheng Zhang, Xi Chen, Wai-Leong Tam, Leng-Siew Yeap, Pin Li, et al. "Reciprocal Transcriptional Regulation of Pou5f1 and Sox2 via the Oct4/Sox2 Complex in Embryonic Stem Cells." Molecular and Cellular Biology 25, no. 14 (July 2005): 6031–46. http://dx.doi.org/10.1128/mcb.25.14.6031-6046.2005.

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ABSTRACT Embryonic stem cells (ESCs) are pluripotent cells that can either self-renew or differentiate into many cell types. Oct4 and Sox2 are transcription factors essential to the pluripotent and self-renewing phenotypes of ESCs. Both factors are upstream in the hierarchy of the transcription regulatory network and are partners in regulating several ESC-specific genes. In ESCs, Sox2 is transcriptionally regulated by an enhancer containing a composite sox-oct element that Oct4 and Sox2 bind in a combinatorial interaction. It has previously been shown that Pou5f1, the Oct4 gene, contains a distal enhancer imparting specific expression in both ESCs and preimplantation embryos. Here, we identify a composite sox-oct element within this enhancer and show that it is involved in Pou5f1 transcriptional activity in ESCs. In vitro experiments with ESC nuclear extracts demonstrate that Oct4 and Sox2 interact specifically with this regulatory element. More importantly, by chromatin immunoprecipitation assay, we establish that both Oct4 and Sox2 bind directly to the composite sox-oct elements in both Pou5f1 and Sox2 in living mouse and human ESCs. Specific knockdown of either Oct4 or Sox2 by RNA interference leads to the reduction of both genes' enhancer activities and endogenous expression levels in addition to ESC differentiation. Our data uncover a positive and potentially self-reinforcing regulatory loop that maintains Pou5f1 and Sox2 expression via the Oct4/Sox2 complex in pluripotent cells.

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Mischnik, Marcel, Verdon Taylor, Jens Timmer, Wolfgang Driever, and Daria Onichtchouk. "Rescue-potential of chimeric Pou5f1 transcription factors." Developmental Biology 344, no. 1 (August 2010): 454. http://dx.doi.org/10.1016/j.ydbio.2010.05.168.

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Ковалёва, К. Д., Г. С. Бисмилдина, А. Толегенкызы, and З. С. Качиева. "RESEARCH ON POLYMORPHIC VARIANTS CANDIDATE GENES FOR PSORIASIS." Vestnik, no. 1 (June 17, 2021): 202–7. http://dx.doi.org/10.53065/kaznmu.2021.74.41.043.

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В статье рассмотрены результаты исследования полиморфных вариантов генов предрасположенности к псориазу PSORS1C1, POU5F1, IL23R. Исследованы 192 образца ДНК, из них 116 образцов (77 больных псориазом и 39 человек, без признаков данной патологии) соответствовали стандартным требованиям к пробам ДНК. Полученные результаты сравнивались с референсной версией генома человека CRch 37. Распределение частот генотипов полиморфных вариантов трех генов соответствовали уравнению Харди-Вайнберга. Установлены 17 полиморфных вариантов локуса PSORS1C1, один вариант полиморфизма гена POU5F1 и два варианта полиморфизма гена IL23R, представленных в международной базе данных Ensembl. Впервые, выявлен еще один полиморфный вариант гена IL23R, ранее не аннотированный в базе данных Ensembl. The article discusses the results of the study of polymorphic variants of the PSORS1C1, POU5F1, IL23R genes which predisposition to psoriasis. 192 DNA samples were examined, in which 116 samples (77 patients with psoriasis and 39 people without signs of this pathology) met the standard requirements for DNA samples. Obtained results were compared with the reference version of the human genome CRch 37. The distribution of the genotype frequencies of the polymorphic variants of the three genes corresponded to the Hardy-Weinberg equation. Identified 17 polymorphic variants of the PSORS1C1 locus, one variant of the POU5F1 gene polymorphism and two variants of the IL23R gene polymorphism, which presented in the international database Ensembl. For the first time, identified another polymorphic variant of the IL23R gene, which had not been previously annotated in the Ensembl database.

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Javaid, Nasir, Thuong L. H. Pham, and Sangdun Choi. "Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells." International Journal of Molecular Sciences 22, no. 1 (January 1, 2021): 397. http://dx.doi.org/10.3390/ijms22010397.

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Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.

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Izadyar, Fariborz, Francis Pau, Joel Marh, Natalia Slepko, Tracy Wang, Rafael Gonzalez, Thomas Ramos, Kyle Howerton, Chauncey Sayre, and Francisco Silva. "Generation of multipotent cell lines from a distinct population of male germ line stem cells." REPRODUCTION 135, no. 6 (June 2008): 771–84. http://dx.doi.org/10.1530/rep-07-0479.

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Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

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Popovic, Mina, Monika Bialecka, Maria Gomes Fernandes, Jasin Taelman, Margot Van Der Jeught, Petra De Sutter, Björn Heindryckx, and Susana M. Chuva De Sousa Lopes. "Human blastocyst outgrowths recapitulate primordial germ cell specification events." Molecular Human Reproduction 25, no. 9 (June 18, 2019): 519–26. http://dx.doi.org/10.1093/molehr/gaz035.

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Abstract Our current knowledge of the mechanisms leading to human primordial germ cell (PGC) specification stems solely from differentiation experiments starting from human pluripotent stem cells. However, information regarding the origin of PGCs in vivo remains obscure. Here we apply an improved system for extended in vitro culture of human embryos to investigate the presence of PGC-like cells (PGCLCs) 12 days post fertilization (dpf). Good quality blastocysts (n = 141) were plated at 6 dpf and maintained in hypoxia, in medium supplemented with Activin A until 12 dpf. We primarily reveal that 12 dpf outgrowths recapitulate human peri-implantation events and demonstrate that blastocyst quality significantly impacts both embryo viability at 12 dpf, as well as the presence of POU5F1+ cells within viable outgrowths. Moreover, detailed examination of 12 dpf blastocyst outgrowths revealed a population of POU5F1+, SOX2– and SOX17+ cells that may correspond to PGCLCs, alongside POU5F1+ epiblast-like cells and GATA6+ endoderm-like cells. Our findings suggest that, in human, PGC precursors may become specified within the epiblast and migrate either transiently to the extra-embryonic mesoderm or directly to the dorsal part of the yolk sac endoderm around 12 dpf. This is a descriptive analysis and as such the conclusion that POU5F1+ and SOX17+ cells represent bona fide PGCs can only be considered as preliminary. In the future, other PGC markers may be used to further validate the observed cell populations. Overall, our findings provide insights into the origin of the human germline and may serve as a foundation to further unravel the molecular mechanisms governing PGC specification in human.

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Onichtchouk, Daria. "Pou5f1/oct4 in pluripotency control: Insights from zebrafish." genesis 50, no. 2 (January 6, 2012): 75–85. http://dx.doi.org/10.1002/dvg.20800.

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Kim, Kee-Pyo, You Wu, Juyong Yoon, Kenjiro Adachi, Guangming Wu, Sergiy Velychko, Caitlin M. MacCarthy, et al. "Reprogramming competence of OCT factors is determined by transactivation domains." Science Advances 6, no. 36 (September 2020): eaaz7364. http://dx.doi.org/10.1126/sciadv.aaz7364.

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OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components.

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Goel, Sandeep, Mayako Fujihara, Naojiro Minami, Masayasu Yamada, and Hiroshi Imai. "Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis." REPRODUCTION 135, no. 6 (June 2008): 785–95. http://dx.doi.org/10.1530/rep-07-0476.

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Gonocytes are primitive germ cells that are present in the neonatal testis and are committed to male germline development. Gonocytes differentiate to spermatogonia, which establish and maintain spermatogenesis in the postnatal testis. However, it is unknown whether large animal species have pluripotency-specific proteins in the testis. Nanog and Pou5f1 (Oct3/4) have been identified as transcription factors essential for maintaining pluripotency of embryonic stem cells in mice. Here, we show that NANOG protein was expressed in the germ cells of neonatal pig testes, but was progressively lost with age. NANOG was expressed in most of the lectin Dolichos biflorus agglutinin- and ZBTB16-positive gonocytes, which are known gonocyte-specific markers in pigs. NANOG was also expressed in Sertoli and interstitial cells of neonatal testes. Interestingly, POU5F1 expression was not detected at either the transcript or the protein level in neonatal pig testis. In the prepubertal testis, NANOG and POU5F1 proteins were primarily detected in differentiated germ cells, such as spermatocytes and spermatids, and rarely in undifferentiated spermatogonia. By using a testis transplantation assay, we found that germ cells from 2- to 4-day-old pigs could colonize and proliferate in the testes of the recipient mice, suggesting that primitive germ cells from neonatal pig testes have stem cell potential.

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Verdelli, Chiara, Annamaria Morotti, Giulia Stefania Tavanti, Rosamaria Silipigni, Silvana Guerneri, Stefano Ferrero, Leonardo Vicentini, Valentina Vaira, and Sabrina Corbetta. "The Core Stem Genes SOX2, POU5F1/OCT4, and NANOG Are Expressed in Human Parathyroid Tumors and Modulated by MEN1, YAP1, and β-Catenin Pathways Activation." Biomedicines 9, no. 6 (June 2, 2021): 637. http://dx.doi.org/10.3390/biomedicines9060637.

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Tumors of the parathyroid glands are the second most common endocrine neoplasia. Epigenetic studies revealed an embryonic signature involved in parathyroid tumorigenesis. Here, we investigated the expression of the stem core genes SOX2, POU5F1/OCT4, and NANOG. Rare cells within normal parathyroid glands expressed POU5F1/OCT4 and NANOG, while SOX2 was undetectable. Nuclear SOX2 expression was detectable in 18% of parathyroid adenomas (PAds, n = 34) involving 5–30% of cells, while OCT4 and NANOG were expressed at the nuclear level in a more consistent subset of PAds involving 15–40% of cells. Most parathyroid carcinomas expressed the core stem genes. SOX2-expressing cells co-expressed parathormone (PTH). In PAds-derived primary cultures, silencing of the tumor suppressor gene MEN1 induced the expression of SOX2, likely through a MEN1/HAR1B/SOX2 axis, while calcium-sensing receptor activation increased SOX2 mRNA levels through YAP1 activation. In addition, inducing nuclear β-catenin accumulation in PAds-derived primary cultures by short-term incubation with lithium chloride (LiCl), SOX2 and POU5F1/OCT4 expression levels increased, while NANOG transcripts were reduced, and LiCl long-term incubation induced an opposite pattern of gene expression. In conclusion, detection of the core stem genes in parathyroid tumors supports their embryogenic signature, which is modulated by crucial genes involved in parathyroid tumorigenesis.

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Leichsenring, Manuel, Julia Maes, Rebecca Mössner, Wolfgang Driever, and Daria Onichtchouk. "Pou5f1 Transcription Factor Controls Zygotic Gene Activation In Vertebrates." Science 341, no. 6149 (August 15, 2013): 1005–9. http://dx.doi.org/10.1126/science.1242527.

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The development of multicellular animals is initially controlled by maternal gene products deposited in the oocyte. During the maternal-to-zygotic transition, transcription of zygotic genes commences, and developmental control starts to be regulated by zygotic gene products. In Drosophila, the transcription factor Zelda specifically binds to promoters of the earliest zygotic genes and primes them for activation. It is unknown whether a similar regulation exists in other animals. We found that zebrafish Pou5f1, a homolog of the mammalian pluripotency transcription factor Oct4, occupies SOX-POU binding sites before the onset of zygotic transcription and activates the earliest zygotic genes. Our data position Pou5f1 and SOX-POU sites at the center of the zygotic gene activation network of vertebrates and provide a link between zygotic gene activation and pluripotency control.

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Schultz, Sherri S., Sabrina C. Desbordes, Zhuo Du, Settapong Kosiyatrakul, Inna Lipchina, Lorenz Studer, and Carl L. Schildkraut. "Single-Molecule Analysis Reveals Changes in the DNA Replication Program for the POU5F1 Locus upon Human Embryonic Stem Cell Differentiation." Molecular and Cellular Biology 30, no. 18 (July 20, 2010): 4521–34. http://dx.doi.org/10.1128/mcb.00380-10.

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ABSTRACT Human embryonic stem cells (hESCs), due to their pluripotent nature, represent a particularly relevant model system to study the relationship between the replication program and differentiation state. Here, we define the basic properties of the replication program in hESCs and compare them to the programs of hESC-derived multipotent cells (neural rosette cells) and primary differentiated cells (microvascular endothelial cells [MECs]). We characterized three genomic loci: two pluripotency regulatory genes, POU5F1 (OCT4) and NANOG, and the IGH locus, a locus that is transcriptionally active specifically in B-lineage cells. We applied a high-resolution approach to capture images of individual replicated DNA molecules. We demonstrate that for the loci studied, several basic properties of replication, including the average speed of replication forks and the average density of initiation sites, were conserved among the cells analyzed. We also demonstrate, for the first time, the presence of initiation zones in hESCs. However, significant differences were evident in other aspects of replication for the DNA segment containing the POU5F1 gene. Specifically, the locations of centers of initiation zones and the direction of replication fork progression through the POU5F1 gene were conserved in two independent hESC lines but were different in hESC-derived multipotent cells and MECs. Thus, our data identify features of the replication program characteristic of hESCs and define specific changes in replication during hESC differentiation.

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Alanis-Lobato, Gregorio, Jasmin Zohren, Afshan McCarthy, Norah M. E. Fogarty, Nada Kubikova, Emily Hardman, Maria Greco, Dagan Wells, James M. A. Turner, and Kathy K. Niakan. "Frequent loss of heterozygosity in CRISPR-Cas9–edited early human embryos." Proceedings of the National Academy of Sciences 118, no. 22 (April 9, 2021): e2004832117. http://dx.doi.org/10.1073/pnas.2004832117.

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CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9–targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4–20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

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Canon, E., L. Jouneau, T. Blachère, N. Peynot, N. Daniel, L. Boulanger, L. Maulny, et al. "Progressive methylation of POU5F1 regulatory regions during blastocyst development." Reproduction 156, no. 2 (August 2018): 145–61. http://dx.doi.org/10.1530/rep-17-0689.

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ThePOU5F1gene encodes one of the ‘core’ transcription factors necessary to establish and maintain pluripotency in mammals. Its function depends on its precise level of expression, so its transcription has to be tightly regulated. To date, few conserved functional elements have been identified in its 5′ regulatory region: a distal and a proximal enhancer, and a minimal promoter, epigenetic modifications of which interfere withPOU5F1expression and function inin vitro-derived cell lines. Also, its permanent inactivation in differentiated cells depends onde novomethylation of its promoter. However, little is known about the epigenetic regulation ofPOU5F1expression in the embryo itself. We used the rabbit blastocyst as a model to analyze the methylation dynamics of thePOU5F15′ upstream region, relative to its regulated expression in different compartments of the blastocyst over a 2-day period of development. We evidenced progressive methylation of the 5′ regulatory region and the first exon accompanying differentiation and the gradual repression ofPOU5F1. Methylation started in the early trophectoderm before complete transcriptional inactivation. Interestingly, the distal enhancer, which is known to be active in naïve pluripotent cells only, retained a very low level of methylation in primed pluripotent epiblasts and remained less methylated in differentiated compartments than the proximal enhancer. This detailed study identified CpGs with the greatest variations in methylation, as well as groups of CpGs showing a highly correlated behavior, during differentiation. Moreover, our findings evidenced few CpGs with very specific behavior during this period of development.

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Onichtchouk, Daria, Geier Florian, Bozena Polok, Daniel M. Messerschmidt, Rebecca Mössner, Verdon Taylor, Jens Timmer, and Wolfgang Driever. "Oct4/Pou5f1 controls differentiation timing in early zebrafish embryo." Developmental Biology 344, no. 1 (August 2010): 415. http://dx.doi.org/10.1016/j.ydbio.2010.05.033.

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Antonescu, Cristina R., Narasimhan P. Agaram, Yun‐Shao Sung, Lei Zhang, and Brendan C. Dickson. "Undifferentiated round cell sarcomas with novel SS18‐POU5F1 fusions." Genes, Chromosomes and Cancer 59, no. 11 (July 6, 2020): 620–26. http://dx.doi.org/10.1002/gcc.22879.

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Arakawa, Tatsuhiko, Tomohiko Yoshimi, Masayuki Azuma, and Taro Tachibana. "Production of a Monoclonal Antibody Specific for Pou5f1/Oct4." Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 32, no. 3 (June 2013): 229–31. http://dx.doi.org/10.1089/mab.2013.0004.

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Albert, S., J. Ehmcke, J. Wistuba, K. Eildermann, R. Behr, S. Schlatt, and J. Gromoll. "Germ cell dynamics in the testis of the postnatal common marmoset monkey (Callithrix jacchus)." REPRODUCTION 140, no. 5 (November 2010): 733–42. http://dx.doi.org/10.1530/rep-10-0235.

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The seminiferous epithelium in the nonhuman primate Callithrix jacchus is similarly organized to man. This monkey has therefore been used as a preclinical model for spermatogenesis and testicular stem cell physiology. However, little is known about the developmental dynamics of germ cells in the postnatal primate testis. In this study, we analyzed testes of newborn, 8-week-old, and adult marmosets employing immunohistochemistry using pluripotent stem cell and germ cell markers DDX4 (VASA), POU5F1 (OCT3/4), and TFAP2C (AP-2γ). Stereological and morphometric techniques were applied for quantitative analysis of germ cell populations and testicular histological changes. Quantitative RT-PCR (qRT-PCR) of testicular mRNA was applied using 16 marker genes establishing the corresponding profiles during postnatal testicular development. Testis size increased during the first 8 weeks of life with the main driver being longitudinal outgrowth of seminiferous cords. The number of DDX4-positive cells per testis doubled between birth and 8 weeks of age whereas TFAP2C- and POU5F1-positive cells remained unchanged. This increase in DDX4-expressing cells indicates dynamic growth of the differentiated A-spermatogonial population. The presence of cells expressing POU5F1 and TFAP2C after 8 weeks reveals the persistence of less differentiated germ cells. The mRNA and protein profiles determined by qRT-PCR and western blot in newborn, 8-week-old, and adult marmosets corroborated the immunohistochemical findings. In conclusion, we demonstrated the presence of distinct spermatogonial subpopulations in the primate testis exhibiting different dynamics during early testicular development. Our study demonstrates the suitability of the marmoset testis as a model for human testicular development.

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Lin, Zachary Yu-Ching, Masanori Imamura, Chiaki Sano, Ryusuke Nakajima, Tomoko Suzuki, Rie Yamadera, Yuji Takehara, Hirotaka James Okano, Erika Sasaki, and Hideyuki Okano. "Molecular signatures to define spermatogenic cells in common marmoset (Callithrix jacchus)." REPRODUCTION 143, no. 5 (May 2012): 597–609. http://dx.doi.org/10.1530/rep-11-0215.

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Germ cell development is a fundamental process required to produce offspring. The developmental program of spermatogenesis has been assumed to be similar among mammals. However, recent studies have revealed differences in the molecular properties of primate germ cells compared with the well-characterized mouse germ cells. This may prevent simple application of rodent insights into higher primates. Therefore, thorough investigation of primate germ cells is necessary, as this may lead to the development of more appropriate animal models. The aim of this study is to define molecular signatures of spermatogenic cells in the common marmoset, Callithrix jacchus. Interestingly, NANOG, PRDM1, DPPA3 (STELLA), IFITM3, and ZP1 transcripts, but no POU5F1 (OCT4), were detected in adult marmoset testis. Conversely, mouse testis expressed Pou5f1 but not Nanog, Prdm1, Dppa3, Ifitm3, and Zp1. Other previously described mouse germ cell markers were conserved in marmoset and mouse testes. Intriguingly, marmoset spermatogenic cells underwent dynamic protein expression in a developmental stage-specific manner; DDX4 (VASA) protein was present in gonocytes, diminished in spermatogonial cells, and reexpressed in spermatocytes. To investigate epigenetic differences between adult marmoset and mice, DNA methylation analyses identified unique epigenetic profiles to marmoset and mice. Marmoset NANOG and POU5F1 promoters in spermatogenic cells exhibited a methylation status opposite to that in mice, while the DDX4 and LEFTY1 loci, as well as imprinted genes, displayed an evolutionarily conserved methylation pattern. Marmosets have great advantages as models for human reproductive biology and are also valuable as experimental nonhuman primates; thus, the current study provides an important platform for primate reproductive biology, including possible applications to humans.

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Fujino, Takashi, Kimie Nomura, Yuichi Ishikawa, Hatsune Makino, Akihiro Umezawa, Hiroyuki Aburatani, Koichi Nagasaki, and Takuro Nakamura. "Function of EWS-POU5F1 in Sarcomagenesis and Tumor Cell Maintenance." American Journal of Pathology 176, no. 4 (April 2010): 1973–82. http://dx.doi.org/10.2353/ajpath.2010.090486.

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Simmet, Kilian, Valeri Zakhartchenko, Julia Philippou-Massier, Helmut Blum, Nikolai Klymiuk, and Eckhard Wolf. "OCT4/POU5F1 is required for NANOG expression in bovine blastocysts." Proceedings of the National Academy of Sciences 115, no. 11 (February 26, 2018): 2770–75. http://dx.doi.org/10.1073/pnas.1718833115.

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Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated OCT4 knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.

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Nazarov, I. B., V. A. Krasnoborova, A. G. Mitenberg, E. V. Chikhirzhina, A. P. Davidov-Sinitzin, M. A. Liskovykh, and A. N. Tomilin. "Transcription regulation of Oct4 (Pou5F1) gene by its distal enhancer." Cell and Tissue Biology 8, no. 1 (January 2014): 27–32. http://dx.doi.org/10.1134/s1990519x14010106.

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Lippok, Bernadette, Sungmin Song, and Wolfgang Driever. "Pou5f1 protein expression and posttranslational modification during early zebrafish development." Developmental Dynamics 243, no. 3 (November 21, 2013): 468–77. http://dx.doi.org/10.1002/dvdy.24079.

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Eiselleova, Livia, Viktor Lukjanov, Simon Farkas, David Svoboda, Karel Stepka, and Irena Koutna. "The Role of RNA Polymerase II Contiguity and Long-Range Interactions in the Regulation of Gene Expression in Human Pluripotent Stem Cells." Stem Cells International 2019 (February 3, 2019): 1–12. http://dx.doi.org/10.1155/2019/1375807.

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The eukaryotic nucleus is a highly complex structure that carries out multiple functions primarily needed for gene expression, and among them, transcription seems to be the most fundamental. Diverse approaches have demonstrated that transcription takes place at discrete sites known as transcription factories, wherein RNA polymerase II (RNAP II) is attached to the factory and immobilized while transcribing DNA. It has been proposed that transcription factories promote chromatin loop formation, creating long-range interactions in which relatively distant genes can be transcribed simultaneously. In this study, we examined long-range interactions between the POU5F1 gene and genes previously identified as being POU5F1 enhancer-interacting, namely, CDYL, TLE2, RARG, and MSX1 (all involved in transcriptional regulation), in human pluripotent stem cells (hPSCs) and their early differentiated counterparts. As a control gene, RUNX1 was used, which is expressed during hematopoietic differentiation and not associated with pluripotency. To reveal how these long-range interactions between POU5F1 and the selected genes change with the onset of differentiation and upon RNAP II inhibition, we performed three-dimensional fluorescence in situ hybridization (3D-FISH) followed by computational simulation analysis. Our analysis showed that the numbers of long-range interactions between specific genes decrease during differentiation, suggesting that the transcription of monitored genes is associated with pluripotency. In addition, we showed that upon inhibition of RNAP II, long-range associations do not disintegrate and remain constant. We also analyzed the distance distributions of these genes in the context of their positions in the nucleus and revealed that they tend to have similar patterns resembling normal distribution. Furthermore, we compared data created in vitro and in silico to assess the biological relevance of our results.

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Xie, Fang, Christopher L. Anderson, Kelsey R. Timme, Scott G. Kurz, Samodha C. Fernando, and Jennifer R. Wood. "Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice." Endocrinology 157, no. 4 (February 16, 2016): 1630–43. http://dx.doi.org/10.1210/en.2015-1851.

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Abstract RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients.

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Arai, Hiroyuki, Joshua Millstein, Fotios Loupakis, Sebastian Stintzing, Jingyuan Wang, Francesca Battaglin, Natsuko Kawanishi, et al. "Polymorphisms of pluripotency transcription factors for predicting cetuximab efficacy in metastatic colorectal cancer: Data from FIRE-3 and TRIBE trials." Journal of Clinical Oncology 39, no. 3_suppl (January 20, 2021): 98. http://dx.doi.org/10.1200/jco.2021.39.3_suppl.98.

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98 Background: Cancer stem cells are subpopulation of cancer cells characterized by the self-renewal ability, and primarily maintained by a network of pluripotency transcription factors (PTFs). Despite their chemo-resistant phenotype, recent clinical trials data show cetuximab (Cet) is more beneficial for a subset of metastatic colorectal cancer (mCRC) categorized in a mesenchymal/stem-like subtype based on transcriptomic classifications. We investigated whether single nucleotide polymorphisms (SNPs) in PTF encoding genes have predictive values for the efficacy of Cet in mCRC patients. Methods: Genomic and clinical data from three independent cohorts of patient treated with first-line chemotherapy ( n = 451, in total) were tested: Cet cohort (FOLFIRI+Cet arm in FIRE-3 trial, n = 129); bevacizumab (Bev) cohort 1 (FOLFIRI+Bev arm in FIRE-3 trial, n = 107) and Bev cohort 2 (FOLFIRI+Bev arm in TRIBE trial, n = 215), as controls. Genomic DNA extracted from blood samples was genotyped using an OncoArray (Illumina, Inc., San Diego, CA, USA). Eight SNPs in PTF encoding genes ( NANOG rs11055786, NANOG rs11055767, NANOGP8 rs2168958, NANOGP8 rs9944179, POU5F1 rs3130501, POU5F1 rs3130932, SOX2 rs11915160, and MYC rs3891248) were tested for association with progression-free survival (PFS) and overall survival (OS), using Cox proportional hazards model. Results: In the Cet cohort, three SNPs were significantly associated with PFS in univariate analysis: NANOG rs11055767 (C/C vs any A allele, hazard ratio [HR] = 0.62, 95% confidence interval [CI] = 0.42–0.94, log-rank p = 0.02), NANOGP8 rs2168958 (A/A vs any C allele, HR = 2.12, 95% CI = 1.36–3.29, log-rank p < 0.01), and NANOGP8 rs9944179 (G/G vs any A allele, HR = 3.68, 95% CI = 1.46–9.31, log-rank p < 0.01). Multivariate analysis confirmed the significance in NANOGP8 rs2168958 and NANOGP8 rs9944179. Furthermore, two SNPs were significantly associated with OS in multivariate analysis: POU5F1 rs313051 (G/G vs any A allele, HR = 0.46, 95% CI = 0.22–0.99, adjusted p = 0.04) and POU5F1 rs3130932 (A/A vs any C allele, HR = 0.46, 95% CI = 0.22–0.93, adjusted p = 0.03). Both in the Bev cohorts 1 and 2, no significant associations between the SNPs and clinical outcomes were observed. Conclusions: The current findings suggest that polymorphisms in the PTF genes could be predictive markers for Cet in patients with mCRC. Further studies are warranted to validate the predictive utility.

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Ganeshan, L., and C. O'Neill. "145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO." Reproduction, Fertility and Development 21, no. 9 (2009): 63. http://dx.doi.org/10.1071/srb09abs145.

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The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

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Ståhlberg, Anders, Martin Bengtsson, Martin Hemberg, and Henrik Semb. "Quantitative Transcription Factor Analysis of Undifferentiated Single Human Embryonic Stem Cells." Clinical Chemistry 55, no. 12 (December 1, 2009): 2162–70. http://dx.doi.org/10.1373/clinchem.2009.131433.

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Abstract Background: Human embryonic stem cells (hESCs) require expression of transcription factor genes POU5F1 (POU class 5 homeobox 1), NANOG (Nanog homeobox), and SOX2 [SRY (sex determining region Y)-box 2] to maintain their capacity for self-renewal and pluripotency. Because of the heterogeneous nature of cell populations, it is desirable to study the gene regulation in single cells. Large and potentially important fluctuations in a few cells cannot be detected at the population scale with microarrays or sequencing technologies. We used single-cell gene expression profiling to study cell heterogeneity in hESCs. Methods: We collected 47 single hESCs from cell line SA121 manually by glass capillaries and 57 single hESCs from cell line HUES3 by flow cytometry. Single hESCs were lysed and reverse-transcribed. Reverse-transcription quantitative real-time PCR was then used to measure the expression POU5F1, NANOG, SOX2, and the inhibitor of DNA binding genes ID1, ID2, and ID3. A quantitative noise model was used to remove measurement noise when pairwise correlations were estimated. Results: The numbers of transcripts per cell varied >100-fold between cells and showed lognormal features. POU5F1 expression positively correlated with ID1 and ID3 expression (P < 0.05) but not with NANOG or SOX2 expression. When we accounted for measurement noise, SOX2 expression was also correlated with ID1, ID2, and NANOG expression (P < 0.05). Conclusions: We demonstrate an accurate method for transcription profiling of individual hESCs. Cell-to-cell variability is large and is at least partly nonrandom because we observed correlations between core transcription factors. High fluctuations in gene expression may explain why individual cells in a seemingly undifferentiated cell population have different susceptibilities for inductive cues.

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Urrego, R., S. M. Bernal-Ulloa, N. A. Chavarría, E. Herrera-Puerta, A. Lucas-Hahn, D. Herrmann, S. Winkler, D. Pache, H. Niemann, and N. Rodriguez-Osorio. "Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro." Zygote 25, no. 2 (January 31, 2017): 131–40. http://dx.doi.org/10.1017/s096719941600040x.

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SummaryBovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

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Montserrat, Nuria, Lorena De Oñate, Elena Garreta, Federico Gonzãlez, Antonio Adamo, Cristina Eguizãbal, Sophia Häfner, Rita Vassena, and Juan Carlos Izpisua Belmonte. "Generation of Feeder-Free Pig Induced Pluripotent Stem Cells without Pou5f1." Cell Transplantation 21, no. 5 (May 2012): 815–25. http://dx.doi.org/10.3727/096368911x601019.

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Onichtchouk, Daria, Florian Geier, Bozena Polok, Daniel M. Messerschmidt, Rebecca Mössner, Björn Wendik, Sungmin Song, Verdon Taylor, Jens Timmer, and Wolfgang Driever. "Zebrafish Pou5f1‐dependent transcriptional networks in temporal control of early development." Molecular Systems Biology 6, no. 1 (January 2010): 354. http://dx.doi.org/10.1038/msb.2010.9.

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Miyoshi, Norikatsu, Shiki Fujino, Masayuki Ohue, Masayoshi Yasui, Yusuke Takahashi, Keijiro Sugimura, Akira Tomokuni, et al. "The POU5F1 gene expression in colorectal cancer: a novel prognostic marker." Surgery Today 48, no. 7 (February 27, 2018): 709–15. http://dx.doi.org/10.1007/s00595-018-1644-9.

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Li, Xuyan, Tianfei Yu, Ming Li, Youqi Wang, Bo Meng, and Yanshuang Mu. "NANOG improves type I collagen expression in human fetal scleral fibroblasts." Archives of Biological Sciences 71, no. 1 (2019): 63–70. http://dx.doi.org/10.2298/abs180711048l.

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Human fetal scleral fibroblasts (HFSFs) are components of the sclera and play important roles in its structure and function. In myopia, scleral remodeling reduces collagen fibers and the sclera begins to thin. NANOG is a key transcription factor essential for pluripotent and self-renewing phenotypes of undifferentiated embryonic stem cells. To determine whether NANOG improves human fetal scleral fibroblast quality and the underlying mechanisms in these cells, we established stable NANOG-overexpressing HFSFs. We studied type I collagen (COL1A 1) and Rho-associated coiled-coil protein kinase 1 (ROCK1) expression in transfected cells. We also investigated POU5F1, SOX2, KLF4, MYC and SALL4 expression in NANOG stably-overexpressed fibroblasts. Our data show that NANOG expression increased proliferation rates in fibroblasts. When compared to controls, expression of COL1A 1 in transfected fibroblasts was increased and the expression of ROCK1 was decreased. Similarly, the expression of POU5F1, SOX2 and KLF4 was downregulated, the expression of MYC was upregulated and there was no significant change in the expression of SALL4 in transfected fibroblasts. Our results suggest that in fibroblasts, NANOG regulates ROCK1 expression and improves COL1A 1 expression to delay scleral remodeling.

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Wang, Jing, Binbin Wang, Junjie Song, Peisu Suo, Feng Ni, Beili Chen, Xu Ma, and Yunxia Cao. "New candidate gene POU5F1 associated with premature ovarian failure in Chinese patients." Reproductive BioMedicine Online 22, no. 3 (March 2011): 312–16. http://dx.doi.org/10.1016/j.rbmo.2010.11.008.

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Sedlic, Filip, Fran Seiwerth, Ana Sepac, Suncana Sikiric, Marina Cindric, Marija Milavic, Lovorka Batelja Vuletic, Marko Jakopovic, and Sven Seiwerth. "Mitochondrial ROS Induce Partial Dedifferentiation of Human Mesothelioma via Upregulation of NANOG." Antioxidants 9, no. 7 (July 10, 2020): 606. http://dx.doi.org/10.3390/antiox9070606.

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The expression of pluripotency factors is a key regulator of tumor differentiation status and cancer stem cells. The purpose of this study was to examine the expression of pluripotency factors and differentiation status of human mesothelioma and the role of mitochondria in their regulation. We tested the expression of OCT4/POU5F1, NANOG, SOX2, PI3K-AKT pathway and BCL2 genes and proteins in 65 samples of human mesothelioma and 19 samples of normal mesothelium. Mitochondrial membrane potential, reactive oxygen species (ROS) generation and expression of pluripotency factors were also tested in human mesothelioma cell line. Human mesothelium and mesothelioma expressed SOX2, NANOG, PI3K and AKT genes and proteins and POU5F1 gene, whereby NANOG, SOX2 and phosphorylated (activated) AKT were upregulated in mesothelioma. NANOG protein expression was elevated in less differentiated samples of human mesothelioma. The expression of genes of PI3K-AKT pathway correlated with pluripotency factor genes. Mesothelioma cells had functional, but depolarized mitochondria with large capacity to generate ROS. Mitochondrial ROS upregulated NANOG and mitoTEMPO abrogated it. In conclusion, human mesothelioma displays enhanced expression of NANOG, SOX2 and phosphorylated AKT proteins, while elevated NANOG expression correlates with poor differentiation of human mesothelioma. Mitochondria of mesothelioma cells have a large capacity to form ROS and thereby upregulate NANOG, leading to dedifferentiation of mesothelioma.

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Castillo-Martín, Miriam, Marc Yeste, Albert Soler, Roser Morató, and Sergi Bonet. "Addition of L-ascorbic acid to culture and vitrification media of IVF porcine blastocysts improves survival and reduces HSPA1A levels of vitrified embryos." Reproduction, Fertility and Development 27, no. 7 (2015): 1115. http://dx.doi.org/10.1071/rd14078.

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The aim of the present study was to determine the effect of l-ascorbic acid on embryo quality and gene expression of porcine blastocysts after supplementations of in vitro culture medium and/or vitrification–warming media. Embryo quality, in terms of total cell number (TCN), DNA fragmentation and peroxide levels, together with the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), POU class 5 homeobox 1 (POU5F1) and heat shock protein 70 (HSPA1A), was analysed. In Experiment 1, gene expression and embryo quality of fresh blastocysts were evaluated after culture with or without l-ascorbic acid; no significant differences were observed between the groups. In Experiment 2, blastocysts cultured with or without l-ascorbic acid were vitrified using two different vitrification solutions, supplemented or not with l-ascorbic acid. Supplementation of culture and vitrification media significantly enhanced survival rates and reduced peroxide levels. No significant differences in TCN, DNA fragmentation and BAX, BCL2L1 and POU5F1 expression were found in vitrified blastocysts among experimental groups. Vitrification procedures increase HSPA1A transcript abundance, but this increase was significantly lower in embryos cultured and/or vitrified with l-ascorbic acid. Thus, supplementing culture and/or vitrification media with l-ascorbic acid enhances survival rates of porcine blastocysts, suggesting a relationship with HSPA1A expression.

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Verma, Shiv, Eswar Shankar, F. Naz Cemre Kalayci, Amrita Mukunda, Malek Alassfar, Vaibhav Singh, E. Ricky Chan, Gregory T. MacLennan, and Sanjay Gupta. "Androgen Deprivation Induces Transcriptional Reprogramming in Prostate Cancer Cells to Develop Stem Cell-Like Characteristics." International Journal of Molecular Sciences 21, no. 24 (December 16, 2020): 9568. http://dx.doi.org/10.3390/ijms21249568.

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Enzalutamide, an antiandrogen, is approved for therapy of castration resistant prostate cancer. Clinical applications have shown that approximately 30% of patients acquire resistance after a short period of treatment. However, the molecular mechanisms underlying this resistance is not completely understood. To identify transcriptomic signatures associated with acquisition of drug resistance we profiled gene expression of paired enzalutamide sensitive and resistant human prostate cancer LNCaP (lymph node carcinoma of the prostate) and C4-2B cells. Overlapping genes differentially regulated in the enzalutamide resistant cells were ranked by Ingenuity Pathway Analysis and their functional validation was performed using ingenuity knowledge database followed by confirmation to correlate transcript with protein expression. Analysis revealed that genes associated with cancer stem cells, such as POU5F1 (OCT4), SOX2, NANOG, BMI1, BMP2, CD44, SOX9, and ALDH1 were markedly upregulated in enzalutamide resistant cells. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with RUNX2, hedgehog, integrin signaling, and molecules associated with elastic fibers. Further examination of a patient cohort undergoing ADT and its comparison with no-ADT group demonstrated high expression of POU5F1 (OCT4), ALDH1, and SOX2 in ADT specimens, suggesting that they may be clinically relevant therapeutic targets. Altogether, our approach exhibits the potential of integrative transcriptomic analyses to identify critical genes and pathways of antiandrogen resistance as a promising approach for designing novel therapeutic strategies to circumvent drug resistance.

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Goel, Sandeep, Mayako Fujihara, Kazuo Tsuchiya, Yuji Takagi, Naojiro Minami, Masayasu Yamada, and Hiroshi Imai. "Multipotential ability of primitive germ cells from neonatal pig testis cultured in vitro." Reproduction, Fertility and Development 21, no. 5 (2009): 696. http://dx.doi.org/10.1071/rd08176.

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Gonocytes are progenitor-type germ cells that arise from primordial germ cells and differentiate further into spermatogonia, thereby initiating spermatogenesis. In the present study, freshly isolated gonocytes were found to have either weak or no expression of pluripotency determining transcription factors, such as POU5F1, SOX2 and C-MYC. Interestingly, the expression of these transcription factors, as well as other vital transcription factors, such as NANOG, KLF4 and DAZL, were markedly upregulated in cultured cells. Cells in primary cultures expressed specific germ cell and pluripotency markers, such as lectin Dolichos biflorus agglutinin (DBA), KIT, ZBTB16, stage-specific embryonic antigen (SSEA-1), NANOG and POU5F1. Using a monoclonal antibody to specifically identify porcine germ cells, the stem cell potential of fresh and cultured cells was determined with a testis xenotransplantation assay. Colonised porcine germ cells were detected only in mouse testes that were either transplanted with fresh testicular cells or with cells from primary cultures. Interestingly, testes transplanted with cells from primary cultures showed colonisation of germ cells in the interstitial space, reflecting their tumourigenic nature. The formation of teratomas with tissues originating from the three germinal layers following the subcutaneous injection of cells into nude mice from primary cultures confirmed their multipotency. The results of the present study may provide useful information for the establishment of multipotent germ stem cell lines from neonatal pig testis.

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Javed, Awais, Pierre Mattar, Suying Lu, Kamil Kruczek, Magdalena Kloc, Anai Gonzalez-Cordero, Rod Bremner, Robin R. Ali, and Michel Cayouette. "Pou2f1 and Pou2f2 cooperate to control the timing of cone photoreceptor production in the developing mouse retina." Development 147, no. 18 (September 2, 2020): dev188730. http://dx.doi.org/10.1242/dev.188730.

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ABSTRACTMultipotent retinal progenitor cells (RPCs) generate various cell types in a precise chronological order, but how exactly cone photoreceptor production is restricted to early stages remains unclear. Here, we show that the POU-homeodomain factors Pou2f1/Pou2f2, the homologs of Drosophila temporal identity factors nub/pdm2, regulate the timely production of cones in mice. Forcing sustained expression of Pou2f1 or Pou2f2 in RPCs expands the period of cone production, whereas misexpression in late-stage RPCs triggers ectopic cone production at the expense of late-born fates. Mechanistically, we report that Pou2f1 induces Pou2f2 expression, which binds to a POU motif in the promoter of the rod-inducing factor Nrl to repress its expression. Conversely, conditional inactivation of Pou2f2 in RPCs increases Nrl expression and reduces cone production. Finally, we provide evidence that Pou2f1 is part of a cross-regulatory cascade with the other temporal identity factors Ikzf1 and Casz1. These results uncover Pou2f1/2 as regulators of the temporal window for cone genesis and, given their widespread expression in the nervous system, raise the possibility of a general role in temporal patterning.This article has an associated ‘The people behind the papers’ interview.

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Journal articles on the topic 'Pou5f1'

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