Pou5f1 Promoter Research Articles

Abstract P1145: All-in-one AAV CRISPR/dCas9VPR Vectors Enable Endogenous Gene Activation In Cardiomyocytes

The transcriptional landscape of cardiomyocytes harbors the cellular adaptation upon stress, but remains out of scope for therapies preventing heart failure progression. CRISPR/Cas9 mediated endogenous gene activation (CRISPRa) using enzymatically inactive Cas9 (dCas9) fused to transcription activation domains (VPR) allows for precise and fine-tuned gene activity control when targeted to promoter or enhancer regions of interest via guide RNAs (gRNAs). Transgenic mouse models and human induced pluripotent stem cell (hiPSC) lines were established for proof-of-concept (PoC) gene activation studies in cardiomyocytes. However, the size of the commonly used Streptococcus pyogenes Cas9 exceeds the packaging limits of gene therapy relevant adeno-associated virus (AAV), hampering the therapeutic use of the technology. We aimed to engineer AAV vectors based on smaller, enzymatically inactive Cas9 variants fused to a minimalized VPR-transactivation domain co-expressing a single gRNA for all-in-one gene activation strategies. We rendered mini-Cas9s enzymatically inactive by mutating the evolutionary conserved RUVC and HNH regions and C-terminally attached a minimal VPR domain as well as a HA-tag. As PoC, we expressed mini-dCas9VPR under the CMV promoter in HEK293T cells and confirmed expression of the Cas9 variants via immunoblotting. When we targeted mini-dCas9VPR to the anti-hypertrophic transcription factor KLF15 promoter region or the PoC target POU5F1 promoter, we could confirm significant gene activation compared to non-transfected and non-targeted gRNA controls (KLF15: 3-fold change, POU5F1: 4-fold change, n ≤ 4). We next exchanged the CMV promoter by the TNNT2 promoter to allow cardiomyocyte-specific expression of mini-dCas9VPR. Upon targeting the KLF15 promoter with these vectors transfected in hiPSC-cardiomyocytes, we observed significantly elevated KLF15 mRNA levels compared to controls (1.5-fold change, n ≤ 4) and robust expression of the mini-CRISPRa system, respectively. In conclusion, we present novel vectors for AAV mediated cardiomyocyte-specific endogenous gene regulation which are currently tested in hiPSC-based models, patient-derived cardiac slices and for in vivo gene activation.

Sumoylation regulates functional properties of the oocyte transcription factors SOHLH1 and NOBOX.

SOHLH1 and NOBOX are oocyte-expressed transcription factors with critical roles in ovary development and fertility. In mice, Sohlh1 and Nobox are essential for fertility through their regulation of the oocyte transcriptional network and cross-talk to somatic cells. Sumoylation is a posttranslational modification that regulates transcription factor function, and we previously showed that mouse oocytes deficient for sumoylation had an altered transcriptional landscape that included significant changes in NOBOX target genes. Here, we show that mouse SOHLH1 is modified by SUMO2/3 at lysine 345 and mutation of this residue alters SOHLH1 nuclear to cytoplasmic localization. In NOBOX, we identify a non-consensus SUMO site, K97, that eliminates NOBOX mono-SUMO2/3 conjugation, while a point mutation at K125 had no effect on NOBOX sumoylation. However, NOBOXK97R/K125R double mutants showed loss of mono-SUMO2/3 and altered higher molecular weight modifications, suggesting cooperation between these lysine's. NOBOXK97R and NOBOXK97R/K125R differentially regulated NOBOX promoter targets, with increased activity on the Gdf9 promoter, but no effect on the Pou5f1 promoter. These data implicate sumoylation as a novel regulatory mechanism for SOHLH1 and NOBOX, which may prove useful in refining their roles during oogenesis as well as their function during reprogramming to generate de novo germ cells.

Establishing a Cell Model for Studying the Interaction of the Pou5f1 (Oct4) Promoter with the Genetic Environment

Pou5f1 (Oct4), a key gatekeeper of cellular pluripotency, has little been studied with regard to roles of its regulatory cis-elements in transcriptional control of neighboring genes. In this study, bi-allelic deletion of the Pou5f1 promoter with simultaneous rescue of mouse embryonic stem cell (ESC) self-renewal by a 9.8-kb fragment of Pou5f1 knocked into the Rosa26 locus has been performed. The results confirm the previously established Pou5f1 gene boundaries, whereas the generated ESCs will be indispensable for further studies of the Pou5f1 promoter in transcriptional regulation of neighbor genes.

Induced DNA hypomethylation by Folic Acid Deprivation in Bovine Fibroblast Donor Cells Improves Reprogramming of Somatic Cell Nuclear Transfer Embryos

Aberrant patterns of DNA methylation are consistent events in SCNT derived embryos and mechanistically are believed to be related to abnormal development. While some epigenetic drugs have been used in attempts to improve SCNT efficiency but some concerns remained toward the safety of these drugs on the health of future offspring. Folate is an essential cofactor in one‐carbon cycle for conversion of homocysteine to methionine, thereby ensuring supply of SAM, the universal methyl donor for many biological methylation reactions including DNA methylation. Therefore, in vitro DNA hypo-methylation can be induced by folate deprivation and this study aims at deciphering the role of folic acid deprivation in culture medium of BFFs for 6 days on SCNT efficiency. Our data revealed that culture of fibroblast cells in folate− medium containing 0.5% FBS did not alter the cell cycle compared to other groups. Flowcytometric analysis revealed that DNA methylation (5-mC level) in folate deprived cells cultured in 0.5% serum was decreased compared to folate+ group. The result of bisulfite sequencing was in accordance with flowcytometric analysis, which indicated a decrease in DNA methylation of POU5F1 promoter. Gene expression analysis revealed an increase in expression of POU5F1 gene in folate− group. The nuclear area of the cells in folate− group was significantly larger than folate+ group. Induced DNA hypomethylation by folate deprivation in the folate− group significantly improved blastocyst rate compared to the folate+ group. DNA methylation level in POU5F1 promoter and ICR of H19 and IGF2 of SCNT derived embryos in the folate− group was similar to the IVF derived blastocysts. In conclusion, our results proposes a promising “non-chemical” instead of “chemical” approach using inhibitors of epigenetic modifier enzymes for improving mammalian SCNT efficiency for agricultural and biomedical purposes.

DNA methylation regulated by ascorbic acids in yak preimplantation embryo helps to improve blastocyst quality.

DNA methylation as an important, essential epigenetic modification is critical for the successful development of mammalian embryos. In recent years, the important role of ascorbic acid (AA) as an irreplaceable cofactor for epigenetic regulation has been confirmed. However, the effect of AA on DNA methylation in preimplantation embryo development of plateau yak remains unknown. In this study, we explored whether AA can help regulates DNA methylation in yak preimplantation embryos to improve the blastocyst quality. First, our results indicate that the preimplantation of the yak still follows the classical pattern of DNA demethylation and remethylation, however, remethylation occurs in the blastocyst stage. Second, the unique expression pattern of the ten-eleven translocation enzyme (TET3) in the cytoplasm plays a key role in the demethylation mechanism. Third, in the blastocyst stage, the pluripotency gene CDX2 promoter region was in a hypomethylated state, and the POU5F1, SOX2, and NANOG promoter regions were in moderate methylation states. In addition, treatment with 50 μg/ml AA mainly improved the expression levels of DNMT1, DNMT3a, and TET3, ensured the establishment, maintenance and transition of 5-methylcytosine. After AA treatment, the methylation level of the pluripotency genes NANOG promoter regions was significantly reduced, and the mRNA transcript abundance of the pluripotency genes NANOG, POU5F1, and CDX2 was upregulated. In conclusion, our findings suggest that AA could increase blastocyst cell numbers by regulating DNA methylation of yak preimplantation embryos .

Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency.

BackgroundPluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive.MethodsWe utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR).ResultsThe results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8.ConclusionsIn summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells.

Millions of cis-regulatory elements are predicted in the human genome, but direct evidence for their biological function is still scarce. Here we report a high-throughput method, Cis-Regulatory Element Scan by Tiling-deletion and sequencing (CREST-seq), for unbiased discovery and functional assessment of cis regulatory sequences in the genome. We use it to interrogate the 2Mbp POU5F1 locus in the human embryonic stem cells and identify 45 cis-regulatory elements of POU5F1. A majority of these elements display active chromatin marks, DNase hypersensitivity and occupancy by multiple transcription factors, confirming the utility of chromatin signatures in cis elements mapping. Notably, 17 of them are previously annotated promoters of functionally unrelated genes, and like typical enhancers, they form extensive spatial contacts with the POU5F1 promoter. Taken together, these results support the utility of CREST-seq for large-scale cis regulatory element discovery and point to commonality of enhancer-like promoters in the human genome.

Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice.

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.

Sperm DNA methylation analysis in swine reveals conserved and species-specific methylation patterns and highlights an altered methylation at the GNAS locus in infertile boars.

Male infertility is an increasing health issue in today's society for both human and livestock populations. In livestock, male infertility slows the improvement of animal selection programs and agricultural productivity. There is increasing evidence that epigenetic marks play an important role in the production of good-quality sperm. We therefore screened for specific or common epigenetic signatures of livestock infertility. To do so, we compared DNA methylation level in sperm DNA from fertile and infertile boars. We evaluated first the global level of sperm DNA methylation and found no difference between the two groups of boars. We then selected 42 loci of interest, most of them known to be imprinted in human or mice, and assessed the imprinting status of five of them not previously described in swine tissues: WT1, CNTN3, IMPACT, QPCT, and GRB10. DNA methylation level was then quantified in fertile and infertile boars at these 42 loci. Results from fertile boars indicated that the methylation level of the selected loci is highly conserved between pig, human, and mice, with a few exceptions, including the POU5F1 (OCT4) promoter and RTL1. Comparison between fertile and infertile boars revealed that one imprinted region, the GNAS locus, shows an increase in sperm DNA methylation in three out of eight infertile boars with low semen quality. This increase in DNA methylation is associated with an altered expression of the genes belonging to the GNAS locus, suggesting a new role for GNAS in the proper formation of functional gametes.

Characteristics of Bovine Inner Cell Mass-Derived Cell Lines and Their Fate in Chimeric Conceptuses1

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

The Pou5f1 distal enhancer is sufficient to drive Pou5f1 promoter-EGFP expression in embryonic stem cells

The POU5F1 transcription factor is the gatekeeper of the pluripotent state in mammals. It is essential for epigenetic reprogramming events and also for germ cell viability. Pou5f1 gene expression is tightly controlled during embryogenesis, but its regulatory regions are not fully deciphered. The GOF18ΔPE-EGFP transgene, harboring the enhanced green fluorescence protein reporter gene inserted into a 17- kilobase long mouse Pou5f1 genomic sequence, has been widely used to visualize pluripotent embryonic cells and primordial germ cells in the mouse and other mammalian species. This construct includes the Pou5f1 promoter under the control of the distal enhancer and also includes the Pou5f1 gene body and flanking sequences. In search of the essential regulatory regions of Pou5f1, we generated four shorter forms of this construct. We found that the shortest form, containing the Pou5f1 promoter and distal enhancer but lacking the gene body and upstream flanking sequences, correctly expressed EGFP in transiently transformed undifferentiated ES cells, correctly switched it off upon ES cell differentiation, and correctly kept it silenced in differentiated Hep3B cells. Similarly to the original GOF18ΔPE-EGFP, this shortest form was expressed in the fetal mouse gonad. Our data suggest that the Pou5f1 distal enhancer and proximal promoter may be sufficient to specify transgene expression in pluripotent cells.

The EP300, KDM5A, KDM6A and KDM6B Chromatin Regulators Cooperate with KLF4 in the Transcriptional Activation of POU5F1

POU5F1 is essential for maintaining pluripotency in embryonic stem cells (ESCs). It has been reported that the constitutive activation of POU5F1 is sustained by the core transcriptional regulatory circuitry in ESCs; however, the means by which POU5F1 is epigenetically regulated remains enigmatic. In this study a fluorescence-based reporter system was used to monitor the interplay of 5 reprogramming-associated TFs and 17 chromatin regulators in the transcription of POU5F1. We show the existence of a stoichiometric effect for SOX2, POU5F1, NANOG, MYC and KLF4, in regulating POU5F1 transcription. Chromatin regulators EP300, KDM5A, KDM6A and KDM6B cooperate with KLF4 in promoting the transcription of POU5F1. Moreover, inhibiting HDAC activities induced the expression of Pou5f1 in mouse neural stem cells (NSCs) in a spatial- and temporal- dependent manner. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) shows that treatment with valproic acid (VPA) increases the recruitment of Kdm5a and Kdm6a to proximal promoter (PP) and proximal enhancer (PE) of Pou5f1 whereas enrichment of Ep300 and Kdm6b was seen in PP but not PE of Pou5f1 promoter. These findings reveal the interplay between the chromatin regulators and histone modifications in the expression of POU5F1.

Histone methylation is a critical regulator of the abnormal expression of POU5F1 and RASSF1A in testis cancer cell lines

DNA and histone methylation are epigenetic modifications functioning in transcriptional control and have been implicated in the deregulation of gene expression in cancer. As a first step to determine if histone methylation could be involved in testis cancer pathogenesis, we performed immunofluorescent localization of histone H3 methylation at lysine 4 (H3-K4; gene activating) and lysine 9 (H3-K9; gene silencing) in healthy testis tissue and in samples of non-seminoma germ-cell tumours. In healthy testis, the distribution of histone H3 methylation was dependent on the developmental stage of spermatogenic cells and in non-seminoma, histone H3-K4 and K9 methylation was detected in all histological subtypes. This suggested that histone H3-K4 and K9 methylation could be associated with abnormal gene expression in non-seminoma. To determine the gene-specific function of histone H3 methylation, we proceeded to define the epigenetic status of key genes implicated in the pathogenesis of non-seminoma, namely the proto-oncogene POU5F1, which is overexpressed in testis cancer, and the tumour suppressor RASSF1A, which is aberrantly silenced. Cell lines representative of non-seminoma were treated with the chromatin-modifying drug, 5-aza-2'-deoxycytidine (5-aza-dC). Chromatin immunoprecipitation and real-time polymerase chain reaction analyses revealed that treatment with 5-aza-dC restored RASSF1A expression through a loss of gene silencing H3-K9 methylation and by retention of gene activating H3-K4 tri-methylation in the promoter region. In contrast, the expression of POU5F1 was reduced by 5-aza-dC and was associated with a loss of gene activating H3-K4 di-methylation in the promoter region. Analysis of DNA methylation revealed a slight reduction in DNA hypermethylation at the RASSF1A promoter, whereas the POU5F1 promoter remained mostly unmethylated and unaffected. Our results indicate that the effects of 5-aza-dC on histone methylation profiles are gene-specific and that aberrant histone modifications may serve as a principal means of misregulation of RASSF1A and POU5F1 expression in testis cancer.

Platypus Pou5f1 reveals the first steps in the evolution of trophectoderm differentiation and pluripotency in mammals

Uterine nourishment of embryos by the placenta is a key feature of mammals. Although a variety of placenta types exist, they are all derived from the trophectoderm (TE) cell layer of the developing embryo. Egg-laying mammals (platypus and echidnas) are distinguished by a very short intrauterine embryo development, in which a simple placenta forms from TE-like cells. The Pou5f1 gene encodes a class V POU family transcription factor Oct3/4. In mice, Oct3/4 together with the highly conserved caudal-related homeobox transcription factor Cdx2, determines TE fate in pre-implantation development. In contrast to Cdx2, Pou5f1 has only been identified in eutherian mammals and marsupials, whereas, in other vertebrates, pou2 is considered to be the Pou5f1 ortholog. Here, we show that platypus and opossum genomes contain a Pou5f1 and pou2 homolog, pou2-related, indicating that these two genes are paralogues and arose by gene duplication in early mammalian evolution. In a complementation assay, we found that platypus or human Pou5f1, but not opossum or zebrafish pou2, restores self-renewal in Pou5f1-null mouse ES cells, showing that platypus possess a fully functional Pou5f1 gene. Interestingly, we discovered that parts of one of the conserved regions (CR4) is missing from the platypus Pou5f1 promoter, suggesting that the autoregulation and reciprocal inhibition between Pou5f1 and Cdx2 evolved after the divergence of monotremes and may be linked to the development of more elaborate placental types in marsupial and eutherian mammals.

Quantitative Monitoring of Pluripotency Gene Activation after Somatic Cloning in Cattle1

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.