POU Domain Transcription Factor Oct-1 Research Articles

Critical POU domain residues confer Oct4 uniqueness in somatic cell reprogramming.

The POU domain transcription factor Oct4 plays critical roles in self-renewal and pluripotency of embryonic stem cells (ESCs). Together with Sox2, Klf4 and c-Myc, Oct4 can reprogram any other cell types to pluripotency, in which Oct4 is the only factor that cannot be functionally replaced by other POU family members. To investigate the determinant elements of Oct4 uniqueness, we performed Ala scan on all Ser, Thr, Tyr, Lys and Arg of murine Oct4 by testing their capability in somatic cell reprogramming. We uncovered a series of residues that are important for Oct4 functionality, in which almost all of these key residues are within the POU domains making direct interaction with DNA. The Oct4 N- and C-terminal transactivation domains (TADs) are not unique and could be replaced by the Yes-associated protein (YAP) TAD domain to support reprogramming. More importantly, we uncovered two important residues that confer Oct4 uniqueness in somatic cell reprogramming. Our systematic structure-function analyses bring novel mechanistic insight into the molecular basis of how critical residues function together to confer Oct4 uniqueness among POU family for somatic cell reprogramming.

Germ cell nuclear factor regulates gametogenesis in developing gonads.

Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.

HIF‐2α/ARNT complex regulates hair development via induction of p21 Waf1/Cip1 and p27 Kip1

The hypoxia-inducible factors HIF-1α or HIF-2α form heterodimeric complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-1α/ARNT and HIF-2α/ARNT complexes activate hypoxia-inducible genes that play critical roles in angiogenesis, anaerobic metabolism, and other processes in response to O2 deprivation. HIF-2α is known to regulate the function and/or differentiation of stem cells by activating the POU domain transcription factor Oct4; however, the precise underlying mechanism is unknown. This study examined the role of HIF-2α/ARNT in hair development using conditional-knockout mice, in which Arnt was specifically deleted in keratinocytes. In wild-type mice, HIF-2α and ARNT were highly expressed in the precortex above the hair matrix, an area containing differentiating stem cells. An analysis of hair size and type in these mice showed that loss of ARNT decreased the production of zigzag hairs, corresponding to reduced expression of HIF-2α and induction of the mammalian cyclin-dependent kinase inhibitors p21(Waf1/Cip1) and p27 (Kip1). The results suggest that the HIF-2α/ARNT complex regulates hair follicle differentiation via induction of p21(Waf1/Cip1) and possibly p27(Kip1), as p27(Kip1) expression was not altered in ARNT knockout mice. The findings provide insight into a possible mechanism underlying hair growth disorders and can be useful for future studies on hair follicle response to insults, such as chemotherapy and ionizing radiation.

A Conserved Oct4/POUV-Dependent Network Links Adhesion and Migration to Progenitor Maintenance

SummaryBackgroundThe class V POU domain transcription factor Oct4 (Pou5f1) is a pivotal regulator of embryonic stem cell (ESC) self-renewal and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. Oct4 is also an important evolutionarily conserved regulator of progenitor cell differentiation during embryonic development.ResultsHere we examine the function of Oct4 homologs in Xenopus embryos and compare this to the role of Oct4 in maintaining mammalian embryo-derived stem cells. Based on a combination of expression profiling of Oct4/POUV-depleted Xenopus embryos and in silico analysis of existing mammalian Oct4 target data sets, we defined a set of evolutionary-conserved Oct4/POUV targets. Most of these targets were regulators of cell adhesion. This is consistent with Oct4/POUV phenotypes observed in the adherens junctions in Xenopus ectoderm, mouse embryonic, and epiblast stem cells. A number of these targets could rescue both Oct4/POUV phenotypes in cellular adhesion and multipotent progenitor cell maintenance, whereas expression of cadherins on their own could only transiently support adhesion and block differentiation in both ESC and Xenopus embryos.ConclusionsCurrently, the list of Oct4 transcriptional targets contains thousands of genes. Using evolutionary conservation, we identified a core set of functionally relevant factors that linked the maintenance of adhesion to Oct4/POUV. We found that the regulation of adhesion by the Oct4/POUV network occurred at both transcriptional and posttranslational levels and was required for pluripotency.

Reactive oxygen species downregulate catalase expression via methylation of a CpG Island in the Oct-1 promoter

Reactive oxygen species (ROS) caused oxidative stress plays a key role in carcinogenesis. The POU domain transcription factor Oct-1 and catalase is closely associated with ROS. However, a correlation between these two key proteins has not been demonstrated before. In this report, we show that Oct-1 acts as an activator of catalase, by binding to the catalase promoter in hepatocellular carcinoma (HCC) cell lines. In addition, we suggest that Oct-1 is downregulated by ROS via CpG island methylation in its promoter. These findings contribute to a better understanding of the epigenetic changes induced by ROS in the process of carcinogenesis.

Direct Conversion of Skin Cells into Blood: Alchemy or Science?

Our neighborhood blood bank just announced an emergency Thanksgiving-season blood drive. For years, the field of stem cell biology has described an elusive but commonly understood goal: the elimination of a need for blood drives. Many years of work have led to methods for generation of bona fide hematopoietic cells from human and mouse embryonic stem cells, but problems with efficiency as well as maturation of the blood cells persist, impeding the utility of these methods in manufacturing blood. Now, in the era of “direct reprogramming,” we can imagine a goal of converting an adult cell, such as a skin cell, into a hematopoietic stem cell that can be used for autologous blood replacement (Figure 1). In an article recently published in Nature, Szabo et al. report a huge step toward that goal.1 The authors describe the generation of differentiated hematopoietic cells following the enforced expression of the POU-domain transcription factor OCT4 in adult or neonatal human skin fibroblasts.

Regulation of Adipose Tissue Stromal Cells Behaviors by Endogenic Oct4 Expression Control

BackgroundTo clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages.Methodology/Principal Findings and ConclusionsHere, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities.

Identification of karyopherin-alpha 2 as an Oct4 associated protein

The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem (ES) cells. To further explore the functional network of Oct4, the yeast two-hybrid system was used to search for Oct4 interacting proteins. PH domain (containing POU domain and homeodomain) of human OCT4 was used as a bait. From the human testis cDNA library, we identified a strong interaction between OCT4 and karyopherin-alpha 2 (KPNA-2). KPNA2 is involved in active nuclear import of proteins. This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins. In addition, we studied nuclear localization signal (NLS) of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization. Thus, our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.

Absence of OCT4 Expression in Somatic Tumor Cell Lines

The POU-domain transcription factor OCT4 is associated with the pluripotent state of cells comprising the inner cell mass of pre-implantation embryos and has been known to play a critical role in the maintenance of pluripotency of embryonic stem cells. Reactivation of OCT4 expression is postulated to occur in differentiated cells that have undergone carcinogenesis, or tumor formation. In contrast to earlier studies, recent reports describe OCT4 expression in several human tumor cell lines. To resolve the apparent discrepancy in OCT4 expression between earlier and recent studies, we determined OCT4 expression in the cervical carcinoma cell line HeLa and the breast cancer cell line MCF7 in comparison with the human teratoma cell line nTera by immunofluorescence, Western blot, and RT-PCR analyses. We were unable to detect staining of the OCT4 transcription factor in the nucleus of HeLa and MCF7 cells by immunofluorescence using two different monoclonal antibodies. Faint cytoplasmic staining in HeLa and MCF7 cells was observed; however, no OCT4 signal could be detected by Western blot analysis. In addition, we were unable to detect significant levels of OCT4 mRNA in HeLa and in MCF7 cells by RT-PCR. Furthermore, the OCT4 promoter region is highly methylated in HeLa and MCF7 cells. We argue that recent reports of OCT4 expression in these and other cancer cell lines could actually be attributed to OCT4 pseudogene expression or misinterpretation of background signals in immunofluorescence experiments. In conclusion, we emphasize the need for adequate controls in investigations of OCT4 expression in somatic cell lines by immunofluorescence and RT-PCR.

PIAS Proteins as Repressors of Oct4 Function

The POU domain transcription factor Oct4 plays essential functions in the maintenance of pluripotent embryonic and germ cells of mammals. Molecular mechanisms of Oct4 action remain poorly understood. To isolate modulators of Oct4 activity, we performed a yeast two-hybrid screen with the Oct4 POU domain as a bait and isolated PIASy as an Oct4-interacting protein. Oct4 and PIASy interact in vivo via their POU domain and SAP-domain-containing N terminus, respectively. PIASy does not enhance Oct4 sumoylation but acts as a potent inhibitor of Oct4-mediated transcriptional activation, sequestering Oct4 protein from the vicinity of Cajal bodies and splicing speckles to the nuclear periphery. These modes of PIASy action are uncoupled from its sumoylation activity. Other PIAS family members, PIAS1 and PIAS3, can also interact with Oct4 in vivo and target Oct4 to the nuclear periphery, depending on cellular context. We propose that Oct4 inhibition, mediated by this new class of transcriptional partners, might be instrumental during mammalian development.

Post‐translational modification of POU domain transcription factor Oct‐4 by SUMO‐1

POU domain transcription factor Oct-4 plays a crucial role in maintaining self-renewal and pluripotency of embryonic stem (ES) cells in a concentration-dependent manner. However, the molecular mechanism controlling Oct-4 levels in ES cells remains largely unknown. To explore the molecular mechanism regulating Oct-4 function, we constructed a mouse ES cell cDNA library and performed yeast two-hybrid screening using the POU domain of Oct-4 as bait. Here, we present novel evidence for Oct-4 interaction with Ubc9, an E2 conjugation enzyme for SUMO modification, and its modification by SUMO-1. The SUMO acceptor site was identified at lysine residue 118. Importantly, disruption of Oct-4 sumoylation reduced Oct-4 protein stability and self-renewal capacity in ES cells. Interestingly, expression of cYes was found to reduce when Oct-4 sumoylation was disrupted or Oct-4 expression downregulated in ES cells. We further demonstrate that Oct-4 was recruited to the cYes promoter region, suggesting that cYes might be a novel downstream gene of Oct-4. Taken together, we first demonstrate the post-translational modification of endogenous Oct-4 by SUMO and the role of sumoylation in regulating Oct-4 protein stability and function. Our findings provide new evidence for the important role of post-translational modification in controlling Oct-4 function in ES cells.

Regulation of NF-κB-dependent Gene Expression by the POU Domain Transcription Factor Oct-1

Maintenance of the cells of the vessel wall in a quiescent state is an important aspect of normal vascular physiology. Transcriptional repressors are widely believed to regulate this process, yet the exact factors involved and the mechanism of repression are not known. Here, we report that the POU domain transcription factor Oct-1 represses the expression of E-selectin and vascular cell adhesion molecule (VCAM-1), two cytokine-inducible, NF-kappaB-dependent endothelial-leukocyte adhesion molecules that participate in the leukocyte recruitment phase of the inflammatory response. Co-transfection and microinjection studies demonstrate that Oct-1 blocks tumor necrosis factor alpha-stimulated E-selectin and VCAM-1 expression. Gene expression arrays indicate that control of tumor necrosis factor alpha-induced, NF-kappaB-dependent gene expression by Oct-1 is promoter-specific. A DNA-binding mutant of Oct-1 represses NF-kappaB-dependent reporter gene expression. Biochemically, Oct-1 interacts with p65, suggesting that Oct-1 is involved in the regulation of NF-kappaB transactivation function. NF-kappaB-dependent gene expression is more pronounced in Oct-1-deficient than in wild-type murine embryonic fibroblasts, and reintroduction of human Oct-1 abolishes these differences. Finally, the cytokine interleukin-6 induces Oct-1 gene expression, providing a biologically relevant means by which NF-kappaB-dependent gene expression can be selectively reverted by Oct-1 to quiescent levels.

Analysis of Oct4-Dependent Transcriptional Networks Regulating Self-Renewal and Pluripotency in Human Embryonic Stem Cells

The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference-mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4-dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4-associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans.

The Octamer Binding Transcription Factor Oct-1 Is a Stress Sensor

The POU-domain transcription factor Oct-1 is widely expressed in adult tissues and has been proposed to regulate a large group of target genes. Microarray expression profiling was used to evaluate gene expression changes in Oct-1-deficient mouse fibroblasts. A number of genes associated with cellular stress exhibited altered expression. Consistent with this finding, Oct-1-deficient fibroblasts were hypersensitive to gamma radiation, doxorubicin, and hydrogen peroxide and harbored elevated reactive oxygen species. Expression profiling identified a second group of genes dysregulated in Oct-1-deficient fibroblasts following irradiation, including many associated with oxidative and metabolic stress. A number of these genes contain octamer sequences in their immediate 5' regulatory regions, some of which are conserved in human. These results indicate that Oct-1 modulates the activity of genes important for the cellular response to stress.

Ectopic Expression of Oct-4 Blocks Progenitor-Cell Differentiation and Causes Dysplasia in Epithelial Tissues

The POU-domain transcription factor Oct-4 is normally expressed in pluripotent cells of the mammalian embryo. In addition, germ-cell tumors and a few somatic tumors show detectable expression of Oct-4. While Oct-4's role during preimplantation development is to maintain embryonic cells in a pluripotent state, little is known about its potential oncogenic properties. Here we investigate the effect of ectopic Oct-4 expression on somatic tissues of adult mice using a doxycycline-dependent expression system. Activation of Oct-4 results in dysplastic growths in epithelial tissues that are dependent on continuous Oct-4 expression. Dysplastic lesions show an expansion of progenitor cells and increased beta-catenin transcriptional activity. In the intestine, Oct-4 expression causes dysplasia by inhibiting cellular differentiation in a manner similar to that in embryonic cells. These data show that certain adult progenitors remain competent to interpret key embryonic signals and support the notion that progenitor cells are a driving force in tumorigenesis.

Mouse Germline Restriction of Oct4 Expression by Germ Cell Nuclear Factor

The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.

DNA recognition by the herpes simplex virus transactivator VP16: a novel DNA-binding structure.

Upon infection, the herpes simplex virus (HSV) transcriptional activator VP16 directs the formation of a multiprotein-DNA complex-the VP16-induced complex-with two cellular proteins, the host cell factor HCF-1 and the POU domain transcription factor Oct-1, on TAATGARAT-containing sequences found in the promoters of HSV immediate-early genes. HSV VP16 contains carboxy-terminal sequences important for transcriptional activation and a central conserved core that is important for VP16-induced complex assembly. On its own, VP16 displays little, if any, sequence-specific DNA-binding activity. We show here that, within the VP16-induced complex, however, the VP16 core has an important role in DNA binding. Mutation of basic residues on the surface of the VP16 core reveals a novel DNA-binding surface with essential residues which are conserved among VP16 orthologs. These results illuminate how, through association with DNA, VP16 is able to interpret cis-regulatory signals in the DNA to direct the assembly of a multiprotein-DNA transcriptional regulatory complex.

Oct-1 promoter region contains octamer sites and TAAT motifs recognized by Oct proteins

The 5′-upstream region (1.3 kb) of the gene encoding the POU domain transcription factor Oct-1 was cloned and sequenced. CAT reporter gene analysis of this region has detected a functionally active promoter. This region contains 24 TAAT-core sites, arranged in five clusters (four to six sites in one cluster); two octamer sites (ATGCAAAT) are located in the first and second clusters; in the second one the CCAAT-box adjacent to the octamer overlaps with the TAAT-core site. As shown by gel retardation assay, Oct-1, Oct-2, and some unknown proteins from myeloma cell line NS/0 interact with the TAAT-core sites of these clusters. The results suggest autoregulation of Oct-1 gene expression that may also be controlled by other POU proteins, homeodomain proteins and CCAAT trans-action factors.

OCA-B is a functional analog of VP16 but targets a separate surface of the Oct-1 POU domain.

OCA-B is a B-cell-specific coregulator of the broadly expressed POU domain transcription factor Oct-1. OCA-B associates with the Oct-1 POU domain, a bipartite DNA-binding structure containing a POU-specific (POU[S]) domain joined by a flexible linker to a POU homeodomain (POU[H]). Here, we show that OCA-B alters the activity of Oct-1 in two ways. It provides a transcriptional activation domain which, unlike Oct-1, activates an mRNA-type promoter effectively, and it stabilizes Oct-1 on the Oct-1-responsive octamer sequence ATGCAAAT. These properties of OCA-B parallel those displayed by the herpes simplex virus Oct-1 coregulator VP16. OCA-B, however, interacts with a different surface of the DNA-bound Oct-1 POU domain, interacting with both the POU(S) and POU(H) domains and the center of the ATGCAAAT octamer sequence. The OCA-B and VP16 interactions with the Oct-1 POU domain are sufficiently different to permit OCA-B and VP16 to bind the Oct-1 POU domain simultaneously. These results emphasize the structural versatility of the Oct-1 POU domain in its interaction with coregulators.

Characterization of a Trimeric Complex Containing Oct-1, SNAPc, and DNA

The human small nuclear (sn) RNA promoters contain a proximal sequence element (PSE), which recruits the basal transcription factor SNAPc, and a distal sequence element characterized by an octamer sequence, which recruits the POU domain transcription factor Oct-1. The Oct-1 POU domain and SNAPc bind cooperatively to probes containing a PSE and an octamer sequence, and this effect contributes to efficient transcription in vitro. In vivo, however, Oct-1 regions outside of the POU domain can activate snRNA gene transcription. Here, we have examined whether the role of these regions is to contribute to cooperative binding with SNAPc. We find that they indeed improve cooperative binding, but most of the effect is nevertheless mediated by just the POU domain. This suggests that Oct-1 activates transcription of snRNA genes in at least two steps, recruitment of SNAPc mediated primarily by the POU domain, and a later step mediated by regions outside of the POU domain. We also show that a PSE-binding complex observed in nuclear extracts consists of Oct-1 and SNAPc. Although Oct-1 cannot bind effectively to the PSE probe on its own, in the complex it contacts DNA. Thus, in a nuclear extract, SNAPc can recruit Oct-1 to a probe to which Oct-1 cannot bind on its own.