Oct-1 Binding Site Research Articles

Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9.

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.

Circulating Tumor Cell Clustering Shapes DNA Methylation to Enable Metastasis Seeding

SummaryThe ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, we profile the DNA methylation landscape of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer.

Multiple Interactions of the Oct-1 (POU2F1) Transcription Factor with PORE and MORE Sites

The Oct-1 (POU2F1) transcription factor is one of the most important regulatory proteins in humans and other mammals. An increase in Oct-1 aids the resistance to oxidative and cytotoxic stresses and radiation exposure. A high level of Oct-1 is found in many human tumors and correlates with low survival. Oct-1 interacts with its binding sites as a monomer, a homodimer, or a multimer. The nucleotide sequence of the Oct-1 binding site determines the character of interaction and the conformation of Oct-1 on target DNA, thus influencing the binding of Oct-1 co-repressors and co-activators. Nucleotide substitutions were introduced in all positions of the PORE and MORE sequences and tested for effect on the Oct-1 capability of forming monomeric and dimeric DNA-protein complexes. The position and nature of nucleotide substitutions were found to affect the type of Oct-1 binding to DNA. Several substitutions suppressed the formation of dimers, while others stimulated the process. Certain nucleotide substitutions completely prevented the binding of both monomers and dimers. The Oct-1 concentration in the cell is another factor that affects the character of DNA-protein interactions. Based on the results, the nature and affinity of interaction with Oct-1 is possible to predict from the nucleotide sequence for PORE and MORE sites of the human genome.

Three newly identified Immediate Early Genes of Bovine herpesvirus 1 lack the characteristic Octamer binding motif- 1

Only three immediate early genes (IE) BICP0, BICP4 and BICP22 of Bovine herpesvirus 1 (BoHV-1) are known. These genes are expressed coordinately and their promoters are well characterized. We provide evidence for expression of three additional IE genes of BoHV-1 i.e. UL21, UL33 and UL34. These genes are expressed in the presence of cycloheximide (CH) at the same time as known IE genes. Surprisingly, the promoters of newly identified IE genes (UL21, UL33, UL34) lack the OCT-1 binding site, a considered site of transactivation of the BoHV-1 IE genes. The other difference in the promoters of the newly identified IE genes is the presence of TATA box at near optimal site. However, all the IE genes have similar spatial placements of C/EBPα, DPE and INR elements.

Heterodimer formation by Oct4 and Smad3 differentially regulates epithelial-to-mesenchymal transition-associated factors in breast cancer progression

The multifunctional cytokine TGF-β crucially participates in breast cancer (BCa) metastasis and works differently in the disease stages, thus contributing in BCa progression. We address connections between TGF-β and the stem cell-related transcription factor (TF) Oct4 in BCa. In 147 BCa patients with infiltrating duct carcinoma, we identified a significantly higher number of cases with both moderate/high Oct4 expression and high TGF-β in late stages compared to early stages of the disease. In vitro studies showed that TGF-β elevated Oct4 expression, which in turn, regulated Epithelial-to-Mesenchymal transition (EMT)-regulatory gene (Snail and Slug) expression, migratory ability, chemotactic invasiveness and extracellular matrix (ECM) degradation potential of BCa cells. Putative binding sites for Oct4 on the snail, slug and cxcl13 promoters and for Smad3 on the snail and slug promoters were identified. Promoter activities of snail and slug were greater in dual-treated cells than only TGF-β-treated or Oct4-overexpressing cells. CXCL13 mRNA fold changes, however, were low in cells induced with TGF-β, compared to dual-treated or Oct4-overexpressing cells. Our co-IP studies confirmed that Oct4 and Smad3 form heterodimers that recognize specific promoter sequences to promote Snail and Slug expression, but which in turn, indirectly inhibits Smad3-mediated repression of CXCL13 expression, allowing Oct4 to act as a positive TF for CXCL13. Taken together, these data suggest that TGF-β signaling and Oct4 cooperate to induce expression of EMT-related genes Snail, Slug and CXCL13, which accelerates disease progression, particularly in the late stages, and may indicate a poor prognosis for BCa patients.

The expression of the embryonic gene Cripto-1 is regulated by OCT4 in human embryonal carcinoma NCCIT cells.

Cripto-1 and OCT4, expressed in stem cells and cancers, play important roles in tumorigenesis. Here, we demonstrate that Cripto-1 expression is regulated by OCT4 in human embryonic carcinoma NCCIT cells. The endogenous expression of Cripto-1 and OCT4 is significantly reduced during differentiation. Cripto-1 expression is increased by OCT4 overexpression, but decreased by shRNA-mediated OCT4 knockdown. OCT4 overexpression significantly activates Cripto-1 transcriptional activity. A 5'-upstream minimal promoter sequence in the gene-encoding Cripto-1 is significantly activated by OCT4 overexpression. Mutation of the putative OCT4-binding site abolishes OCT4-mediated activation of the Cripto-1 promoter. The OCT4 transactivation domains mediate transcriptional activity of the Cripto-1 minimal promoter through direct interaction. Taken together, OCT4 plays an important role as a transcriptional activator of Cripto-1 expression in NCCIT cells.

Oct1 and Oca-B Regulate the Myeloid Leukemic Phenotype

Oct1, encoded by the Pou2f1 gene, is a widely expressed transcription factor that is associated with metabolic control, stress response, stem cell phenotypes and malignant transformation. Oct1 associates with a number of cofactors including the activating cofactor OCA-B encoded by Pou2af1. OCA-B is normally expressed specifically in B cells, but is misexpressed in about 30% of AML cases in myeloid cells and is a poor prognostic indicator. We observed a conserved Oct1 binding site in the promoter of Cdx2, a homeodomain transcription factor that is normally expressed solely in the gastrointestinal tract of adult humans, but has been shown to misexpressed in blood cells in almost all cases of AML. Our study looks at the interaction of Oct1 with Cdx2, and the effects of Oct1 and OCAB-B knockout in a mouse model of AML.Using ChIP we determined that Oct1 is bound to the Cdx2 promoter in normal patient samples and AML patient samples. However, in normal patient samples a repressive cofactor (the NuRD complex) is also bound. In AML patient samples an activating cofactor (Jmjd1a) is bound. This suggests that in AML Oct1 is bound to Cdx2 and is associating with Jmjd1a, which can occur through ERK signaling or OCA-B expression, and activating expression of Cdx2.We next wanted to determine the effect of Oct1 knockout in AML using a MLL-AF9 mouse model. Our lab has developed a conditional Oct1 allele, as Oct1 germline knockout is embryonic lethal. We harvested bone marrow from Oct1+/fl;MX1-Cre, Oct1fl/D;MX1-Cre, and Oct1+/+;MX1-Cre mice and transduced the bone marrow with virus expressing MLL-AF9. Recipient mice were lethally irradiated and transplanted with the transduced bone marrow. Mice were injected with Poly IC at 5 weeks post-transplant to delete Oct1 foxed alleles. Mice that were wildtype for Oct1 developed leukemia around 7 weeks. Mice with one allele of Oct1 survived longer but still developed leukemia. Mice with no Oct1 survived longer still, but eventually suffered from bone marrow failure.In order to assess whether the lethality in Oct1 deficient mice with MLL-AF9-induced leukemia was due to the presence of the MLL-AF9 oncoprotein or if Oct1 deletion is lethal in adult mice under normal conditions, we treated non-leukemic mice that were Oct1+/fl;MX1-Cre or Oct1fl/D;MX1-Cre with Poly IC to delete Oct1 at 5 weeks. Following Poly IC injection, mice with and without Oct1 appeared normal and healthy by examination and by blood counts. At 20 weeks, all mice were injected with 5-flurouracil (5-FU). We observed that mice that were deficient in Oct1, namely Oct1fl/D;MX1-Cre mice injected with Poly IC, died within 2 weeks of 5-FU treatment. Put together, our data suggests that Oct1 is not necessary for normal hematopoiesis, as adult mice with no Oct1 appear normal under homeostatic conditions, but Oct1 is necessary for the hematopoietic proliferative stress response.The protective effect of Oct1 deletion against leukemia in mice suggested an important role for Oct1 in regulating the leukemic phenotype. However, since Oct1 deletion is also lethal in the leukemic context, we looked at the effect of deletion of OCA-B in leukemia. OCA-B germline deficient mice are viable and the only detectable phenotype is the lack of germinal center formation. We harvested OCA-B germline deficient and wildtype control bone marrow and followed the same protocol as outlined above with the transduction of the bone marrow with MLL-AF9. Mice with no OCA-B did not develop leukemia and had 100% survival. This striking result suggests that OCA-B could be a therapeutic target in MLL-AF9 AML. DisclosuresNo relevant conflicts of interest to declare.

HLA-C Level Is Regulated by a Polymorphic Oct1 Binding Site in the HLA-C Promoter Region

Differential HLA-C levels influence several human diseases, but the mechanisms responsible are incompletely characterized. Using a validated prediction algorithm, we imputed HLA-C cell surface levels in 228 individuals from the 1000 Genomes dataset. We tested 68,726 SNPs within the MHC for association with HLA-C level. The HLA-C promoter region variant, rs2395471, 800bp upstream of the transcription start site, gave the most significant association with HLA-C levels (p = 4.2× 10-66). This imputed expression quantitative trait locus, termed impeQTL, was also shown to associate with HLA-C expression in a genome-wide association study of 273 donors in which HLA-C mRNA expression levels were determined by quantitative PCR (qPCR) (p = 1.8× 10-20) and in two cohorts where HLA-C cell surface levels were determined directly by flow cytometry (n = 369 combined, p < 10-15). rs2395471 is located in an Oct1 transcription factor consensus binding site motif where the A allele is predicted to have higher affinity for Oct1 than the G allele. Mobility shift electrophoresis demonstrated that Oct1 binds to both alleles invitro, but decreased HLA-C promoter activity was observed in a luciferase reporter assay for rs2395471_G relative to rs2395471_A on a fixed promoter background. The rs2395471 variant accounts for up to 36% of the explained variation of HLA-C level. These data strengthen our understanding of HLA-C transcriptional regulation and provide a basis for understanding the potential consequences of manipulating HLA-C levels therapeutically.

Ubiquitously expressed genes participate in cell-specific functions via alternative promoter usage.

How do different cell types acquire their specific identities and functions is a fundamental question of biology. Previously significant efforts have been devoted to search for cell-type-specifically expressed genes, especially transcription factors, yet how do ubiquitously expressed genes participate in the formation or maintenance of cell-type-specific features remains largely unknown. Here, we have identified 110 mouse embryonic stem cell (mESC) specifically expressed transcripts with cell-stage-specific alternative transcription start sites (SATS isoforms) from 104 ubiquitously expressed genes, majority of which have active epigenetic modification- or stem cell-related functions. These SATS isoforms are specifically expressed in mESCs, and tend to be transcriptionally regulated by key pluripotency factors through direct promoter binding. Knocking down the SATS isoforms of Nmnat2 or Usp7 leads to differentiation-related phenotype in mESCs. These results demonstrate that cell-type-specific transcription factors are capable to produce cell-type-specific transcripts with alternative transcription start sites from ubiquitously expressed genes, which confer ubiquitously expressed genes novel functions involved in the establishment or maintenance of cell-type-specific features.

IL18 Gene Variants Influence the Susceptibility to Chagas Disease.

Chagas disease is a parasitic disorder caused by the infection with the flagellated protozoan Trypanosoma cruzi. According to the World Health Organization, more than six million people are currently infected in endemic regions. Genetic factors have been proposed to influence predisposition to infection and development of severe clinical phenotypes like chronic Chagas cardiomyopathy (CCC). Interleukin 18 (IL18) encodes a proinflammatory cytokine that has been proposed to be involved in controlling T. cruzi infection. In this study, we analyzed the possible role of six IL18 gene variants (rs5744258, rs360722, rs2043055, rs187238, rs1946518 and rs360719), which cover most of the variation within the locus, in the susceptibility to infection by T. cruzi and/or CCC. In total, 1,171 individuals from a Colombian region endemic for Chagas disease, classified as seronegative (n = 595), seropositive asymptomatic (n = 175) and CCC (n = 401), were genotyped using TaqMan probes. Significant associations with T. cruzi infection were observed when comparing seronegative and seropositive individuals for rs187238 (P = 2.18E-03, OR = 0.77), rs360719 (P = 1.49E-03, OR = 0.76), rs2043055 (P = 2.52E-03, OR = 1.29), and rs1946518 (P = 0.0162, OR = 1.22). However, dependence analyses suggested that the association was mainly driven by the polymorphism rs360719. This variant is located within the promoter region of the IL18 gene, and it has been described that it creates a binding site for the transcription factor OCT-1 affecting IL-18 expression levels. In addition, no evidence of association was observed between any of the analyzed IL18 gene polymorphisms and the development of CCC. In summary, our data suggest that genetic variation within the promoter region of IL18 is directly involved in the susceptibility to infection by T. cruzi, which provides novel insight into disease pathophysiology and adds new perspectives to achieve a more effective disease control.

Computer analysis of co-localization of transcription factor binding sites in genome by ChIP-seq data

Statistical features of the distribution of transcription factor binding sites in the mouse genome that are obtained by ChIP-seq experiments in embryonic stem cells have been considered. Clusters of sites that contain four or more different transcription factor binding sites in the mouse genome have been defined, also their location relatively to the regulatory regions of genes has been described. The presence of two types of site co-localization has been shown: clusters containing binding sites for factors Oct4, Nanog, Sox2, located in the distal regions, and clusters containing binding sites n-Myc, c-Myc, mainly located in the promoter regions of mouse genes. Analysis of new ChIPseq data about binding of transcription factors Nr5a2, Tbx3 in the same cell type has confirmed the division of clusters of transcription factors binding sites into two types: those containing the binding sites of regulators of pluripotency (Oct4, Nanog, and others) and those not. The computer program of the statistical data processing of gene location and chromatin domains that analyzes experimental data of site localization obtained by ChIP-seq in the mouse genome and the human genome has been developed. The presence of preferences at position of transcription factor binding sites of various types has been revealed, the distances between the nearest groups of TF binding sites Oct4, Nanog, Sox2 and TF binding sites n-Myc and c-Myc have been calculated using this program. The presence of nucleotide motifs of transcription factor binding sites in the selected areas of ChIP-seq has been estimated, nucleotide motifs have been refined. A correlation between the presence of motifs and the intensity of ChIPseq binding has been shown. Computer methods for estimating the clustering of different transcription factors binding sites for new data ChIP-seq have been developed. Programs are available upon the request to the authors.

In Silico Methods to Identify Exapted Transposable Element Families.

Transposable elements (TEs) have recently been shown to have many regulatory roles within the genome. In this chapter, we will examine two in silico methods for analyzing TEs and identifying families that may have acquired such functions. The first method will look at how the overrepresentation of a repeat family in a set of genomic features can be discovered. The example situation of OCT4 binding sites originating from LTR7 TE sequences will be used to show how this method could be applied. The second method will describe how to determine if a TE family exhibits a cell type-specific expression pattern. As an example, we will look at the expression of HERV-H, an endogenous retrovirus known to act as an lncRNA in embryonic stem cells. We will use this example to demonstrate how RNA-seq data can be used to compare cell type expression of repeats.

Glucose-Based Regulation of miR-451/AMPK Signaling Depends on the OCT1 Transcription Factor

In aggressive, rapidly growing solid tumors such as glioblastoma multiforme (GBM), cancer cells face frequent dynamic changes in their microenvironment, including the availability of glucose and other nutrients. These challenges require that tumor cells have the ability to adapt in order to survive periods of nutrient/energy starvation. We have identified a reciprocal negative feedback loop mechanism in which the levels of microRNA-451 (miR-451) are negatively regulated through the phosphorylation and inactivation of its direct transcriptional activator OCT1 by 5' AMP-activated protein kinase (AMPK), which is activated by glucose depletion-induced metabolic stress. Conversely, in a glucose-rich environment, unrestrained expression of miR-451 suppresses AMPK pathway activity. These findings uncover miR-451 as a major effector of glucose-regulated AMPK signaling, allowing tumor cell adaptation to variations in nutrient availability in the tumor microenvironment.

Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3.

As a prompt response against invasion of various viruses, interferon regulatory factor-3 (IRF-3) is initially phosphorylated to become activated and upregulates mainly Type I Interferons (IFN-I) in most cell types. We previously reported that IRF-3-dependent host innate immune responses partially interfere in infection of prions. Here, we found that stable infection of prion suppressed IRF-3 gene-expression. The decreased promoter activity of IRF-3 was significantly restored along with treatment of anti-prion drugs in the prion-infected cells, suggesting that infection of prion directly influence the regulation of IRF-3 transcription. We further investigated promoter activity of 5′- flanking region of murine IRF-3 using a luciferase reporter system and found that the nucleotides -119 to -1 were indispensable for the promoter activity. Within this region, mutations in the Oct-1 binding site significantly reduced the promoter activity and chromatin immunoprecipitation (ChIP) assay revealed that Oct-1 indeed binds to the region. In addition, overexpression of Oct-1 increased the promoter activity of IRF-3. Intriguingly, Oct-1 protein was significantly reduced in prion-infected cells and mice brains compared with uninfected groups. Taken together, we concluded that prion infection could interfere in the function of Oct-1, resulting in the down-regulation of IRF-3.

Oct-1 Regulates IL-17 Expression by Directing Interchromosomal Associations in Conjunction with CTCF in T Cells

Interchromosomal associations can regulate gene expression, but little is known about the molecular basis of such associations. In response to antigen stimulation, naive Tcells can differentiate into Th1, Th2, and Th17 cells expressing IFN-γ, IL-4, and IL-17, respectively. We previously reported that in naive Tcells, the IFN-γ locus is associated with the Th2 cytokine locus. Here we show that the Th2 locus additionally associates with the IL-17 locus. This association requires a DNase I hypersensitive region (RHS6) at the Th2 locus. RHS6 and the IL-17 promoter both bear Oct-1 binding sites. Deletion of either of these sites or Oct-1 gene impairs the association. Oct-1 and CTCF bind their cognate sitescooperatively, and CTCF deficiency similarly impairs the association. Finally, defects in the association lead to enhanced IL-17 induction. Collectively, our data indicate Th17 lineage differentiation is restrained by the Th2 locus via interchromosomal associations organized by Oct-1 and CTCF.

Oct4/Sox2 Binding Sites Contribute to Maintaining Hypomethylation of the Maternal Igf2/H19 Imprinting Control Region

A central question in genomic imprinting is how parental-specific DNA methylation of imprinting control regions (ICR) is established during gametogenesis and maintained after fertilization. At the imprinted Igf2/H19 locus, CTCF binding maintains the unmethylated state of the maternal ICR after the blastocyst stage. In addition, evidence from Beckwith-Wiedemann patients and cultured mouse cells suggests that two Sox-Oct binding motifs within the Igf2/H19 ICR also participate in maintaining hypomethylation of the maternal allele. We found that the Sox and octamer elements from both Sox-Oct motifs were required to drive hypomethylation of integrated transgenes in mouse embryonic carcinoma cells. Oct4 and Sox2 showed cooperative binding to the Sox-Oct motifs, and both were present at the endogenous ICR. Using a mouse with mutations in the Oct4 binding sites, we found that maternally transmitted mutant ICRs acquired partial methylation in somatic tissues, but there was little effect on imprinted expression of H19 and Igf2. A subset of mature oocytes also showed partial methylation of the mutant ICR, which suggested that the Sox-Oct motifs provide some protection from methylation during oogenesis. The Sox-Oct motifs, however, were not required for erasure of paternal methylation in primordial germ cells, which indicated that the oocyte methylation was acquired post-natally. Maternally inherited mutant ICRs were unmethylated in blastocysts, which suggested that at least a portion of the methylation in somatic tissues occurred after implantation. These findings provide evidence that Sox-Oct motifs contribute to ICR hypomethylation in post-implantation embryos and maturing oocytes and link imprinted DNA methylation with key stem cell/germline transcription factors.

A novel de novo point mutation of the OCT‐binding site in the IGF2/H19‐imprinting control region in a Beckwith–Wiedemann syndrome patient

The IGF2/H19-imprinting control region (ICR1) functions as an insulator to methylation-sensitive binding of CTCF protein, and regulates imprinted expression of IGF2 and H19 in a parental origin-specific manner. ICR1 methylation defects cause abnormal expression of imprinted genes, leading to Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS). Not only ICR1 microdeletions involving the CTCF-binding site, but also point mutations and a small deletion of the OCT-binding site have been shown to trigger methylation defects in BWS. Here, mutational analysis of ICR1 in 11 BWS and 12 SRS patients with ICR1 methylation defects revealed a novel de novo point mutation of the OCT-binding site on the maternal allele in one BWS patient. In BWS, all reported mutations and the small deletion of the OCT-binding site, including our case, have occurred within repeat A2. These findings indicate that the OCT-binding site is important for maintaining an unmethylated status of maternal ICR1 in early embryogenesis.

The Regulation of Downstream Target Gene AP-2γ by Transcription Factor OCT-4

In recent years,OCT-4 and AP-2γ have been widely used as clinical markers of testicular germ cell tumors.Transcription factor OCT-4,which plays an important role in embryonic development,gives full play to a variety of biological functions in different developmental periods and differentiations.The effect of OCT-4 is realized through the regulation of the target genes.In this study,we found OCT-4 binding sites within the sequence of AP-2γ promoter region by a variety of bionemerics.AP-2γ,a novel target gene of OCT-4,was identified by chromatin immunoprecipitation(ChIP)-PCR.A combination of sequence analysis,reporter gene assays,Western blot,immunofluorescence assay and mouse cryptorchidism model experiment further confirmed that AP-2γ was the target gene of OCT-4.OCT-4 inhibited the transcriptive activity of AP-2γ.The expression of AP-2γ gene was confirmed to be significantly altered by silencing or overexpression of OCT-4.This new discovery is conductive to study the malignant process of germ cell tumors at the molecular level.

HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency

BackgroundCertain post-translational modifications to histones, including H3K4me3, as well as binding sites for the transcription factor STAT1, predict the site of integration of exogenous gamma-retroviruses with great accuracy and cell-type specificity. Statistical methods that were used to identify chromatin features that predict exogenous gamma-retrovirus integration site selection were exploited here to determine whether cell type-specific chromatin markers are enriched in the vicinity of endogenous retroviruses (ERVs).ResultsAmong retro-elements in the human genome, the gamma-retrovirus HERV-H was highly associated with H3K4me3, though this association was only observed in embryonic stem (ES) cells (p < 10-300) and, to a lesser extent, in induced pluripotent stem (iPS) cells. No significant association was observed in nearly 40 differentiated cell types, nor was any association observed with other retro-elements. Similar strong association was observed between HERV-H and the binding sites within ES cells for the pluripotency transcription factors NANOG, OCT4, and SOX2. NANOG binding sites were located within the HERV-H 5′LTR itself. OCT4 and SOX2 binding sites were within 1 kB and 2 kB of the 5′LTR, respectively. In keeping with these observations, HERV-H RNA constituted 2% of all poly A RNA in ES cells. As ES cells progressed down a differentiation pathway, the levels of HERV-H RNA decreased progressively. RNA-Seq datasets showed HERV-H transcripts to be over 5 kB in length and to have the structure 5′LTR-gag-pro-3′LTR, with no evidence of splicing and no intact open reading frames.ConclusionThe developmental regulation of HERV-H expression, the association of HERV-H with binding sites for pluripotency transcription factors, and the extremely high levels of HERV-H RNA in human ES cells suggest that HERV-H contributes to pluripotency in human cells. Proximity of HERV-H to binding sites for pluripotency transcription factors within ES cells might be due to retention of the same chromatin features that determined the site of integration of the ancestral, exogenous, gamma-retrovirus that gave rise to HERV-H in the distant past. Retention of these markers, or, alternatively, recruitment of them to the site of the established provirus, may have acted post-integration to fix the provirus within the germ-line of the host species. Either way, HERV-H RNA provides a specific marker for pluripotency in human cells.

Effects of SNPs in &lt;I&gt;POU&lt;/I&gt;1&lt;I&gt;F&lt;/I&gt;1 promoter on growth traits in largemouth bass(&lt;I&gt;Micropterus salmoides&lt;/I&gt;)

In the present study,the promoter sequence of largemouth bass POU1F1 was cloned which is 1 629 bp in length,predicted the existence of four octamer transcription factor 1(Oct-1)binding sites,one homeobox transcription factors sites,two cAMP-responsive element binding proteins sites and a TATA box.Using directly sequencing,two nucleotides mutations were identified which were located at the-18 and-183 sites,and only two haplotypes were identified(haplotype A and haplotype B).The different genotype individuals and the mutant alleles in stochastic population(126 individuals)were examined.The frequency of A haplotype and B haplotype was 0.246 and 0.754 respectively.The population correlation analysis showed that the AA and AB genotypes have significantly correlated with BB genotype individuals' weight,body length,full length,body depth and body width(P0.05),but there is no significant correlation between AA genotype individuals and AB genotype individuals.Identified growth traits markers for growth traits could be implemented through marker-assisted selection(MAS),with the aim of producing rapidly growing largemouth bass.