Transcriptional regulation of human genes by endogenous retroviral elements

Human endogenous retroviruses (HERVs) and other long terminal repeat (LTR)- containing elements comprise a significant portion (8%) of the human genome and are likely vestiges of retroviral infections during primate evolution. Although the vast majority of HERVs are now unable to code for retroviral proteins, an unknown number have retained functional transcriptional elements within their LTRs and some of these regulatory sequences have been shown to participate in the transcription of nearby genes. The overall objective of my thesis was to further understand the role of HERVs in human gene regulation by investigating LTRs that provide alternative promoters to cellular genes. When I began my study, three putative endogenous retroviral promoters were identified by screening sequence databases for chimeric (viral-cellular) transcripts. These searches revealed fusion transcripts containing the LTRs of three HERV-E elements linked to the endothelin B receptor (EDNRB), the apolipoprotein C-l (APOC1) and the Opitz syndrome gene, midline 1. To confirm the authenticity of the chimeric transcript and to establish that the mRNAs were transcribed from the retroviral LTRs, we performed 5'RACE and determined the genomic organization for each gene. Our results indicated that the chimeric transcripts were alternatively promoted by the retroviral elements, as they initiated within HERV-E LTRs but spliced into the downstream coding sequence of the cellular genes. To determine the expression pattern and the relative contribution of the retroviral promoter, we quantified the percentage of transcripts which were chimeric in various tissues using real-time PCR. While chimeric APOC1 transcripts could be detected in several tissues tested, the retroviral promoter of EDNRB and MIDI appeared to be placenta-specific. Transient transfection studies supported a role for the EDNRB and MIDI LTRs as strong promoters in placenta and suggested a function for the LTRs as enhancers. Further deletion and hybrid constructs delineated regions within both LTRs necessary for strong promoter activity. Finally, to further characterize the APOC1, EDNRB and MIDI genes, the non-retro viral (native) promoters of these three genes were also analysed. These findings provide further evidence that some endogenous retroviruses have evolved a biological function as transcriptional regulatory elements by contributing alternative promoters to human genes.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Gfi1 Expression Is Controlled by Five Distinct Regulatory Regions Spread over 100 Kilobases, with Scl/Tal1, Gata2, PU.1, Erg, Meis1, and Runx1 Acting as Upstream Regulators in Early Hematopoietic Cells▿

The growth factor independence 1 (Gfi1) gene was originally discovered in the hematopoietic system, where it functions as a key regulator of stem cell homeostasis, as well as neutrophil and T-cell development. Outside the blood system, Gfi1 is essential for inner-ear hair and intestinal secretory cell differentiation. To understand the regulatory hierarchies within which Gfi1 operates to control these diverse biological functions, we used a combination of comparative genomics, locus-wide chromatin immunoprecipitation assays, functional validation in cell lines, and extensive transgenic mouse assays to identify and characterize the complete ensemble of Gfi1 regulatory elements. This concerted effort identified five distinct regulatory elements spread over 100kb each driving expression in transgenic mice to a subdomain of endogenous Gfi1. Detailed characterization of an enhancer 35 kb upstream of Gfi1 demonstrated activity in the dorsal aorta region and fetal liver in transgenic mice, which was bound by key stem cell transcription factors Scl/Tal1, PU.1/Sfpi1, Runx1, Erg, Meis1, and Gata2. Taken together, our results reveal the regulatory regions responsible for Gfi1 expression and importantly establish that Gfi1 expression at the sites of hematopoietic stem cell (HSC) emergence is controlled by key HSC regulators, thus integrating Gfi1 into the wider HSC regulatory networks

<em>Itga2b</em> Regulation at the Onset of Definitive Hematopoiesis and Commitment to Differentiation

<div><p>Product of the <em>Itga2b</em> gene, CD41 contributes to hematopoietic stem cell (HSC) and megakaryocyte/platelet functions. CD41 expression marks the onset of definitive hematopoiesis in the embryo where it participates in regulating the numbers of multipotential progenitors. Key to platelet aggregation, CD41 expression also characterises their precursor, the megakaryocyte, and is specifically up regulated during megakaryopoiesis. Though phenotypically unique, megakaryocytes and HSC share numerous features, including key transcription factors, which could indicate common sub-regulatory networks. In these respects, <em>Itga2b</em> can serve as a paradigm to study features of both developmental-stage and HSC- versus megakaryocyte-specific regulations. By comparing different cellular contexts, we highlight a mechanism by which internal promoters participate in <em>Itga2b</em> regulation. A developmental process connects epigenetic regulation and promoter switching leading to CD41 expression in HSC. Interestingly, a similar process can be observed at the <em>Mpl</em> locus, which codes for another receptor that defines both HSC and megakaryocyte identities. Our study shows that <em>Itga2b</em> expression is controlled by lineage-specific networks and associates with H4K8ac in megakaryocyte or H3K27me3 in the multipotential hematopoietic cell line HPC7. Correlating with the decrease in H3K27me3 at the <em>Itga2b</em> Iocus, we find that following commitment to megakaryocyte differentiation, the H3K27 demethylase Jmjd3 up-regulation influences both <em>Itga2b</em> and <em>Mpl</em> expression.</p> </div

Encode project: RNA-seq and ChIp-seq data from CD41- cells.

<p>(A) Transcription levels assayed by RNA-seq and histone modification (H3K4me3 and H3K9ac) ChIP-seq on <i>ITGA2B, MPL,</i> and <i>GPIBA</i> in ES (H1-hESC), EC (HUVEC) and B lymphocytic (GM12878) cells. Plots are aligned to the transcript representations. CpG Islands are indicated by green boxes.</p

Transcriptional activity at the <i>Itga2b</i> locus in CD41- cells.

<p>(A) Cis-element cluster analysis at the <i>Itga2b</i> locus. Vertical lines indicate probabilities that regulatory factors bind to cis-elements at these positions. The overlain curve indicates the overall probability of being within a cluster of cis-elements bound by their factors. (B) RNA polymerase II (PolII) recruitment on the <i>Itga2b</i> locus in endothelial cells (MS1). Antibody against PolII was used to perform ChIP on chip. The fold enrichments are plotted on a logarithmic scale against the position across the <i>Itga2b</i> locus and aligned to the cluster predictive plot. (C) Schematic representation of the alternative <i>Itga2b</i> TSS deduced from 5¢ RACE PCR in MS1 cells. The lower panel indicates the full-length <i>Itga2b</i> exons and the exons cloned by RACE PCR (full sequences available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043300#pone.0043300.s003" target="_blank">Figure S3</a>). (D) Cis-element cluster analysis at the <i>Mpl</i> locus. (E) Relative levels of transcription assayed by Q-PCR for <i>Itga2b</i> exons 8–9–10, 11–12 and 29–30 and for <i>Mpl</i> exons 6–7 and 11–12 in MS1 and ES, and HPC7 cells. PCR results were normalized to HPRT and compared to the level measured in HPC7 cells<b>.</b> Error bars reflect standard error or the mean (SEM).</p