13 research outputs found
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
<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