Epigenetic Regulation of the Dlk1-Meg3 Imprinted Locus in Human Islets

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease characterized by inadequate insulin secretion by the pancreatic β-cell in response to increased blood glucose levels. Despite compelling evidence that T2DM has a high rate of familial aggregation, known genetic risk variants account for less than 10% of the observed heritability. Consequently, post-transcriptional regulators of gene expression, including microRNAs and other noncoding RNAs, have been implicated in the etiology of T2DM, in part due to their ability to simultaneously regulate the expression of hundreds of targets. To determine if microRNAs are involved in the pathogenesis of human T2DM, I sequenced the small RNAs of human islets from diabetic and non-diabetic organ donors. From this screen, I identified the maternally-expressed genes in the imprinted DLK1-MEG3 locus as highly- and specifically-expressed in human β-cells, but repressed in T2DM islets. Repression of this noncoding transcript was strongly correlated with hyper-methylation of the promoter that drives transcription of all the maternal noncoding RNAs including the long noncoding RNA MEG3, several microRNAs and snoRNAs. Additionally, I identified disease-relevant targets of DLK1-MEG3 microRNAs in vivo using HITS-CLIP, a technique to detect targets of RNA binding proteins. My results provide strong evidence for a role of microRNAs and epigenetic modifications, such as DNA methylation, in the pathogenesis of T2DM. In addition, my data set catalogs human islet microRNAs relevant to human T2DM pathogenesis and characterizes their target transcriptomes. Despite being associated with T2DM and several other diseases, very little is known about the regulation of imprinting of the MEG3-DLK1 locus. Hence, I interrogated a newly described enhancer in this locus, as enhancers are known mediators of mono-allelic expression at other imprinted loci. I discovered allele-specific binding of this enhancer by critical islet transcription factors, including FOXA2 in human islets. In addition, I mapped long-range interactions of this enhancer in human islets using 4C-Seq. Overall, my findings provide novel insights into the regulation of an imprinted locus critical to β-cell health and function

Epigenetic deregulation of micrornas in rhabdomyosarcoma and neuroblastoma and translational perspectives

Gene expression control mediated by microRNAs and epigenetic remodeling of chromatin are interconnected processes often involved in feedback regulatory loops, which strictly guide proper tissue differentiation during embryonal development. Altered expression of microRNAs is one of the mechanisms leading to pathologic conditions, such as cancer. Several lines of evidence pointed to epigenetic alterations as responsible for aberrant microRNA expression in human cancers. Rhabdomyosarcoma and neuroblastoma are pediatric cancers derived from cells presenting features of skeletal muscle and neuronal precursors, respectively, blocked at different stages of differentiation. Consistently, tumor cells express tissue markers of origin but are unable to terminally differentiate. Several microRNAs playing a key role during tissue differentiation are often epigenetically downregulated in rhabdomyosarcoma and neuroblastoma and behave as tumor suppressors when re-expressed. Recently, inhibition of epigenetic modulators in adult tumors has provided encouraging results causing re-expression of anti-tumor master gene pathways. Thus, a similar approach could be used to correct the aberrant epigenetic regulation of microRNAs in rhabdomyosarcoma and neuroblastoma. The present review highlights the current insights on epigenetically deregulated microRNAs in rhabdomyosarcoma and neuroblastoma and their role in tumorigenesis and developmental pathways. The translational clinical implications and challenges regarding modulation of epigenetic chromatin remodeling/microRNAs interconnections are also discusse

Small RNA-Dependent Gene Silencing in the Green Alga \u3ci\u3eChlamydomonas reinhardtii\u3c/i\u3e: Functions and Mechanisms

Small RNAs (sRNAs), ~20-30 nucleotides in length, are non-coding RNAs that play essential roles in the regulation of gene expression in eukaryotes. They lead to inactivation of cognate sequences at the post-transcriptional level via a variety of mechanisms involved in translation inhibition and/or RNA degradation. In the Chlorophyta Chlamydomonas reinhardtii, however, the molecular machinery responsible for sRNA-mediated translational repression remains unclear. To address the mechanisms of translation inhibition by sRNA, we have isolated an RNAi defective mutant (Mut26), which contains a deletion of the gene encoding the homolog of CCR4 in Chlamydomonas. We investigated the expression of both an exogenous siRNA target and endogenous miRNA target. Additionally, the pattern of poly(A) tailing in diagnostic mRNAs was examined with the G/I tailing assay and CCR4 partner proteins were identified through affinity purification. Our overall results are consistent with the role of CCR4 in sRNA-dependent translational repression without target mRNA degradation in Chlamydomonas. The biological function(s) of miRNAs in responses to nutrient deprivation in Chlamydomonas reinhardtii were also explored. Transcriptome analysis using cells grown under various trophic conditions revealed that several miRNAs were differentially expressed, but their predicted targets showed no changes in transcript abundance. Collective evidence suggests that miRNAs may not play an essential role in endogenous gene regulation in Chlamydomonas. Advisor: Heriberto Cerutt

GENOME-WIDE ANALYSIS OF CHICKEN MIRNAS AND DNA METHYLATION AND THEIR ROLES IN MAREK'S DISEASE RESISTANCE AND SUSCEPTIBILITY

Marek's disease (MD) is a T cell lymphoma in chickens and causes high mortality and morbidity in productive chickens. Two inbred chicken lines, resistant line 63 and susceptible line 72, with the same MHC haplotype, showed distinct disease outcomes after MDV infection. The current studies aimed to illustrate the role of microRNA (miRNAs) and DNA methylation in MD resistance and susceptibility in chickens. First, to ascertain the function of miRNAs, miRNA microarray experiments were used to identify miRNAs sensitive to MDV infection in the 2 lines. Most miRNAs were repressed in line 72 after MDV infection, while their transcription was steady in line 63. The miRNA target genes were identified in chickens. Cellular miRNA gga-miR-15b and gga-let-7iwere reduced in infected line 72 chickens and MD tumors. The downregulation of the two miRNAs increased the expression of ATF2 (activating transcription factor 2) and DNMT3a (DNA methyltransferase 3a) in infected line 72. These results indicated that miRNAs may play antiviral functions through modulating target gene expression. Next, to characterize the role of miRNAs in MDV infection, the selected chicken miRNAs were overexpressed in MDV infected DF-1 cells. The overexpressions of chicken miRNA gga-miR-15b and gga-let-7i, by using the retroviral based vector, significantly restricted MDV replications in vitro. MDV oncoprotein was repressed, suggesting that chicken miRNAs may limit MDV propagation. Finally, we found deregulation of transcription of DNA methyltransfereases (DNMTs) in lines 63 and 72 after MDV infection, which coordinated with the methylation alterations in the 2 lines. Infection induced differential methylation regions (iDMRs) that were identified through genome-wide DNA methylation quantification. Genes overlapping line-specific iDMRs were related with pathways of different functions in these two lines, implying the involvement of DNA methylation in MD- resistance and susceptibility. An in vitro study showed that DNA methylation inhibitor repressed viral spread and viral replication. In conclusion, the observed variations of miRNA expression and DNA methylation may be associated with disease predisposition in chickens

In sickness and in health: the role of methyl-CpG binding protein 2 in the central nervous system

The array of specialized neuronal and glial cell types that characterize the adult central nervous system originates from neuroepithelial proliferating precursor cells. The transition from proliferating neuroepithelial precursor cells to neuronal lineages is accompanied by rapid global changes in gene expression in coordination with epigenetic modifications at the level of the chromatin structure. A number of genetic studies have begun to reveal how epigenetic deregulation results in neurodevelopmental disorders such as mental retardation, autism, Rubinstein–Taybi syndrome and Rett syndrome. In this review we focus on the role of the methyl-CpG binding protein 2 (MeCP2) during development of the central nervous system and its involvement in Rett syndrome. First, we present recent findings that indicate a previously unconsidered role of glial cells in the development of Rett syndrome. Next, we discuss evidence of how MeCP2 deficiency or loss of function results in aberrant gene expression leading to Rett syndrome. We also discuss MeCP2's function as a repressor and activator of gene expression and the role of its different target genes, including microRNAs, during neuronal development. Finally, we address different signaling pathways that regulate MeCP2 expression at both the post-transcriptional and post-translational level, and discuss how mutations in MeCP2 may result in lack of responsiveness to environmental signals