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

A Retrospective Study of Cochlear Re-Implantations - Experience from a Large Centre in India

Objective: Cochlear re-implantation (CRI) is becoming increasingly common throughout the world. However, studies regarding CRI incidence and etiology are lacking from developing countries like ours. The aim of this study was to present the Indian experience with CRI based on our experience. Objectives were to determine the incidence and the indications of CRI and the cumulative survival rate (CSR) of cochlear implantation (CI).Methods: Our study was a retrospective one, conducted at a tertiary care centre in southern India. 1,500 consecutive cochlear implanted ears from 1997 to 2016 were studied. All patients who underwent CRI during this period were included in the study.Results: There were a total of 53 ears (31 male and 22 female ears) who underwent CRI. This gives an incidence of 3.53%. The most common indication of CRI was device failure in 39 ears contributing to 73.6% of the total CRI. The overall CSR of CI in pediatric population was 96.5% over a 20-year period.Conclusion:The CRI incidence and etiology at our centre appears to reflect the findings of the literature. Cochlear implant centres across the world should report the CSR of devices used at their respective centres so that it can be made an important criterion in choice of implant

Dynamic recruitment of microRNAs to their mRNA targets in the regenerating liver.

BACKGROUND: Validation of physiologic miRNA targets has been met with significant challenges. We employed HITS-CLIP to identify which miRNAs participate in liver regeneration, and to identify their target mRNAs. RESULTS: miRNA recruitment to the RISC is highly dynamic, changing more than five-fold for several miRNAs. miRNA recruitment to the RISC did not correlate with changes in overall miRNA expression for these dynamically recruited miRNAs, emphasizing the necessity to determine miRNA recruitment to the RISC in order to fully assess the impact of miRNA regulation. We incorporated RNA-seq quantification of total mRNA to identify expression-weighted Ago footprints, and developed a microRNA regulatory element (MRE) prediction algorithm that represents a greater than 20-fold refinement over computational methods alone. These high confidence MREs were used to generate candidate \u27competing endogenous RNA\u27 (ceRNA) networks. CONCLUSION: HITS-CLIP analysis provide novel insights into global miRNA:mRNA relationships in the regenerating liver

Additional file 4: of The BisPCR2 method for targeted bisulfite sequencing

Table S2. PCR#2 primer sequences for library amplification and barcoding. Amplification with PCR#2 primers adds the remainder of adapter sequence and unique barcodes for up to 48 samples. A common forward primer, “Library_Primer1,” is used in combination with each unique barcoding reverse primer

Additional file 3: of The BisPCR2 method for targeted bisulfite sequencing

Table S1. PCR#1 primer sequences for amplification of bisulfite-converted genomic DNA. Forward and reverse target enrichment primers were modified with adapter overhangs. Locus-specific portion of primer sequences are in bold text and common adapter overhangs are in plain text

Molecular basis of CTCF binding polarity in genome folding.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns

Molecular basis of CTCF binding polarity in genome folding.

Current models propose that boundaries of mammalian topologically associating domains (TADs) arise from the ability of the CTCF protein to stop extrusion of chromatin loops by cohesin. While the orientation of CTCF motifs determines which pairs of CTCF sites preferentially stabilize loops, the molecular basis of this polarity remains unclear. By combining ChIP-seq and single molecule live imaging we report that CTCF positions cohesin, but does not control its overall binding dynamics on chromatin. Using an inducible complementation system, we find that CTCF mutants lacking the N-terminus cannot insulate TADs properly. Cohesin remains at CTCF sites in this mutant, albeit with reduced enrichment. Given the orientation of CTCF motifs presents the N-terminus towards cohesin as it translocates from the interior of TADs, these observations explain how the orientation of CTCF binding sites translates into genome folding patterns