Project description:Ewing sarcoma is a bone malignancy of children and young adults, frequently harboring the EWS/FLI t(11;22)(q24;q12) chromosomal translocation. The resulting fusion protein is an aberrant transcription factor that uses highly repetitive GGAA-containing elements (microsatellites) to activate and repress thousands of target genes mediating oncogenesis. However, the mechanisms of EWS/FLI interaction with microsatellites and regulation of target genes expression is not clearly understood. Here, we profile genome-wide protein binding and gene expression. Using a combination of unbiased genome-wide computational and experimental analysis, we define GGAA-microsatellites in a Ewing sarcoma context. Our study identifies two distinct classes of GGAA-microsatellites and demonstrates that EWS/FLI responsiveness is dependent on microsatellite length. At close range (within 5 kb) “promoter-like” microsatellites, EWS/FLI binding and subsequent target genes activation is highly dependent on the number of GGAA-motifs. “Enhancer-like” microsatellites demonstrate a positive correlation with length-dependent EWS/FLI binding, but minimal correlation for activated and none for repressed target genes. Our data suggest that EWS/FLI binds to “promoter-like” and “enhancer-like” microsatellites to mediate activation and repression of target genes through different regulatory mechanisms. Such characterization contributes valuable insight to EWS/FLI transcription factor biology and clarifies the role of GGAA-microsatellites on a global genomic scale. This may provide a unique perspective on the role of non-coding DNA in cancer susceptibility and therapeutic development.
Project description:We identified global DNA binding properties of EWS-FLI1 in mouse Ewing sarcoma. GGAA microsatellites were found as binding sites of EWS-FLI1 but with less frequency than that in human Ewing sarcoma, and genomic distribution is not conserved between human and mouse.
Project description:Gene expression during spermatogenesis undergoes significant changes due to a demanding sequence of mitosis, meiosis and differentiation. Round spermatids have to prepare for DNA condensation as well as synthesize transcripts of genes required for the differentiation process. However, the mechanisms of gene activation and silencing in round spermatids remain largely unknown. We employed ChIP-seq to figure out the correlation of H3K4me3 and H3K9me3 marks with transcriptional activation and silencing in round spermatids. Out of about 2800 genes identified by H3K4me3 chipseq, transcriptome sequencing in purified round spermatids showed the presence of transcripts corresponding to about 64% of the genes. On the other hand, only about 25% of H3K9me3 enriched genes showed transcription in round spermatids, that too at very low levels. In conclusion, H3K4me3 enrichment in round spermatids correlates significantly with gene expression and H3K9me3 correlates with gene silencing, both of which are equally important for successful spermatogenesis.
Project description:We generated RRBS data to analyse the DNA methylation profiling among WT-AG-haESCs, DKO-AG-haESCs and round spermatids, we found deletion of H19 and Gtl2 DMRs do not change the methylation patterns in AG-haESCs base on all detected CpG sites. We used round spermatids as control and analysed the DNA methylation profiles of all the cell lines were by RRBS.
Project description:The goal of this study was to identify transcriptional resonses to acute heat stress following differential acclimation in the highly eurythermal goby fish, Gillichthys mirabilis. G. mirabilis were acclimated to 3 temperatures (9C, 19C and 28C) for one month and then exposed to an acute heat ramp at 4C/hr and sampled during the timecourse. Gill tissues were dissected and immediately flash frozen in liquid nitrogen. Total RNA was extracted, reverse transcribed into cDNA, labeled with cy5 and hybridized against a common reference (cy3) on 2-color cDNA microarray developed for this species. Significant genes were determined by expression differences between groups (one-way ANOVA, FDR<0.05) . Common reference design