Project description:R-loops are three-stranded nucleic acid structures consisting of a DNA-RNA hybrid and a displaced single-stranded DNA (ssDNA). R-loops are facilitators of gene expression and genome stability that play both regulatory and potentially deleterious roles in cells. To elucidate the protein-based mechanisms of R-loop regulation, we developed an APEX-based proximity proteomics using a catalytically inactive mutant of RNase H1 (APEX-RNH1D210N) to profile the proximal proteome of R-loops in cells. Our proteomics results identified a list of known and potential R-loop-binding proteins with diverse molecular functions, and confirmed YY1 as a novel R-loop-binding protein through a series of in-vitro binding assays. YY1 is a DNA-binding protein recognizing a consensus motif or G-quadruplex (G4) structure. Our binding results indicated YY1's notably stronger affinity for R-loops compared to RNA/DNA hybrid, DNA G4 and DNA without a consensus motif, implying R-loops' role in YY1 recruitment to genomic regions lacking a YY1-binding motif. To investigate YY1-R-loop interaction in cells, we introduced R-loop structures into specific genomic regions vis CRISPR-dCas9 targeting, followed by DRIP-qPCR, YY1-ChIP-qPCR and RT-qPCR experiments. Increased R-loop levels corresponded with heightened YY1 occupancy and differential gene expression, suggesting YY1-R-loop interactions contribute to transcriptional regulation. More importantly, our RNA-seq and YY1-ChIP-seq results revealed that the interactions between YY1 and promoter R-loops were involved in global transcriptional regulation, especially in the positive regulation of transcription. Together, we identified YY1 as a novel R-loop-binding protein and uncovered a mechanism wherein YY1-R-loop interactions regulate gene expression.
Project description:We mesured YY1 binding in isolated mouse crypt epihtelium using ChIP-seq Jejunal crypt epithelia were isolated and processed for ChIP using YY1 antibody Santa Cruz, SC-1703, lot E0511
Project description:We use comprehensive and unsupervised transcriptome analyses to provide molecular classifications of sensory neurons in the mouse geniculate ganglion. 96 neurons were isolated on a C1 Fluodigm chip, underwent RNA-Seq, and iteratively clustered into sub-classes.
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other
Project description:We report the application of Chip-seq to study the binding properties of C-maf. We determine the genetic and non genetic regions with the largest Chip-seq peaks in C-maf_ab compared to Isotype control antibody (Iso) samples.