Project description:ChIP seq of Cfp-1 and H3K4me3 in C. elegans late embryos The coding region of F52B11.1a (cfp-1) was PCR amplified from N2 genomic DNA using Phusion polymerase (Finnzymes) and Gateway cloned into pDONR221. The cfp-1 coding region was then recombined into the MosSCI compatible vector pCFJ201 (which targets Mos site Mos1(cxTi10882) chrIV ) downstream of the dpy-30 promoter and upstream of gfp::tbb-2 3’UTR (Zeiser et al. 2011) to generae strain JA1597 expressing GFP tagged Cfp-1 protein. Late embryos were obtained by aging embryos collected by hypochlorite treatment 3.5 hrs prior to flash freezing in liquid nitrogen. Formaldehyde-fixed chromatin extracts and chromatin immunoprecipitations were as in (Kolasinska-Zwierz et al. 2009) except that DNA was sonicated to a size range of 200-400bp. ChIP assays were performed in 1 ml extract (1 mg protein) in FA buffer with 10 micrograms of anti-GFP rabbit serum (Abcam ab290) and anti-H3K4me3 (Abcam ab8580) individually. DNA sequencing libraries were constructed according using the Illumina Truseq sequencing kit and were sequenced on the Illumina platform.
Project description:Most vertebrate promoters lie in unmethylated CpG-dense islands, whereas methylation of the more sparsely distributed CpGs in the remainder of the genome is thought to contribute to transcriptional repression. Nonmethylated CG dinucleotides are recognized by CXXC finger protein 1 (CXXC1, also known as CFP1), which recruits SETD1A (also known as Set1) methyltransferase for trimethylation of histone H3 lysine 4, an active promoter mark. Genomic regions enriched for CpGs are thought to be either absent or irrelevant in invertebrates that lack DNA methylation, such as C. elegans; however, a CXXC1 ortholog (CFP-1) is present. Here we demonstrate that C. elegans CFP-1 targets promoters with high CpG density, and these promoters are marked by high levels of H3K4me3. Furthermore, as for mammalian promoters, high CpG content is associated with nucleosome depletion irrespective of transcriptional activity. We further show that highly occupied target (HOT) regions identified by the binding of a large number of transcription factors are CpG-rich promoters in C. elegans and human genomes, suggesting that the unusually high factor association at HOT regions may be a consequence of CpG-linked chromatin accessibility. Our results indicate that nonmethylated CpG-dense sequence is a conserved genomic signal that promotes an open chromatin state, targeting by a CXXC1 ortholog, and H3K4me3 modification in both C. elegans and human genomes.
Project description:The CFP1 CXXC zinc finger protein targets the SET1/COMPASS complex to nonmethylated CpG rich promoters to implement tri-methylation of histone H3 Lys4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss vary, suggesting additional chromatin factors contribute to context dependent effects. Using a proteomics approach, we identified CFP1 associated proteins and an unexpected direct link between C. elegans CFP-1 and an Rpd3/Sin3 small (SIN3S) histone deacetylase complex.
Project description:The highly conserved CFP1 CXXC zinc finger protein targets SET1 family complexes to promoter regions to implement methylation of histone H3 Ly4 (H3K4me), a mark that correlates with gene expression depending on the chromatin context. Whether CFP1 physically interacts with additional chromatin modifying proteins on chromatin has not been investigated. By applying interaction proteomics coupled to mass spectrometry to identify factors that interact with C. elegans CFP-1, we establish a link between CFP-1 and the Rpd3/Sin3 histone deacetylase (HDAC) small complex (Rpd3/Sin3S) in embryos. We show that CFP-1 directly interacts with SIN-3 through a highly conserved C-terminal domain, and that the two proteins extensively colocalize with H3K4me3 at promoter regions genome-wide. Animals lacking sin-3, cfp-1 or the catalytic H3K4 methyltransferase subunit set-2/SET1 show misregulation of common genes in early embryos, reduction in fertility, and defects in the division of intestinal cells in adults, consistent with common developmental functions. Our results suggest that in addition to its well characterized role as a central subunit of the SET-2/SET1 complex, CFP-1 interacts with the Sin3S/HDAC complex at promoter regions to influence gene expression in a developmental context. This has implications for the coordinate regulation of gene expression by chromatin associated complexes with distinct activities.
Project description:The CFP1/CXXC protein targets SET1/COMPASS complexes to non-methylated CpG rich promoter regions to implement tri-methylation of histone H3 Ly4 (H3K4me3). Although H3K4me3 is widely associated with gene expression, the effects of CFP1 loss depend on chromatin context. Little is known about how CFP1 binding is coordinated with other chromatin factors. Using a proteomics approach, we establish a link between C. elegans CFP1 and a small Rpd3/Sin3 histone deacetylase (HDAC) complex (Sin3S). Mutants of CFP-1, SIN-3, and the catalytic subunit SET-2/SET1 have similar phenotypes and misregulate the expression of common genes. CFP-1 directly interacts with SIN-3 through a region including the conserved PAH1 domain, and contributes to recruitment of SIN-3 and the Sin3S subunit HDA-1 to H3K4me3 enriched promoters. Our results reveal a novel role for CFP-1 in mediating interaction between SET1/COMPASS and a Sin3 HDAC small complex at promoters, with implications for the coordinate regulation of gene expression by chromatin- associated complexes with distinct activities.
Project description:In this study, we profiled genome-wide pattern of tri-methylation of lysine 4 on histone H3 (H3K4me3) in the somatic cells of young and old C. elegans to investigate the changes of H3K4me3 during aging. In addition, we profiled H3K4me3 pattern in developmental stage L3. We found that H3K4me3 pattern in L3 is largely maintained in adults and the age-dynamic H3K4me3 are mainly adult stage specific. We profiled gene expression in young and old C. elegans somatic cells with ribo-RNAseq. The changes of H3K4me3 and RNA abundance are positively correlated.
Project description:Aim: The goal of this study was to comprehensively interrogate and map DNA methylation across 16 CpG-dense regions previously associated with oral and pharyngeal squamous cell carcinoma (OPSCC). Materials & methods: Targeted multiplex bisulfite amplicon sequencing was performed on four OPSCC cell lines and primary non-neoplastic oral epithelial cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for a subset of associated genes. Results: There was clear differential methylation between one or more OPSCC cell lines and control cells for the majority of CpG-dense regions. Conclusion: Targeted multiplex bisulfite amplicon sequencing allowed us to efficiently map methylation across the entire region of interest with a high degree of sensitivity and helps shed light on novel differentially methylated regions that may have value as biomarkers of OPSCC.
Project description:Loss of function during ageing is accompanied by transcriptional drift, altering gene expression and contributing to a variety of age-related diseases. CREB-regulated transcriptional coactivators (CRTCs) have emerged as key regulators of gene expression that might be targeted to promote longevity. Here, we define the role of the Caenorhabditis elegans CRTC-1 in the epigenetic regulation of longevity. Endogenous CRTC-1 binds chromatin factors, including components of the COMPASS complex, which trimethylates lysine 4 on histone H3 (H3K4me3). CRISPR editing of endogenous CRTC-1 reveals that the CREB-binding domain in neurons is specifically required for H3K4me3-dependent longevity. However, this effect is independent of CREB but instead acts via the transcription factor AP-1. Strikingly, CRTC-1 also mediates global histone acetylation levels, and this acetylation is essential for H3K4me3-dependent longevity. Indeed, overexpression of an acetyltransferase enzyme is sufficient to promote longevity in wild-type worms. CRTCs, therefore, link energetics to longevity by critically fine-tuning histone acetylation and methylation to promote healthy ageing.
Project description:Loss of function during ageing is accompanied by transcriptional drift, altering gene expression and contributing to a variety of age-related diseases. CREB-regulated transcriptional coactivators (CRTCs) have emerged as key regulators of gene expression that might be targeted to promote longevity. Here, we define the role of the Caenorhabditis elegans CRTC-1 in the epigenetic regulation of longevity. Endogenous CRTC-1 binds chromatin factors, including components of the COMPASS complex, which trimethylates lysine 4 on histone H3 (H3K4me3). CRISPR editing of endogenous CRTC-1 reveals that the CREB-binding domain in neurons is specifically required for H3K4me3-dependent longevity. However, this effect is independent of CREB but instead acts via the transcription factor AP-1. Strikingly, CRTC-1 also mediates global histone acetylation levels, and this acetylation is essential for H3K4me3-dependent longevity. Indeed, overexpression of an acetyltransferase enzyme is sufficient to promote longevity in wild-type worms. CRTCs, therefore, link energetics to longevity by critically fine-tuning histone acetylation and methylation to promote healthy ageing.