Dataset Information

Life-time study of premodified histone for Identification of chromatinized proteome defined by site-specific histone acylations in living cells

ABSTRACT: C13, N15 isotope labelled heavy lysine bearing the modification group, such as cr and bhb, were site-sepcifically incorporated into histone H3K56 in living cells for measurement of the modification life of H3K56cr and H3K56bhb without iinterference caused by endogenous histone modification. Utilizaing mass spectrometry, we compared with H3K56cr/bhb peptide with the H3K79 peptide as internal standard at different times. The quantitation analysis of H3K56cr and H3K56bhb at different time points can be comparative.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Fangfei Qin

LAB HEAD: Peng R. Chen

PROVIDER: PXD033958 | Pride | 2023-03-06

REPOSITORIES: Pride

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Publications

Linking chromatin acylation mark-defined proteome and genome in living cells.

Qin Fangfei F Li Boyuan B Wang Hui H Ma Sihui S Li Jiaofeng J Liu Shanglin S Kong Linghao L Zheng Huangtao H Zhu Rongfeng R Han Yu Y Yang Mingdong M Li Kai K Ji Xiong X Chen Peng R PR

Cell 20230301 5

A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed di ...[more]

PMID: 36868209

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Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.

			Action	DRS
	H3K56h_Cr_24h_all_result.txt	Txt
	H3K56h_Cr_36h_all_result.txt	Txt
	H3K56h_Cr_48h_all_result.txt	Txt
	H3K56h_bhb-24h.raw	Raw
	H3K56h_bhb-36h.raw	Raw