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Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones using Quantitative Mass Spectrometry

ABSTRACT: Chemical modification of histone proteins by methylation plays a central role in chromatin regulation by recruiting epigenetic ‘readers’ via specialized binding domains. Depending on the degree of methylation, the exact modified amino acid, and the associated reader proteins histone methylations are involved in the regulation of all DNA-based processes, such as transcription, DNA replication, and DNA repair. We have previously established a method that allows the unbiased identification of nuclear proteins which binding to nucleosomes is regulated by the presence of specific histone modifications (1,2). The method is based on an in-vitro reconstitution of semi-synthetic nucleosomes bearing a predefined set of histone modifications which are subsequently used as baits for affinity purification pull-down experiments with nuclear extracts followed by identification and quantification of nucleosome-interacting proteins using LC-MS/MS. Here we provide a representative set of label-free MS results for nucleosome pull-down affinity purification experiments performed using unmodified as well as H3K4me3- and H3K9me3-modified di-nucleosomes and nuclear extract obtained from HeLa S3 cells. 1. Bartke T, Vermeulen M, Xhemalce B, Robson SC, Mann M, Kouzarides T (2010) Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143:470-484 2. Makowski MM, Gräwe C, Foster BM, Nguyen NV, Bartke T, Vermeulen M (2018) Global profiling of protein-DNA and protein-nucleosome binding affinities using quantitative mass spectrometry. Nat Commun 9:1653

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Andrey Tvardovskiy

LAB HEAD: Till Bartke

PROVIDER: PXD027238 | Pride | 2022-08-11

REPOSITORIES: Pride

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Project description:Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted high-throughput nucleosome reconstitution experiments on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The DNA loci from both species contained the genes that encode the serum albumin family, the MYC protein, and the tumor suppressor p53. The results demonstrated that a small subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. We propose that these features serve to enhance the affinity of methylated DNA for the histone octamer and that this effect could be involved in gene regulatory mechanisms such as silencing. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin. For the human data, there were 3 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, and an unmethylated sonicated control. For the mouse data, there were 2 unmethylated nucleosome replicates, 2 methylated nucleosome replicates, an unmethylated MNase control, a methylated MNase control, and an unmethylated sonicated control.

Project description:In mammalian cells transcription factors (TFs) preferentially bind sites contained in regions of high nucleosomal occupancy, as determined by nucleotide-dependent computational analysis. This observation suggests that nucleosomes may act as gatekeepers of TF binding sites. We hypothesized that in mammalian genomes the information controlling nucleosome assembly may partially coincide with the information that enables TFs to recognize cognate sites in cis-regulatory elements while ignoring the myriad of non-functional, randomly occurring consensus binding sites. This way, nucleosome-mediated masking would be coupled to TF binding site functionality. The hematopoietic master regulator Pu.1 maintained nucleosome depletion at macrophage-specific enhancers that were otherwise occupied by nucleosomes in other cell types and in reconstituted chromatin. We identified a minimal set of DNA sequence and shape features that predicted Pu.1 binding with 78% accuracy. The same features predicted nucleosome occupancy in cells where Pu.1 was not expressed with higher accuracy than specifically designed models. Control of nucleosome deposition by DNA sequence and shape features that also specify TF consensus site functionality may allow maintaining the gatekeeper function of nucleosomes during evolution of cis-regulatory elements. Chromatin immuno-precipitations of the transcription factor Pu.1 followed by multiparallel sequencing performed in murine bone marrow-derived macrophages. Experiments carried out in cells infected either with a retroviral vector containing a short hairpin targeting Pu.1 or with the empty vector as control. The shPU.1 hairpin (sequence available upon request) was selected among five designed using a publicly available software (http://katahdin.mssm.edu/siRNA) and was cloned in a modified version of TtRMPVIR inducible retroviral vector (Genbank HQ456318) in which the puromycin resistance gene was inserted. The empty vector, containing an sh-Renilla sequence, was used as control. Chromatin immuno-precipitations of the transcription factor PU.1 binding to in vitro reconstituted chromatin followed by multiparallel sequencing Micrococcal nuclease digestion of chromatin extracted from bone marrow derived macrophages followed by multiparallel sequencing . Experiments were carried out in untreated cells (4 replicates) and cells infected either with a retroviral vector containing a short hairpin targeting Pu.1 or with the empty vector as control (2 replicates). The shPU.1 hairpin (sequence available upon request) was selected among five designed using a publicly available software (http://katahdin.mssm.edu/siRNA) and was cloned in a modified version of TtRMPVIR inducible retroviral vector (Genbank HQ456318) in which the puromycin resistance gene was inserted. The empty vector, containing an sh-Renilla sequence, was used as control. Micrococcal nuclease digestion of in vitro reconstituted chromatin followed by multiparallel sequencing

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Identifying Specific Protein Interactors of Nucleosomes Carrying Methylated Histones using Quantitative Mass Spectrometry

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