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Post-translational modification of H2B C-terminal helix regulates nucleosome binding and chromatin signaling

ABSTRACT: Histone H2B contains a highly conserved C-terminal (H2B C) helix that has been implicated in chromatin interactions and dynamics. The H2B C helix is positioned adjacent to a major site of nucleosome interactions called the acidic patch. Despite individual structural studies highlighting interactions between chromatin proteins and the H2B C helix, the general role of the helix in mediating nucleosome recognition has not been explored. Moreover, many post-translational modifications (PTMs) have been identified within the H2B C helix, but significant gaps exist in our understanding of their regulatory potential. In this study, we employed nucleosome affinity proteomics using a library of nucleosomes with mutations or PTMs of the H2B C helix to investigate contributions to nucleosome binding. Our work uncovers new spatial patterns of H2BC helix engagement across the proteome. We also demonstrate that H2BK120 ubiquitylation (H2BK120ub1) broadly disrupts nucleosome binding, phenocopying mutation of the acidic patch, while differentially regulating acidic patch-dependent chromatin functions. In contrast, lysine acetylation results in more subtle position-specific changes, highlighting a more general role of H2B C helix PTMs in tuning acidic patch recognition.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Dennis Goldfarb

LAB HEAD: Robert McGinty

PROVIDER: PXD063304 | Pride | 2025-08-25

REPOSITORIES: Pride

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			Action	DRS
	MC040_HEK293T_Nucleosomes_TMTpro_Group1.raw	Raw
	MC041_HEK293T_Nucleosomes_TMTpro_Group2.raw	Raw
	MC042_HEK293T_Nucleosomes_TMTpro_Group3.raw	Raw
	MC043_HEK293T_Nucleosomes_TMTpro_Group4.raw	Raw
	MaxQuant_txt.zip	Other

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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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Post-translational modification of H2B C-terminal helix regulates nucleosome binding and chromatin signaling

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