Chromatin condensation and recruitment of PHD finger proteins to histone H3K4me3 are mutually exclusive

Histone post-translational modifications, and specific combinations they create, mediate a wide range of nuclear events. However, the mechanistic bases for recognition of these combinations have not been elucidated. Here, we characterize crosstalk between H3T3 and H3T6 phosphorylation, occurring in mitosis, and H3K4me3, a mark associated with active transcription. We detail the molecular mechanisms by which H3T3ph/K4me3/T6ph switches mediate activities of H3K4me3-binding proteins, including those containing plant homeodomain (PHD) and double Tudor reader domains. Our results derived from nuclear magnetic resonance chemical shift perturbation analysis, orthogonal binding assays and cell fluorescence microscopy studies reveal a strong anti-correlation between histone H3T3/T6 phosphorylation and retention of PHD finger proteins in chromatin during mitosis. Together, our findings uncover the mechanistic rules of chromatin engagement for H3K4me3-specific readers during cell division

An Acetyl-Methyl Switch Drives a Conformational Change in p53

Individual posttranslational modifications (PTMs) of p53 mediate diverse p53-dependent responses, however much less is known about the combinatorial action of adjacent modifications. Here, we describe crosstalk between the early DNA damage response mark p53K382me2 and the surrounding PTMs that modulate binding of p53 co-factors, including 53BP1 and p300. The 1.8 Å resolution crystal structure of the tandem Tudor domain (TTD) of 53BP1 in complex with p53 peptide acetylated at K381 and dimethylated at K382 (p53K381acK382me2) reveals that the dual PTM induces a conformational change in p53. The α-helical fold of p53K381acK382me2 positions the side chains of R379, K381ac, and K382me2 to interact with TTD concurrently, reinforcing a modular design of double PTM mimetics. Biochemical and NMR analyses show that other surrounding PTMs, including phosphorylation of serine/threonine residues of p53, affect association with TTD. Our findings suggest a novel PTM-driven conformation switch-like mechanism that may regulate p53 interactions with binding partners

Predicting Patterns of Distant Metastasis in Breast Cancer Patients following Local Regional Therapy Using Machine Learning

Up to 30% of breast cancer (BC) patients will develop distant metastases (DM), for which there is no cure. Here, statistical and machine learning (ML) models were developed to estimate the risk of site-specific DM following local-regional therapy. This retrospective study cohort included 175 patients diagnosed with invasive BC who later developed DM. Clinicopathological information was collected for analysis. Outcome variables were the first site of metastasis (brain, bone or visceral) and the time interval (months) to developing DM. Multivariate statistical analysis and ML-based multivariable gradient boosting machines identified factors associated with these outcomes. Machine learning models predicted the site of DM, demonstrating an area under the curve of 0.74, 0.75, and 0.73 for brain, bone and visceral sites, respectively. Overall, most patients (57%) developed bone metastases, with increased odds associated with estrogen receptor (ER) positivity. Human epidermal growth factor receptor-2 (HER2) positivity and non-anthracycline chemotherapy regimens were associated with a decreased risk of bone DM, while brain metastasis was associated with ER-negativity. Furthermore, non-anthracycline chemotherapy alone was a significant predictor of visceral metastasis. Here, clinicopathologic and treatment variables used in ML prediction models predict the first site of metastasis in BC. Further validation may guide focused patient-specific surveillance practices.</jats:p

Recognition of cancer mutations in histone H3K36 by epigenetic writers and readers

Histone posttranslational modifications control the organization and function of chromatin. In particular, methylation of lysine 36 in histone H3 (H3K36me) has been shown to mediate gene transcription, DNA repair, cell cycle regulation, and pre-mRNA splicing. Notably, mutations at or near this residue have been causally linked to the development of several human cancers. These observations have helped to illuminate the role of histones themselves in disease and to clarify the mechanisms by which they acquire oncogenic properties. This perspective focuses on recent advances in discovery and characterization of histone H3 mutations that impact H3K36 methylation. We also highlight findings that the common cancer-related substitution of H3K36 to methionine (H3K36M) disturbs functions of not only H3K36me-writing enzymes but also H3K36me-specific readers. The latter case suggests that the oncogenic effects could also be linked to the inability of readers to engage H3K36M

TCF19 Promotes Cell Proliferation through Binding to the Histone H3K4me3 Mark

Transcription factor 19 (TCF19) plays critical roles in type 1 diabetes and the maintenance of pancreatic β cells. Recent studies have also implicated TCF19 in cell proliferation of hepatic carcinoma and non-small cell lung carcinoma; however, the mechanism underlying this regulation remains elusive. At the molecular level, TCF19 contains two modules, the plant homeodomain (PHD) finger and the forkhead-associated (FHA) domain, of unclear function. Here, we show that TCF19 mediates hepatocellular carcinoma HepG2 cell proliferation through its PHD finger that recognizes trimethylated lysine 4 of histone 3 (H3K4me3). W316 of the PHD finger of TCF19 is one of the critical residues eliciting this function. Whole genome microarray analysis and orthogonal cell-based assays identified a large subset of genes involved in cell survival and proliferation that depend on TCF19. Our data suggest that TCF19 acts as a pro-oncogene in hepatocellular carcinoma cells and that its functional PHD finger is critical in cell proliferation

Structural and biophysical characterization of the nucleosome-binding PZP domain

Summary: The core subunit of the MORF acetyltransferase complex BRPF1 contains a unique combination of zinc fingers, including a plant homeodomain (PHD) finger followed by a zinc knuckle and another PHD finger, which together form a PZP domain (BRPF1PZP). BRPF1PZP has been shown to bind to the nucleosome and make contacts with both histone H3 tail and DNA. Here, we describe biophysical and structural methods for characterization of the interactions between BRPF1PZP, H3 tail, DNA, and the intact nucleosome.For complete details on the use and execution of this protocol, please refer to Klein et al. (2020)

PHF20 Readers Link Methylation of Histone H3K4 and p53 with H4K16 Acetylation

PHF20 is a core component of the lysine acetyltransferase complex MOF (male absent on the first)-NSL (non-specific lethal) that generates the major epigenetic mark H4K16ac and is necessary for transcriptional regulation and DNA repair. The role of PHF20 in the complex remains elusive. Here, we report on functional coupling between methylation readers in PHF20. We show that the plant homeodomain (PHD) finger of PHF20 recognizes dimethylated lysine 4 of histone H3 (H3K4me2) and represents an example of a native reader that selects for this modification. Biochemical and structural analyses help to explain this selectivity and the preference of Tudor2, another reader in PHF20, for dimethylated p53. Binding of the PHD finger to H3K4me2 is required for histone acetylation, accumulation of PHF20 at target genes, and transcriptional activation. Together, our findings establish a unique PHF20-mediated link between MOF histone acetyltransferase (HAT), p53, and H3K4me2, and suggest a model for rapid spreading of H4K16ac-enriched open chromatin

Mechanism for autoinhibition and activation of the MORC3 ATPase.

Microrchidia 3 (MORC3) is a human protein linked to autoimmune disorders, Down syndrome, and cancer. It is a member of a newly identified family of human ATPases with an uncharacterized mechanism of action. Here, we elucidate the molecular basis for inhibition and activation of MORC3. The crystal structure of the MORC3 region encompassing the ATPase and CW domains in complex with a nonhydrolyzable ATP analog demonstrates that the two domains are directly coupled. The extensive ATPase:CW interface stabilizes the protein fold but inhibits the catalytic activity of MORC3. Enzymatic, NMR, mutational, and biochemical analyses show that in the autoinhibited, off state, the CW domain sterically impedes binding of the ATPase domain to DNA, which in turn is required for the catalytic activity. MORC3 autoinhibition is released by disrupting the intramolecular ATPase:CW coupling through the competitive interaction of CW with histone H3 tail or by mutating the interfacial residues. Binding of CW to H3 leads to a marked rearrangement in the ATPase-CW cassette, which frees the DNA-binding site in active MORC3 (on state). We show that ATP-induced dimerization of the ATPase domain is strictly required for the catalytic activity and that the dimeric form of ATPase-CW might cooperatively bind to dsDNA. Together, our findings uncovered a mechanism underlying the fine-tuned regulation of the catalytic domain of MORC3 by the epigenetic reader, CW