Structural insight into H4K20 methylation on H2A.Z-nucleosome by SUV420H1.

DNA replication ensures the accurate transmission of genetic information during the cell cycle. Histone variant H2A.Z is crucial for early replication origins licensing and activation in which SUV420H1 preferentially recognizes H2A.Z-nucleosome and deposits H4 lysine 20 dimethylation (H4K20me2) on replication origins. Here, we report the cryo-EM structures of SUV420H1 bound to H2A.Z-nucleosome or H2A-nucleosome and demonstrate that SUV420H1 directly interacts with H4 N-terminal tail, the DNA, and the acidic patch in the nucleosome. The H4 (1-24) forms a lasso-shaped structure that stabilizes the SUV420H1-nucleosome complex and precisely projects the H4K20 residue into the SUV420H1 catalytic center. In vitro and in vivo analyses reveal a crucial role of the SUV420H1 KR loop (residues 214-223), which lies close to the H2A.Z-specific residues D97/S98, in H2A.Z-nucleosome preferential recognition. Together, our findings elucidate how SUV420H1 recognizes nucleosomes to ensure site-specific H4K20me2 modification and provide insights into how SUV420H1 preferentially recognizes H2A.Z nucleosome.

Discovery of a novel 53BP1 inhibitor through AlphaScreen-based high-throughput screening.

Tumor suppressor p53-binding protein 1 (53BP1), a tantem tudor domain (TTD) protein, takes part in DNA Damage Repair (DDR) pathways through the specific recognition of lysine methylation on histones. The dysregulation of 53BP1 is closely related to the development of many diseases including cancer. Moreover, recent studies found that deficiency of 53BP1 could increase the efficiency of precise CRISPR/Cas9 genome editing. Thus, discovery of inhibitor is beneficial to the study of biological functions of 53BP1 and the application of CRISPR/Cas9 genome editing. UNC2170 and its derivatives have been reported as 53BP1 targeted small molecular inhibitors with modest activities. Hence, to discover better 53BP1 inhibitors, we conducted an AlphaScreen assay based high-throughput screening (HTS) and identified a novel and effective 53BP1-TTD inhibitor DP308 which disrupts the binding between 53BP1 and H4K20me2 peptide with an IC50 value of 1.69 ± 0.73 µM. Both Microscale Themophoresis (MST) and Surface Plasmon Resonance (SPR) assays confirmed the direct binding between DP308 and 53BP1-TTD protein with binding affinity (Kd) of about 2.7 µM. Molecular docking studies further suggested that DP308 possibly occupies the H4K20me2 binding pocket of the 53BP1-TTD aromatic cage. These results demonstrated that DP308 is a promising small molecule inhibitor for further optimization towards a more potent chemical probe of 53BP1. Additionally, it could be a potential valuable tool for applying to gene editing therapy by increasing the efficiency of CRISPR/Cas9 genome editing.

Computer-aided screening for suppressor of variegation 4-20 homolog 1 inhibitors and their preliminary activity validation in human osteosarcoma.

Histone methylation/demethylation facilitate to maintain balanced histone methylation levels and underpin gene regulation, playing the key roles in epigenetic regulation. Suppressor of variegation 4-20 homolog 1 (SUV420H1), a member of class Histone Lysine Methyltransferase and a key enzyme in the epigenetic regulation of the pathways controlling metabolism and tumorigenesis, is crucial to maintain cell homeostasis. The inhibition of SUV420H1 has emerged as a promising candidate for drug development and cancer therapy. Herein, two potential and potent SUV420H1 inhibitors (ZINC08398384, ZINC08439608) were identified through in silico approach and in vitro biological experiments. In vitro biological tests demonstrated that these compounds can inhibit the proliferation of U2OS cells and restrict its migration ability. And the level of dimethylation of lysine 20 on histone H4 (H4K20me2) was markedly decreased by these compounds-treatment in a dose-dependent manner. These results indicated that ZINC08398384 and ZINC08439608 are potential SUV420H1 inhibitors and could be developed as promising drug candidates applied to cancer epigenetic therapy.Communicated by Ramaswamy H. Sarma.

HDAC6 is associated with the formation of aortic dissection in human.

BACKGROUND: The pathological features of aortic dissection (AD) include vascular smooth muscle cell (VSMC) loss, elastic fiber fraction, and inflammatory responses in the aorta. However, little is known about the post-translational modification mechanisms responsible for these biological processes. METHODS: A total of 72 aorta samples, used for protein detection, were collected from 36 coronary artery disease (CAD, served as the control) patients and 36 type A AD (TAAD) patients. Chromatin immunoprecipitation (ChIP)-PCR was used to identify the genes regulated by H3K23ac, and tubastatin A, an inhibitor of HDAC6, was utilized to clarify the downstream mechanisms regulated by HDAC6. RESULTS: We found that the protein level of histone deacetylase HDAC6 was reduced in the aortas of patients suffering from TAAD and that the protein levels of H4K12ac, and H3K23ac significantly increased, while H3K18ac, H4K8ac, and H4K5ac dramatically decreased when compared with CAD patients. Although H3K23ac, H3K18ac, and H4K8ac increased in the human VSMCs after treatment with the HDAC6 inhibitor tubastatin A, only H3K23ac showed the same results in human tissues. Notably, the results of ChIP-PCR demonstrated that H3K23ac was enriched in extracellular matrix (ECM)-related genes, including Col1A2, Col3A1, CTGF, POSTN, MMP2, TIMP2, and ACTA2, in the aortic samples of TAAD patients. In addition, our results showed that HDAC6 regulates H4K20me2 and p-MEK1/2 in the pathological process of TAAD. CONCLUSIONS: These results indicate that HDAC6 is involved in human TAAD formation by regulating H3K23ac, H4K20me2 and p-MEK1/2, thus, providing a strategy for the treatment of TAAD by targeting protein post-translational modifications (PTMs), chiefly histone PTMs.

The inhibition of checkpoint activation by telomeres does not involve exclusion of dimethylation of histone H4 lysine 20 (H4K20me2).

DNA double-strand breaks (DSBs) activate the DNA damage checkpoint machinery to pause or halt the cell cycle.  Telomeres, the specific DNA-protein complexes at linear eukaryotic chromosome ends, are capped DSBs that do not activate DNA damage checkpoints.  This "checkpoint privileged" status of telomeres was previously investigated in the yeast  Schizosaccharomyces pombelacking the major double-stranded telomere DNA binding protein Taz1. Telomeric DNA repeats in cells lacking Taz1 are 10 times longer than normal and contain single-stranded DNA regions. DNA damage checkpoint proteins associate with these damaged telomeres, but the DNA damage checkpoint is not activated. This severing of the DNA damage checkpoint signaling pathway was reported to stem from exclusion of histone H4 lysine 20 dimethylation (H4K20me2) from telomeric nucleosomes in both wild type cells and cells lacking Taz1.  However, experiments to identify the mechanism of this exclusion failed, prompting our re-evaluation of H4K20me2 levels at telomeric chromatin.  In this short report, we used an extensive series of controls to identify an antibody specific for the H4K20me2 modification and show that the level of this modification is the same at telomeres and internal loci in both wild type cells and those lacking Taz1.  Consequently, telomeres must block activation of the DNA Damage Response by another mechanism that remains to be determined.

H4K20me2: Orchestrating the recruitment of DNA repair factors in nucleotide excision repair.

The integrity of the genome is maintained by specific DNA repair pathways. The main pathway removing DNA lesions induced by exposure to UV light is nucleotide excision repair (NER). The DNA damage response at chromatin is accompanied by the recruitment of DNA repair factors to the lesion site and the deposition of specific histone marks. The function of these histone marks in NER stays for the most part elusive. We have recently reported that the methyltransferase MMSET catalyzes the dimethylation of histone H4 at lysine 20 (H4K20me2) at the lesion site. The deposition of H4K20me2 at DNA damage sites elicits the recruitment of the NER factor XPA providing evidence for an H4K20me2-dependent DNA repair factor recruitment mechanism during lesion recognition in the global-genomic branch of NER. Here we discuss how H4K20me2 might impact on the chromatin conformation and the DNA damage response.

Contribution of virtual biopsy to the screening of microvascular invasion in hepatocellular carcinoma: A pilot study.

BACKGROUND & AIMS: Microvascular invasion (mVI) is a major prognostic factor in hepatocellular carcinoma (HCC) that cannot be detected before surgery. Predictive biomarkers of mVI are thus urgently needed. We have developed an original approach of virtual biopsy to assess the performance of an immunohistochemical panel comprising three biomarkers of mVI (H4K16ac, H4K20me2, PIVKA-II) for the prediction of mVI in HCC core needle biopsies (CNB). METHODS: A test set of HCC surgical specimens (n = 64) and an independent validation set of HCC CNB (n = 42) were retrospectively constituted. Immunostainings were first quantified in the test set on the whole tissue section, to determine optimal cut-off values for each marker. From the digitised image of the whole section, three virtual biopsies were provided. Immunostainings and accuracy of the panel for the prediction of mVI were further assessed in virtual biopsies and in the validation set of CNB. RESULTS: In virtual biopsies, PIVKA-II/H4K16ac had the best performance for prediction of mVI, with sensitivity, specificity, predictive positive value (PPV), and predictive negative value (PNV) of 30%, 97%, 91%, 56%, respectively. In CNB, PIVKA-II/H4K20me2 showed the best accuracy for prediction of mVI, with sensitivity, specificity, PPV, and NPV of 43%, 95%, 90%, and 62%, respectively. The two panels were independent predictive factors of mVI (PIVKA-II/H4K16ac, P = .037; PIVKA-II/H4K20me2, P = .026). CONCLUSION: This study shows that a panel of two markers is able to predict mVI in HCC CNB, and pave the way for the future development of prognostic biomarkers in HCC that could guide the therapeutic strategy.

Dynamic Control of X Chromosome Conformation and Repression by a Histone H4K20 Demethylase.

Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We have discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C. elegans dosage compensation and demonstrated H4K20me1's pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. The structure and the activity of the dosage compensation complex (DCC) subunit DPY-21 define a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminates H4K20me1 enrichment in somatic cells, elevates X-linked gene expression, reduces X chromosome compaction, and disrupts X chromosome conformation by diminishing the formation of topologically associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression.