Mapping the electrostatic potential of the nucleosome acidic patch

The nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. We here set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy. We present a refined solution structure of the H2A-H2B dimer based on intermolecular distance restraints, displaying a well-defined histone-fold core. We show that the conserved histidines H2B H46 and H106 that line the acidic patch have pKa of 5.9 and 6.5, respectively, and that most acidic patch carboxyl groups have pKa values well below 5.0. For H2A D89 we find strong evidence for an elevated pKa of 5.3. Our data establish that the acidic patch is highly negatively charged at physiological pH, while protonation of H2B H106 and H2B H46 at slightly acidic pH will reduce electronegativity. These results will be valuable to understand the impact of pH changes on nucleosome-protein interactions in vitro, in silico or in vivo.

Collateral Victim or Rescue Worker?—The Role of Histone Methyltransferases in DNA Damage Repair and Their Targeting for Therapeutic Opportunities in Cancer

Disrupted DNA damage signaling greatly threatens cell integrity and plays significant roles in cancer. With recent advances in understanding the human genome and gene regulation in the context of DNA damage, chromatin biology, specifically biology of histone post-translational modifications (PTMs), has emerged as a popular field of study with great promise for cancer therapeutics. Here, we discuss how key histone methylation pathways contribute to DNA damage repair and impact tumorigenesis within this context, as well as the potential for their targeting as part of therapeutic strategies in cancer.

Subtelomeric Chromatin in the Fission Yeast S. pombe

Telomeres play important roles in safeguarding the genome. The specialized repressive chromatin that assembles at telomeres and subtelomeric domains is key to this protective role. However, in many organisms, the repetitive nature of telomeric and subtelomeric sequences has hindered research efforts. The fission yeast S. pombe has provided an important model system for dissection of chromatin biology due to the relative ease of genetic manipulation and strong conservation of important regulatory proteins with higher eukaryotes. Telomeres and the telomere-binding shelterin complex are highly conserved with mammals, as is the assembly of constitutive heterochromatin at subtelomeres. In this review, we seek to summarize recent work detailing the assembly of distinct chromatin structures within subtelomeric domains in fission yeast. These include the heterochromatic SH subtelomeric domains, the telomere-associated sequences (TAS), and ST chromatin domains that assemble highly condensed chromatin clusters called knobs. Specifically, we review new insights into the sequence of subtelomeric domains, the distinct types of chromatin that assemble on these sequences and how histone H3 K36 modifications influence these chromatin structures. We address the interplay between the subdomains of chromatin structure and how subtelomeric chromatin is influenced by both the telomere-bound shelterin complexes and by euchromatic chromatin regulators internal to the subtelomeric domain. Finally, we demonstrate that telomere clustering, which is mediated via the condensed ST chromatin knob domains, does not depend on knob assembly within these domains but on Set2, which mediates H3K36 methylation.

Functional analysis of chromatin assembly genes in tetrahymena thermophila

The basic structural unit of chromatin is the nucleosome composed of ~147 base pairs of DNA wrapped around an octamer of histone proteins. Post-translational modifications such as histone acetylation or the substitution of histone variants in place of core histones have been implicated in various chromatin related processes. There are two distinct chromatin assembly pathways. Replication-dependent mediated by CAF-1 (H3-H4) and replication-independent mediated by HIRA (H3.3-H4). Miss-regulation of chromatin assembly patterns result in the onset of many disease states such as cancer. Tetrahymena thermophila is a useful model for understanding basic questions in chromatin biology due to the segregation of transcriptionally active and silent chromatin into two distinct nuclei. To better characterize replication-dependent and independent chromatin assembly pathways in T. thermophila, I have engineered somatic knockouts (HIRA, CAC2, UBN1 and UBN2) and initiated the functional analysis of these chromatin assembly genes mediated in growth and development. The absence of CAC2 results in larger macronuclei and speculated to be a result of reduced histone H3-H4 deposition onto chromatin during growth.

Functional analysis of chromatin assembly genes in tetrahymena thermophila

The basic structural unit of chromatin is the nucleosome composed of ~147 base pairs of DNA wrapped around an octamer of histone proteins. Post-translational modifications such as histone acetylation or the substitution of histone variants in place of core histones have been implicated in various chromatin related processes. There are two distinct chromatin assembly pathways. Replication-dependent mediated by CAF-1 (H3-H4) and replication-independent mediated by HIRA (H3.3-H4). Miss-regulation of chromatin assembly patterns result in the onset of many disease states such as cancer. Tetrahymena thermophila is a useful model for understanding basic questions in chromatin biology due to the segregation of transcriptionally active and silent chromatin into two distinct nuclei. To better characterize replication-dependent and independent chromatin assembly pathways in T. thermophila, I have engineered somatic knockouts (HIRA, CAC2, UBN1 and UBN2) and initiated the functional analysis of these chromatin assembly genes mediated in growth and development. The absence of CAC2 results in larger macronuclei and speculated to be a result of reduced histone H3-H4 deposition onto chromatin during growth.

Evolution of Epigenome as the Blueprint for Carcinogenesis

Epigenetics “above or over genetics” is the term used for processes that result in modifications which are stably inherited through cell generations, without changing the underlying DNA sequence of the cell. These include DNA methylation, Post-translational histone modification and non-coding RNAs. Over the last two decades, interest in the field of epigenetics has grown manifold because of the realization of its involvement in key cellular and pathological processes beyond what was initially anticipated. Epigenetics and chromatin biology have been underscored to play key roles in diseases like cancer. The landscape of different epigenetic signatures can vary considerably from one cancer type to another, and even from one ethnic group to another in the case of same cancer. This chapter discusses the emerging role of epigenetics and chromatin biology in the field of cancer research. It discusses about the different forms of epigenetic mechanisms and their respective role in carcinogenesis in the light of emerging research.

From bedside to bench: regulation of host factors in SARS-CoV-2 infection

The zoonotic coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), which causes COVID-19 (coronavirus disease-2019), has resulted in a pandemic. This has led to an urgent need to understand the molecular determinants of SARS-CoV-2 infection, factors associated with COVID-19 heterogeneity and severity, and therapeutic options for these patients. In this review, we discuss the role of host factors in SARS-CoV-2 infection and describe variations in host factor expression as mechanisms underlying the symptoms and severity of COVID-19. We focus on two host factors, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), implicated in SARS-CoV-2 infection. We also discuss genetic variants associated with COVID-19 severity revealed in selected patients and based on genome-wide association studies (GWASs). Furthermore, we highlight important advances in cell and chromatin biology, such as single-cell RNA and chromatin sequencing and chromosomal conformation assays, as methods that may aid in the discovery of viral–host interactions in COVID-19. Understanding how regulation of host factor genes varies in physiological and pathological states might explain the heterogeneity observed in SARS-CoV-2 infection, help identify pathways for therapeutic development, and identify patients most likely to progress to severe COVID-19.

REMY: A platform for the rapid interrogation of epigenome modifications on yeast

Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here we describe a versatile and scalable platform termed Rapid interrogation of Epigenome Modifications using Yeast surface display (REMY), which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first used REMY to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, we used REMY to screen a large panel of commercially available anti-acetylation antibodies for their specificities, identifying many suitable and unsuitable reagents. Further, use of REMY enabled efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4, and uncovered previously unreported residue interdependencies affecting p300 activity. Our results show that REMY is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications.