Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1.

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.

Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease.

Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have previously reported histone H3 specific proteolytic clipping and a protein inhibitor in chicken liver. However, the sites of clipping are still not known very well. In this study, we attempt to identify clipping sites in histone H3 and to determine the mechanism of inhibition by stefin B protein, a cysteine protease inhibitor. By employing site-directed mutagenesis and in vitro biochemical assays, we have identified three distinct clipping sites in recombinant human histone H3 and its variants (H3.1, H3.3, and H3t). However, post-translationally modified histones isolated from chicken liver and Saccharomyces cerevisiae wild-type cells showed different clipping patterns. Clipping of histone H3 N-terminal tail at three sites occurs in a sequential manner. We have further observed that clipping sites are regulated by the structure of the N-terminal tail as well as the globular domain of histone H3. We also have identified the QVVAG region of stefin B protein to be very crucial for inhibition of the protease activity. Altogether, our comprehensive biochemical studies have revealed three distinct clipping sites in histone H3 and their regulation by the structure of histone H3, histone modifications marks, and stefin B.

Unexpected histone H3 tail-clipping activity of glutamate dehydrogenase.

Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro.

Identification of a novel histone H3 specific protease activity in nuclei of chicken liver.

Evolutionary conserved histone proteins play a very important role in the regulation of eukaryotic gene expression by undergoing post translational modifications within the tail regions. However, their role in tissue-specific gene expression and development remains unclear. In this study, we provide evidence for in vivo tissue-specific proteolytic cleavage of histone H3 in the liver of adult white Leghorn chickens, which we believe to be regulated by tissue-specific protease activity and epigenetic markers. The cleavage of histone H3 in the liver of adult chickens is very unique, and can serve as a model for studying tissue-specific changes in chromatin organization and gene expression. For the first time, we have identified and partially purified histone H3-specific protease activity that is distinct from histone H3 protease activities recently reported. Together, our data provide evidence of proteolytic processing and identification of protease activity that is specific to histone H3 in the liver of adult chickens, which may be involved in the regulation of gene expression during development, aging, and age-associated diseases.

Zinc ions prevent α-synuclein aggregation by enhancing chaperone function of human serum albumin.

Metal ions present in cellular microenvironment have been implicated as drivers of aggregation of amyloid forming proteins. Zinc (Zn2+) ions have been reported to directly interact with α-synuclein (AS), a causative agent of Parkinson's disease and other neurodegenerative diseases, and promote its aggregation. AS is a small intrinsically disordered protein (IDP) i.e., understanding molecular factors that drive its misfolding and aggregation has been challenging since methods used routinely to study protein structure are not effective for IDPs. Here, we report the atomic details of Zn2+ binding to AS at physiologically relevant conditions using proton-less NMR techniques that can be applied to highly dynamic systems like IDPs. We also examined how human serum albumin (HSA), the most abundant protein in human blood, binds to AS and whether Zn2+ and/or ionic strength affect this. We conclude that Zn2+ enhances the anti-aggregation chaperoning role of HSA that relies on protecting the hydrophobic N-terminal and NAC regions of AS, rather than polar negatively charged C-terminus. This suggested a previously undocumented role of Zn2+ in HSA function and AS aggregation.

Molecular basis of hUHRF1 allosteric activation for synergistic histone modification binding by PI5P.

Chromatin marks are recognized by distinct binding modules, many of which are embedded in multidomain proteins. How the different functionalities of such complex chromatin modulators are regulated is often unclear. Here, we delineated the interplay of the H3 amino terminus- and K9me-binding activities of the multidomain hUHRF1 protein. We show that the phosphoinositide PI5P interacts simultaneously with two distant flexible linker regions connecting distinct domains of hUHRF1. The binding is dependent on both, the polar head group, and the acyl part of the phospholipid and induces a conformational rearrangement juxtaposing the H3 amino terminus and K9me3 recognition modules of the protein. In consequence, the two features of the H3 tail are bound in a multivalent, synergistic manner. Our work highlights a previously unidentified molecular function for PI5P outside of the context of lipid mono- or bilayers and establishes a molecular paradigm for the allosteric regulation of complex, multidomain chromatin modulators by small cellular molecules.

H3 clipping activity of glutamate dehydrogenase is regulated by stefin B and chromatin structure.

Glutamate dehydrogenase has been recently identified as a tissue-specific histone H3-specific clipping enzyme. We have previously shown that it cleaves free as well as chromatin-bound histone H3. However, the physiological significance of this enzyme is still not clear. The present study aimed to improve our understanding of its significance in vivo. Using biochemical and cell biological approaches, we show that glutamate dehydrogenase is primarily associated with euchromatin, and it re-localizes from the nuclear periphery to the nucleolus upon DNA damage. The cysteine protease inhibitor stefin B regulates the H3 clipping activity of the enzyme. Chromatin structure and certain histone modifications influence H3 clipping activity. Interestingly, we also observed that an in vivo truncated form of H3 lacks H3K56 acetylation, which is a code for the DNA damage response. Together, these results suggest that glutamate dehydrogenase is a euchromatin-associated enzyme, and its H3 clipping activity is regulated by chromatin structure, histone modifications and an in vivo inhibitor. In response to DNA damage, it re-localizes to the nuclei, and hence may be involved in regulation of gene expression in vivo.

Anti-cancer drug KP1019 modulates epigenetics and induces DNA damage response in Saccharomyces cerevisiae.

KP1019 comprises a class of ruthenium compounds having promising anticancer activity. Here, we investigated the molecular targets of KP1019 using Saccharomyces cerevisiae as a model organism. Our results revealed that in the absence of the N-terminal tail of histone H3, the growth inhibitory effect of KP1019 was markedly enhanced. Furthermore, H3K56A or rtt109Δ mutants exhibit hypersensitivity for KP1019. Moreover, KP1019 evicts histones from the mononucleosome and interacts specifically with histone H3. We have also shown that KP1019 treatment causes induction of Ribonucleotide Reductase (RNR) genes and degradation of Sml1p. Our results also suggest that DNA damage induced by KP1019 is primarily repaired through double-strand break repair (DSBR). In summary, KP1019 targets histone proteins, with important consequences for DNA damage responses and epigenetics.

Ebselen induces reactive oxygen species (ROS)-mediated cytotoxicity in Saccharomyces cerevisiae with inhibition of glutamate dehydrogenase being a target.

Ebselen is a synthetic, lipid-soluble seleno-organic compound. The high electrophilicity of ebselen enables it to react with multiple cysteine residues of various proteins. Despite extensive research on ebselen, its target molecules and mechanism of action remains less understood. We performed biochemical as well as in vivo experiments employing budding yeast as a model organism to understand the mode of action of ebselen. The growth curve analysis and FACS (florescence activated cell sorting) assays revealed that ebselen exerts growth inhibitory effects on yeast cells by causing a delay in cell cycle progression. We observed that ebselen exposure causes an increase in intracellular ROS levels and mitochondrial membrane potential, and that these effects were reversed by addition of antioxidants such as reduced glutathione (GSH) or N-acetyl-l-cysteine (NAC). Interestingly, a significant increase in ROS levels was noticed in gdh3-deleted cells compared to wild-type cells. Furthermore, we showed that ebselen inhibits GDH function by interacting with its cysteine residues, leading to the formation of inactive hexameric GDH. Two-dimensional gel electrophoresis revealed protein targets of ebselen including CPR1, the yeast homolog of Cyclophilin A. Additionally, ebselen treatment leads to the inhibition of yeast sporulation. These results indicate a novel direct connection between ebselen and redox homeostasis.

Sen1p contributes to genomic integrity by regulating expression of ribonucleotide reductase 1 (RNR1) in Saccharomyces cerevisiae.

Gene expression is a multi-step process which requires recruitment of several factors to promoters. One of the factors, Sen1p is an RNA/DNA helicase implicated in transcriptional termination and RNA processing in yeast. In the present study, we have identified a novel function of Sen1p that regulates the expression of ribonucleotide reductase RNR1 gene, which is essential for maintaining genomic integrity. Cells with mutation in the helicase domain or lacking N-terminal domain of Sen1p displayed a drastic decrease in the basal level transcription of RNR1 gene and showed enhanced sensitivity to various DNA damaging agents. Moreover, SEN1 mutants [Sen1-1 (G1747D), Sen1-2 (Δ1-975)] exhibited defects in DNA damage checkpoint activation. Surprisingly, CRT1 deletion in Sen1p mutants (Sen1-1, Sen1-2) was partly able to rescue the slow growth phenotype upon genotoxic stress. Altogether, our observations suggest that Sen1p is required for cell protection against DNA damage by regulating the expression of DNA repair gene RNR1. Thus, the misregulation of Sen1p regulated genes can cause genomic instability that may lead to neurological disorders and premature aging.