Mark R. Parthun

The Ohio State University

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Michael A. Freitas

The Ohio State University

Prabakaran Nagarajan

The Ohio State University

Guido Marcucci

City of Hope

David Lucas

Centre Hospitalier Régional Universitaire de Brest

Paula A. Agudelo Garcia

The Ohio State University

Melanie E. Davis

Washington University Medical Center

Michael R. Grever

The Ohio State University

Liwen Zhang

Huazhong Agricultural University

Liudmila V. Popova

The Ohio State University

Amy R. Knapp

The Ohio State University

Cooperative Institutions

The Ohio State University

133

The Ohio State University Wexner Medical Center

The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute

Ohio University

Cancer Genetics (United States)

Comprehensive Blood & Cancer Center

Harvard University

National Cancer Institute

Nationwide Children's Hospital

University of Washington

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Discovery of Histone Modification Crosstalk Networks by Stable Isotope Labeling of Amino Acids in Cell Culture Mass Spectrometry (SILAC MS)

Molecular & Cellular Proteomics (2013)

Xiaoyan Guan Neha Rastogi Mark R. Parthun Michael A. Freitas

In this paper we describe an approach that combines stable isotope labeling of amino acids in cells culture, high mass accuracy liquid chromatography tandem mass spectrometry and a novel data analysis approach to accurately determine relative peptide post-translational modification levels. This paper describes the application of this approach to the discovery of novel histone modification crosstalk networks in Saccharomyces cerevisiae. Yeast histone mutants were generated to mimic the presence/absence of 44 well-known modifications on core histones H2A, H2B, H3, and H4. In each mutant strain the relative change in H3 K79 methylation and H3 K56 acetylation were determined using stable isotope labeling of amino acids in cells culture. This approach showed relative changes in H3 K79 methylation and H3 K56 acetylation that are consistent with known histone crosstalk networks. More importantly, this study revealed additional histone modification sites that affect H3 K79 methylation and H3 K56 acetylation.

Crosstalk

10.1074/mcp.m112.026716

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Citations (33)

Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions

bioRxiv (Cold Spring Harbor Laboratory) (2019)

Paula A. Agudelo Garcia Prabakaran Nagarajan Mark R. Parthun

ABSTRACT Lysine acetylation has emerged as one of the most important post-translational modifications, regulating different biological processes. However, its regulation by lysine acetyltransferases is still unclear in most cases. Hat1 is a lysine acetyltransferase originally identified based on its ability to acetylate histones. Using an unbiased proteomics approach, we have determined how loss of Hat1 affects the mammalian acetylome. Hat1 +/+ and Hat1 −/− mouse embryonic fibroblast (MEF) cells lines were grown in both glucose- and galactose-containing media, as Hat1 is required for growth on galactose and Hat1 −/− cells exhibit defects in mitochondrial function. Following trypsin digestion of whole cell extracts, acetylated peptides were enriched by acetyllysine affinity purification and acetylated peptides were identified and analyzed by label-free quantitation. Comparison of the acetylome from Hat1 +/+ cells grown on galactose and glucose demonstrated that there are large carbon source-dependent changes in the mammalian acetylome where the acetylation of enzymes involved in glycolysis was the most affected. Comparisons of the acetylomes from Hat1 +/+ and Hat1 −/− cells identified 65 proteins whose acetylation decreased by at least 2.5-fold in cells lacking Hat1. In Hat1 −/− cells, acetylation of the auto regulatory loop of CBP was the most highly affected, decreasing by up to 20-fold. In addition to proteins involved in chromatin structure, Hat1-dependent acetylation was also found in a number of transcriptional regulators, including p53, and mitochondrial proteins. Hat1 mitochondrial localization suggests that it may be directly involved in the acetylation of mitochondrial proteins.

Acetyltransferases

P300-CBP Transcription Factors

10.1101/825539

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Citations (1)

Differential expression of histone post-translational modifications in acute myeloid and chronic lymphocytic leukemia determined by high-pressure liquid chromatography and mass spectrometry

Journal of the American Society for Mass Spectrometry (2003)

Liwen Zhang Michael A. Freitas Joseph Wickham Mark R. Parthun Marko I. Klisovic

The post-translational modification of the core histones is critical to the regulation of chromatin structure. Traditional methods for the determination of histone modification utilize immunoassay techniques to determine the extent and site of post-translational modification. These methods, though sensitive, require site-specific antibodies. This manuscript describes the application of reverse-phase high-pressure liquid chromatography and mass spectrometry (LC-MS) to analyze global modification levels of core histones. The method is fast, sensitive, and easily automated. Furthermore, the technique gives the global patterns of modification for all four core histones in a single experiment. The LC-MS method was optimized using histones extracted from bovine thymus. These methods were then applied to the characterization of changes in histone modification in acute myeloid leukemia (AML) cell lines treated with histone deacetylase (HDAC) inhibitors. Dose-dependent changes in the distribution of modified core histones were observed. These results were validated in primary leukemia cells from patients with refractory or relapsed AML or chronic lymphocytic leukemia (CLL) treated on a Phase I clinical trial of the HDAC inhibitor depsipeptide. An increase in the relative abundance of specific acetylated forms of histone H4 was readily observable in these patients at intervals of 4 and 24 h after treatment.

Histone deacetylase inhibitor

Histone H4

10.1016/j.jasms.2003.10.001

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Citations (59)

Expanded binding specificity of the human histone chaperone NASP

Nucleic Acids Research (2008)

Huanyu Wang Scott T.R. Walsh Mark R. Parthun

NASP (nuclear autoantigenic sperm protein) has been reported to be an H1-specific histone chaperone. However, NASP shares a high degree of sequence similarity with the N1/N2 family of proteins, whose members are H3/H4-specific histone chaperones. To resolve this paradox, we have performed a detailed and quantitative analysis of the binding specificity of human NASP. Our results confirm that NASP can interact with histone H1 and that this interaction occurs with high affinity. In addition, multiple in vitro and in vivo experiments, including native gel electrophoresis, traditional and affinity chromatography assays and surface plasmon resonance, all indicate that NASP also forms distinct, high specificity complexes with histones H3 and H4. The interaction between NASP and histones H3 and H4 is functional as NASP is active in in vitro chromatin assembly assays using histone substrates depleted of H1.

Chaperone (clinical)

Histone code

10.1093/nar/gkn574

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Citations (62)

The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell Malignancies In Vitro and In Vivo

PLoS ONE (2010)

David Lucas Lapo Alinari Derek A. West Melanie E. Davis Ryan B. Edwards

Background While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies. Principal Findings In mantle cell lymphoma (JeKo-1), Burkitt's lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC50 (50% growth inhibitory concentration) of AR-42 is 0.61 µM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC50 (concentration lethal to 50%) of AR-42 is 0.76 µM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity. Conclusions/Significance Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.

Vorinostat

10.1371/journal.pone.0010941

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Citations (53)

Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis

Nucleic Acids Research (2013)

Rajbir Singh Amir Mortazavi Kelly H. Telu Prabakaran Nagarajan David Lucas

Abstract Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5′ untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis.

Replication timing

10.1093/nar/gkt736

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Citations (31)

Isolation of Proteins on Nascent Chromatin and Characterization by Quantitative Mass Spectrometry

Methods in molecular biology (2019)

Paula A. Agudelo Garcia Miranda L. Gardner Michael A. Freitas Mark R. Parthun

Epigenome

Histone-modifying enzymes

Histone code

10.1007/978-1-4939-9434-2_2

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Citations (3)

Hat2p recognizes the histone H3 tail to specify the acetylation of the newly synthesized H3/H4 heterodimer by the Hat1p/Hat2p complex

Genes & Development (2014)

Yang Li Zhang Li Tingting Liu Chengliang Chai Qianglin Fang

Post-translational modifications of histones are significant regulators of replication, transcription, and DNA repair. Particularly, newly synthesized histone H4 in H3/H4 heterodimers becomes acetylated on N-terminal lysine residues prior to its incorporation into chromatin. Previous studies have established that the histone acetyltransferase (HAT) complex Hat1p/Hat2p medicates this modification. However, the mechanism of how Hat1p/Hat2p recognizes and facilitates the enzymatic activities on the newly assembled H3/H4 heterodimer remains unknown. Furthermore, Hat2p is a WD40 repeat protein, which is found in many histone modifier complexes. However, how the WD40 repeat proteins facilitate enzymatic activities of histone modification enzymes is unclear. In this study, we first solved the high-resolution crystal structure of a Hat1p/Hat2p/CoA/H4 peptide complex and found that the H4 tail interacts with both Hat1p and Hat2p, by which substrate recruitment is facilitated. We further discovered that H3 N-terminal peptides can bind to the Hat2p WD40 domain and solved the structure of the Hat1p/Hat2p/CoA/H4/H3 peptide complex. Moreover, the interaction with Hat2p requires unmodified Arg2/Lys4 and Lys9 on the H3 tail, suggesting a novel model to specify the activity of Hat1p/Hat2p toward newly synthesized H3/H4 heterodimers. Together, our study demonstrated the substrate recognition mechanism by the Hat1p/Hat2p complex, which is critical for DNA replication and other chromatin remodeling processes.

Histone H4

Histone code

Histone octamer

Histone acetyltransferase

10.1101/gad.240531.114

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Citations (36)

A Transcriptionally Active Form of GAL4 Is Phosphorylated and Associated with GAL80

Molecular and Cellular Biology (1992)

Mark R. Parthun Judith A. Jaehning

The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation.

Transcription

gal operon

10.1128/mcb.12.11.4981-4987.1992

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Citations (12)

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells

Leukemia (2004)

David Lucas Melanie E. Davis Mark R. Parthun Andrew P. Mone Shinichi Kitada

Histone deacetylase inhibitor

10.1038/sj.leu.2403388

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Citations (135)