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Functionally Distinct Roles for Replication-Dependent Histone H2A Isoforms in Cell Proliferation and Carcinogenesis Rajbir Singh1, Amir Mortazavi2, Kelly H

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Functionally Distinct Roles for Replication-Dependent Histone H2A Isoforms in Cell Proliferation and Carcinogenesis Rajbir Singh1, Amir Mortazavi2, Kelly H 9284–9295 Nucleic Acids Research, 2013, Vol. 41, No. 20 Published online 16 August 2013 doi:10.1093/nar/gkt736 NAR Breakthrough Article Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis Rajbir Singh1, Amir Mortazavi2, Kelly H. Telu3, Prabakaran Nagarajan1, David M. Lucas2, Jennifer M. Thomas-Ahner2, Steven K. Clinton2, John C. Byrd2, Michael A. Freitas4 and Mark R. Parthun1,* 1Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210, USA, 2Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA, 3Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA, 4Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA Received June 21, 2013; Revised July 22, 2013; Accepted July 25, 2013 ABSTRACT nucleosome consists of 147 base pairs of DNA wrapped around a protein complex called the histone Replication-dependent histones are encoded by octamer that contains two molecules of each of the four multigene families found in several large clusters in core histones (H2A, H2B, H3 and H4). The importance of the human genome and are thought to be functionally chromatin structure for the packaging and regulation of redundant. However, the abundance of specific rep- eukaryotic genomes is evidenced by the extraordinary con- lication-dependent isoforms of histone H2A is altered servation of this structure throughout eukaryotic evolu- in patients with chronic lymphocytic leukemia. Similar tion. The core histones are among the most highly changes in the abundance of H2A isoforms are also conserved eukaryotic proteins with many residues being associated with the proliferation and tumorigenicity completely invariant (1). However, despite this seeming of bladder cancer cells. To determine whether these uniformity, one of the most important characteristics of H2A isoforms can perform distinct functions, expres- chromatin structure is complexity, which is necessary for encoding all of the regulatory information necessary for sion of several H2A isoforms was reduced by siRNA the proper execution of nuclear processes and for epigen- knockdown. Reduced expression of the HIST1H2AC etic inheritance. locus leads to increased rates of cell proliferation and The complexity of chromatin is derived from two main tumorigenicity. We also observe that regulation of sources; the post-translational modification of histones replication-dependent histone H2A expression can and the presence of histone variants. Histones are occur on a gene-specific level. Specific replication- subject to multiple forms of post-translational modifica- dependent histone H2A genes are either up- or tion (2). Further complexity is derived from the fact that downregulated in chronic lymphocytic leukemia the cellular complement of most histones is not homoge- tumor tissue samples. In addition, discreet elements neous but, rather, is composed of multiple primary are identified in the 50 untranslated region of the sequence variants (3–5). HIST1H2AC locus that confer translational repres- Histone variants can be distinguished on a number of levels. The first is the distinction between replication- sion. Taken together, these results indicate that rep- dependent and replication-independent histones. lication-dependent histone isoforms can possess Replication-dependent histones become highly expressed distinct cellular functions and that regulation of just before S-phase and are then repressed at the comple- these isoforms may play a role in carcinogenesis. tion of DNA replication (6). Interestingly, the DNA replication-dependent histone genes are found in several large clusters that contain dozens of histone genes, and INTRODUCTION they are the only protein-coding mRNAs produced in At the most fundamental level, chromatin is composed of mammalian cells that lack a poly(A) tail. Instead of a a repeated structure known as the nucleosome. Each poly(A) tail, these messages contain a short highly *To whom correspondence should be addressed. Tel: +1 614 292 6215; Fax: +1 614 292 4118; Email: [email protected] ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2013, Vol. 41, No. 20 9285 Replication-Dependent Histone H2A Genes and Proteins A B H2A Gene Protein Molecular H2A 1C Name(s) Name Weight H2A 1B/E HIST1H2AH H2A 1H 13,817 (13671) HIST1H2AJ H2A 1J 13,847 (4727) HIST2H2AC H2A 2C 13,899 (4738) HIST2H2AB H2A 2B 13,906 (20560) HIST1H2AG/I/K/L/M H2A 14,002 (4737/4725/4726/4730 /4735) HIST2H2AA H2A 2A 14,006 (4736) HIST1H2AC H2A 1C 14,016 (4733) HIST1H2AD H2A 1D 14,018 (4729) HIST3H2A H2A 3A 14,032 (20507) HIST1H2AB/E H2A 1B/E 14,046 (4734/4724) HIST1H2AA H2A 1A 14,102 (4724) Figure 1. Replication-dependent histone H2A isoforms are altered in bladder cancer cells. (A) The table lists the genes encoding replication- dependent histone H2A isoforms along with nomenclature used to name their respective proteins and the molecular weight of each isoform. The Hugo Gene Naming Consortium (HGNC) identification number is given in parentheses. (B) LC/MS analysis of histone H2A isolated from the indicated cells. Normal bladder is normal human bladder epithelial cells; hTERT is immortalized human bladder epithelial cells; RT4 is non-invasive bladder cancer cells; and T24 is invasive bladder cancer cells. conserved stem–loop structure in their 30 untranslated actually a wide range of primary sequence variations region (UTR), and their processing and stability are within this group (8). To distinguish these histone regulated by the stem–loop binding protein, which variants from the replication-independent histone specifically interacts with this structure (7,8). The DNA variants, they will be referred to here as histone replication-dependent histones are used for the assembly isoforms. Each of the replication-dependent histones is of chromatin structure during DNA replication. Hence, encoded by multiple genes (H2A, 16 genes; H2B, 22 the packaging of genomic DNA with the DNA replica- genes; H3, 14 genes; H4, 14 genes; and H1, 6 genes). tion-dependent histones is the ‘ground state’ at which Hence, the presence of distinct replication-dependent chromatin structure begins. histone isoforms has the potential to significantly expand There are also a large number of core histone genes that the complexity of mammalian chromatin structure. are constitutively expressed throughout the cell cycle and, However, these core histone isoforms have not been hence, are known as replication-independent histone studied in detail, as they have been presumed to encode variants. The replication-independent histones differ in functionally equivalent molecules. primary sequence from the replication-dependent Mass spectrometry-based analysis of the histone H2A histones with these variations ranging from only a complement in HeLa cells indicated that the most handful of amino acid changes to the incorporation of abundant replication-dependent isoforms were the large non-histone domains. Well-characterized examples products of several specific genes (9). The most of replication-independent histone variants include abundant form is encoded by five distinct genes (HIST histones H3.3, H2AX, H2AZ and macroH2A (5). In 1H2AG, HIST1H2AI, HIST1H2AK, HIST1H2AL and addition to changes in protein sequence, the replication- HIST1H2A, see Figure 1A). The second most abundant independent histone genes also differ from their replica- species is encoded by a single gene, HIST1H2AC, and the tion-dependent counterparts in that they are found as third most abundant species is encoded by two genes, single genes dispersed throughout the genome, and they HIST1H2AB and HIST1H2AE. The nomenclature that generate transcripts with normal poly(A) tails. has developed to describe these histone variants has Although the replication-dependent core histones not systematically addressed the naming of these are considered to be the ‘canonical’ histones, there is replication-dependent histone isoforms (8,10). Therefore, 9286 Nucleic Acids Research, 2013, Vol. 41, No. 20 they will be referred to using a nomenclature that is based Grand Island, NY); all supplemented with 10% fetal on the more systematic naming of the genes that encode bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine, these proteins. The names of the replication-dependent and penicillin (10 U/ml) and streptomycin (10 mg/ml) histone genes provide important information about the (GIBCO, Grand Island, NY) and maintained at 37Cin location of the gene. The first part of the gene name a5%CO2/95% air, humidified atmosphere. hTERT and refers to the histone gene cluster in which it resides (i.e. normal human bladder epithelial cells were grown in HIST1, HIST2 or HIST3). The next part of the name Keratinocyte Serum Free Medium (GIBCO, Grand refers to the specific type of histone encoded by the gene Island, NY) with supplements of bovine pituitary extract (i.e. H1, H2A, H2B, H3 or H4). Finally, the multiple and epidermal growth factor plus 30 ng/ml Cholera toxin copies of each histone type are designated alphabetically (Sigma Chemical Company, St. Louis, MO). based
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