Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

and the Processing of Newly Synthesized Histones H3 and H4. PLoS Genet 9(6): e1003518. doi:10.1371/journal.pgen.1003518 Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4 Prabakaran Nagarajan 0 Zhongqi Ge 0 Bianca Sirbu 0 Cheryl Doughty 0 Paula A. Agudelo Garcia 0 Michaela Schlederer 0 Anthony T. Annunziato 0 David Cortez 0 Lukas Kenner 0 Mark R. Parthun 0 Paul D. Kaufman, University of Massachusetts Medical School, United States of America 0 1 Department of Molecular and Cellular Biochemistry, The Ohio State University , Columbus , Ohio, United States of America, 2 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 3 Department of Biology, Boston College , Chestnut Hill, Massachusetts , United States of America, 4 Ludwig Boltzmann Institute for Cancer Research (LBI-CR) , Vienna , Austria , 5 Clinical Institute of Pathology, Medical University of Vienna , Vienna , Austria 1 www.plosgenetics.org Histone acetyltransferase 1 is an evolutionarily conserved type B histone acetyltransferase that is thought to be responsible for the diacetylation of newly synthesized histone H4 on lysines 5 and 12 during chromatin assembly. To understand the function of this enzyme in a complex organism, we have constructed a conditional mouse knockout model of Hat1. Murine Hat1 is essential for viability, as homozygous deletion of Hat1 results in neonatal lethality. The lungs of embryos and pups genetically deficient in Hat1 were much less mature upon histological evaluation. The neonatal lethality is due to severe defects in lung development that result in less aeration and respiratory distress. Many of the Hat12/2 neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw. Hat12/2 mouse embryonic fibroblasts (MEFs) are defective in cell proliferation and are sensitive to DNA damaging agents. In addition, the Hat12/2 MEFs display a marked increase in genome instability. Analysis of histone dynamics at sites of replication-coupled chromatin assembly demonstrates that Hat1 is not only responsible for the acetylation of newly synthesized histone H4 but is also required to maintain the acetylation of histone H3 on lysines 9, 18, and 27 during replication-coupled chromatin assembly. - Funding: This work was supported by grants from the National Institutes of Health (GM62970 to MRP, CA136933 to DC) and by a Pelotonia Predoctoral Fellowship from the Ohio State University Comprehensive Cancer Center (to ZG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The packaging of genomic DNA during replication is a highly orchestrated process that ensures both the necessary compaction of the DNA and the proper transmission of the epigenetic landscape [1,2,3,4,5]. An important aspect of chromatin assembly is the processing of newly synthesized histones for their incorporation into chromatin. The transient acetylation of histone H3 and H4 NH2-terminal tails is a hallmark of this processing. Newly synthesized molecules of histone H4 are predominantly diacetylated. This diacetylation occurs specifically on lysine residues 5 and 12 and this precise pattern is widely conserved throughout eukaryotic evolution. The acetylation of histone H3 occurs on a smaller fraction of the newly synthesized molecules and does not occur in a consistent pattern across eukaryotes. A role for this acetylation in histone deposition was first suggested by the correlation between the presence of these histone marks and active chromatin assembly as H3 and H4 are rapidly modified after their synthesis and then deacetylated following their incorporation into chromatin [6]. However, despite this longstanding correlation, an understanding of the function of histone NH2-terminal tail domain acetylation in chromatin assembly remains elusive. In addition to their NH2-terminal tail domains, evidence from S. cerevisiae indicates that newly synthesized histones are also acetylated in their core domains with H3 acetylated on lysine 56 and H4 acetylated on lysine 91 [7,8,9,10]. H3 lysine 56 lies near the entry/exit point of the nucleosome in close proximity to the DNA. The acetylation of this site occurs specifically in S phase and has been linked to chromatin assembly by a number of observations. First, mutations in yeast that alter H3 lysine 56 cause defects in the reassembly of chromatin structure that accompanies the recombinational repair of a DNA double strand break. Second, H3 lysine 56 mutations influence the binding of histone H3 to the CAF-1 histone chaperone complex that plays a key role in replication coupled chromatin assembly [7,11,12,13, 14,15,16,17]. Histone H4 lysine 91 lies in the interface between H3/H4 tetramers and H2A/H2B dimers where it forms a salt bridge with an aspartic acid residue in histone H2B. Hence, the acetylation of H4 lysine 91 may regulate tetramer-dimer interactions and genetic results are consistent with a role for this modification in chromatin assembly [10,18]. Enzymes known as type B histone acetyltransferases (HATs) catalyze the acetylation of newly synthesized histones. Type B HATs are primarily distinguished from type A HATs by their substrate specificity. As expected for enzymes that modify histones The packaging of genomic DNA during replication is a highly orchestrated process. An important aspect of chromatin assembly is the processing of newly synthesized histones prior to their incorporation into chromatin. The transient acetylation of histone H3 and H4 NH2-terminal tails is a hallmark of this processing with newly synthesized molecules of histone H4 being predominantly diacetylated. This diacetylation occurs specifically on lysine residues 5 and 12 and this precise pattern is widely conserved throughout eukaryotic evolution. The acetylation of newly synthesized histones is catalyzed by type B histone acetyltransferases. Hat1 is the founding member of this class of enzymes and has been proposed to be responsible for the diacetylation of newly synthesized histone H4. Here we describe the development of a mouse knockout model of Hat1. The absence of Hat1 results in neonatal lethality due to developmental defects in the lung. Mouse embryonic fibroblasts derived from Hat12/2 mice are sensitive to DNA damaging agents and display a high level of genome instability. Biochemical analyses provide definitive evidence that Hat1 is the sole enzyme responsible for the acetylation of newly synthesized histone H4. Surprisingly, Hat1 is also necessary for the normal processing of newly synthesized histone H3. prior to their assembly into chromatin, type B HATs are highly specific for free histones. Type B HATs may also function outside of t (...truncated)