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

<div><p>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 Hat1^−/− neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw. Hat1^−/− mouse embryonic fibroblasts (MEFs) are defective in cell proliferation and are sensitive to DNA damaging agents. In addition, the Hat1^−/− 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.</p></div

Hat1 is essential for the acetylation of newly synthesized histones.

<p>Hat1^+/+ and Hat1^−/− MEFs were pulse-labeled with ³H-lysine for 12 minutes. Histones were then isolated and resolved by Acid-Urea (AU) gel electrophoresis. Total protein was visualized with Coomassie blue staining and radio-labeled proteins visualized by fluorography (as indicated). The mobility of each histone is indicated. The brackets indicate the regions of mobility for the acetylated isoforms of histone H4 and histone H3.</p

Developmental lung defects result in neonatal death in the absence of Hat1.

<p>A) Histologic appearance of lungs from newborn pups obtained from a Hat1^+/−+ and Hat1^−/− mice. Staining was with hematoxylin-eosin; magnification 20×, (inlets ×40). The lungs of Hat1^−/− show less aeration, due to thickened mesenchyme resulting in death due to respiratory failure. B) Hat1 is highly expressed in lungs of Hat1^+/+ but not in Hat1^−/− mice; magnification 20×, (inlets ×40) C) Cleaved Caspase3 stained by IHC showed no difference between lungs of Hat1⁺⁺ and Hat1^−/− mice; magnification 20×, (inlets ×40) D) Ki67 stained by IHC shows significantly higher proliferation rates in lungs of Hat1^−/− mice compared to controls; magnification 20×, (inlets ×40). Quantification was done by HistoQuest software.</p

Hat1 is essential for maintaining genome stability.

<p>A) Hat1^+/+ and Hat1^−/− MEFs were stained with either DAPI or α-γH2AX antibodies as indicated (left). Visible γH2AX foci were counted in 12 cells of each genotype (right). B) Metaphase spreads from Hat1^+/+ and Hat1^−/− MEFs were analyzed for chromosome number and the presence of breaks and fusions. The percentage of cells containing the indicated chromosomal abnormality is given. The number of spreads analyzed was 56 (Hat1^+/+) and 112 (Hat1^−/−). C) Metaphase spreads were generated from Hat1^+/+ and Hat1^−/− MEFs. Insets show enlarged views of selected abnormal chromosomes. Red arrows indicate examples of chromosome fusions and blue arrows indicate chromosome breaks.</p

Skeletal defects associated with the loss of Hat1.

<p>A) Micro CT scans of the heads of neonates with the indicated genotype. Top row shows a dorsal view and bottom row shows a ventral view. Arrows indicate defects in the nasal cavity (top) and jaw structures (bottom). B) Micro CT scans of Hat1^+/+ and Hat1^−/− neonates (as indicated) showing a dorsal view of the entire animal. C) Acian blue and Alizarin red stained Hat1^+/+ and Hat1^−/− neonates. D) Hat1^+/+ and Hat1^−/− embryos (12.5 dpc) were stained with α-Hat1 antibodies. E) Cross section of the head and neck of 11.5 dpc Hat1^+/+ and (WT) and Hat1^−/− embryos stained with α-Hat1 antibody. 2.5× magnification.</p

Hat1 is required for the acetylation of histones H3 and H4 deposited during replication-coupled chromatin assembly.

<p>A and B) Hat1^+/+ and Hat1^−/− MEFs were pulse-labeled with EdU and chased with thymidine as schematically depicted at the top of each panel. Samples were isolated at the indicated time points and proteins associated with nascent DNA were resolved by SDS-PAGE following affinity purification of EdU-labeled DNA (iPond). The indicated amounts of the input fractions (prior to affinity purification) are on the left-hand side of each panel. Samples eluted from the affinity purification resin are on the right-hand side of each panel. In all cases, the respective Hat1^+/+ and Hat1^−/− samples were analyzed on the same gel (separated by a MW standard lane that has been removed). Western blots were probed with the antibodies indicated on the right. Numbers below each lane indicate the normalized intensity of the band as determined using Licor software. Each sample was normalized to the level of unmodified histone H3. The intensity observed in the 30′ pulse sample was arbitrarily set to 1.0. No Clck indicates control samples that were not biotin labeled.</p

Hat1^−/− MEFs display cell proliferation and DNA damage repair defects.

<p>A) Hat1^+/+, Hat1^+/− and Hat1^−/− primary MEFs were genotyped by PCR as described in the legend to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003518#pgen-1003518-g001" target="_blank">Figure 1</a>. Whole cell extracts from the indicated MEFs were analyzed by Western blots probed with the indicated antibodies. B) Equal numbers of primary MEFs of the indicated genotype were seeded at time zero. Cell numbers were counted at the indicated time points. C) Primary Hat1^+/+ and Hat1^−/− MEFs were stained with propidium iodide and analyzed by FACS. Fraction of cells in each phase of the cell cycle is indicated. D) Immortalized Hat1^+/+ and Hat1^−/− MEFs were grown under the indicated conditions. Plates were photographed after crystal violet staining.</p

An HDAC3 selective inhibitor triggers apoptosis associated with increased DNA damage and cell cycle defects.

<p>(A) Hut78 cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and apoptosis assessed by Annexin V staining and flow cytometry. Cells were also labeled with propidium iodide to assess DNA content. Untreated (UT) and DMSO treated cells were used as controls. Shown is a representative graph from an experiment performed in duplicate that is consistent with other biological replicates. (B) Western blot analysis of γH2aX levels in Hut78 cells treated with DMSO, 10 nM Depsi, 10 µM 233, or 10 µM 966 for 8 hrs. Untreated and DMSO treated cells were used as controls. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side by side comparison of DMSO and Depsipeptide. (C) Cell cycle status was analyzed using BrdU incorporation and propidium iodide to assess DNA content by flow cytometry. Hut78 cells were treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 for 24 hr and pulsed for an hour and a half with BrdU prior to cell harvest and analysis. Shown are representative flow cytometry plots from an experiment performed in duplicate that is consistent with other biological replicates. (D) Graphical representation of BrdU incorporation from the experiment described in (C). (E) Graphical representation of the percent of S phase cells that did not incorporate BrdU (shown by box in panel (C)). Statistical analysis for both the Annexin V and BrdU experiments was performed using a two-tail T-test and comparing the HDI treated cells to the DMSO treated cells resulting in the following p-values: (A) Depsi: p = 0.0002, 233: p = 0.003, and 966: p = 0.0003. (D) Depsi: p = 0.003, 233: p = 0.01, and 966: p = 0.08. (E) Depsi: p = 0.003, 233: p = 0.003, and 966: p = 0.004.</p

iPOND analysis reveals HDAC3 association with replication forks in Hut78 CTCL cells.

<p>Hut78 cells were pulsed for 15 minutes with EdU followed by either no thymidine chase or a 60 minute thymidine chase. The protein-DNA complexes were then cross-linked, nascent DNA was conjugated to biotin using click chemistry, and then protein-DNA complexes were purified using Streptavidin beads. The eluted proteins were then analyzed using western blot analysis. A no click reaction sample (No Clk) that did not include biotin azide was used as a negative control. 0.1% input samples were included for positive controls of each protein analyzed. PCNA served as a positive control for a replication fork bound protein and H2B served as a loading control and positive control for a chromatin bound protein.</p

HDIs show selective inhibition of HDACs in CTCL cell lines.

<p>(A)Western blot analysis of whole cell lysates from Wild-type (WT) and <i>Hdac3</i>-null livers. Histones H3 and H4 served as loading controls. (B) Upper Panel: Western blot analysis of NIH 3T3 cells following treatment with various HDIs (indicated above each lane). Anti-histone H3 was used as a loading control. Lower panel: Western blot analysis of NIH 3T3 cells treated with either Trichostatin A (TSA) (1 µM), sodium butyrate (NaB) (5 mM), or increasing concentrations of nicotinamide (mM). (C) Western blot analysis of whole cell lysates prepared from cells that were transfected with either non-targeting siRNAs (NT) or siRNAs directed to the indicated Hdacs. (D) Western blot analysis of H3K56ac using whole cell lysates prepared from cells treated with the indicated amounts of RGFP966 for 24 hr. (E & F) Western blot analysis of (E) HH or (F) Hut78 cell lines treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, 10 µM 136, or 10 µM 966. Cells were treated for 24 hr and then harvested for protein isolation. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side-by-side comparison of DMSO and Depsipeptide. Histones H3 and H4 were used as loading controls.</p