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Mark R. Parthun - One of the best experts on this subject based on the ideXlab platform.
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Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability
The Journal of biological chemistry, 2020Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Liudmila V. Popova, Michael A. Freitas, Callie M Lovejoy, Dongju Park, Mark R. ParthunAbstract:The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.
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HAT1-Dependent Lysine Acetylation Targets Diverse Cellular Functions
2019Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Mark R. ParthunAbstract: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.
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Early-onset aging and mitochondrial defects associated with loss of histone acetyltransferase 1 (HAT1).
Aging cell, 2019Co-Authors: Prabakaran Nagarajan, Paula A. Agudelo Garcia, Chitra C. Iyer, Liudmila V. Popova, William Arnold, Mark R. ParthunAbstract:Histone acetyltransferase 1 (HAT1) is responsible for the acetylation of newly synthesized histone H4 on lysines 5 and 12 during the process of chromatin assembly. To understand the broader biological role of HAT1, we have generated a conditional mouse knockout model of this enzyme. We previously reported that HAT1 is required for viability and important for mammalian development and genome stability. In this study, we show that haploinsufficiency of HAT1 results in a significant decrease in lifespan. Defects observed in HAT1+/- mice are consistent with an early-onset aging phenotype. These include lordokyphosis (hunchback), muscle atrophy, minor growth retardation, reduced subcutaneous fat, cancer, and paralysis. In addition, the expression of HAT1 is linked to the normal aging process as HAT1 mRNA and protein becomes undetectable in many tissues in old mice. At the cellular level, fibroblasts from HAT1 haploinsufficient embryos undergo early senescence and accumulate high levels of p21. HAT1+/- mouse embryonic fibroblasts (MEFs) display modest increases in endogenous DNA damage but have significantly higher levels of reactive oxygen species (ROS). Consistently, further studies show that HAT1-/- MEFs exhibit mitochondrial defects suggesting a critical role for HAT1 in mitochondrial function. Taken together, these data show that loss of HAT1 induces multiple hallmarks of early-onset aging.
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Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly
Nucleic acids research, 2017Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Michael E. Hoover, Pei Zhang, Michael A. Freitas, Mark R. ParthunAbstract:Histone acetyltransferase 1 (HAT1) catalyzes the acetylation of newly synthesized histone H4 at lysines 5 and 12 that accompanies replication-coupled chromatin assembly. The acetylation of newly synthesized H4 occurs in the cytoplasm and the function of this acetylation is typically ascribed to roles in either histone nuclear import or deposition. Using cell lines from HAT1+/+ and HAT1-/- mouse embryos, we demonstrate that HAT1 is not required for either histone nuclear import or deposition. We employed quantitative proteomics to characterize HAT1-dependent changes in the composition of nascent chromatin structure. Among the proteins depleted from nascent chromatin isolated from HAT1-/- cells are several bromodomain-containing proteins, including Brg1, Baz1A and Brd3. Analysis of the binding specificity of their bromodomains suggests that HAT1-dependent acetylation of H4 is directly involved in their recruitment. HAT1-/- nascent chromatin is enriched for topoisomerase 2α and 2β. The enrichment of topoisomerase 2 is functionally relevant as HAT1-/- cells are hyper-sensitive to topoisomerase 2 inhibition suggesting that HAT1 is required for proper chromatin topology. In addition, our results indicate that HAT1 is transiently recruited to sites of chromatin assembly, dissociating prior to the maturation of chromatin structure.
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Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4
PLoS genetics, 2013Co-Authors: Prabakaran Nagarajan, Anthony T. Annunziato, Paula A. Agudelo Garcia, Bianca M. Sirbu, Cheryl Doughty, Michaela Schlederer, David Cortez, Lukas Kenner, Mark R. ParthunAbstract: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.
Paula A. Agudelo Garcia - One of the best experts on this subject based on the ideXlab platform.
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Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability
The Journal of biological chemistry, 2020Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Liudmila V. Popova, Michael A. Freitas, Callie M Lovejoy, Dongju Park, Mark R. ParthunAbstract:The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.
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Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability
2020Co-Authors: Paula A. Agudelo Garcia, Lovejoy, Nagarajan, Park, Popova, Freitas, ParthunAbstract:ABSTRACT The replisome functions in a dynamic environment that is at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the action of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12 that accompanies replication-coupled chromatin assembly. Analysis of the role of HAT1 in replication-coupled chromatin assembly demonstrates that HAT1 also physically associates with chromatin near sites of DNA replication. The association of HAT1 with newly replicated DNA is transient but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant as loss of HAT1 results in a decrease in replication fork progression and an increase in replication fork stalling. In addition, in the absence of HAT1, stalled replication forks are unstable and newly synthesized DNA becomes susceptible to Mre11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.
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HAT1-Dependent Lysine Acetylation Targets Diverse Cellular Functions
2019Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Mark R. ParthunAbstract: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.
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Early-onset aging and mitochondrial defects associated with loss of histone acetyltransferase 1 (HAT1).
Aging cell, 2019Co-Authors: Prabakaran Nagarajan, Paula A. Agudelo Garcia, Chitra C. Iyer, Liudmila V. Popova, William Arnold, Mark R. ParthunAbstract:Histone acetyltransferase 1 (HAT1) is responsible for the acetylation of newly synthesized histone H4 on lysines 5 and 12 during the process of chromatin assembly. To understand the broader biological role of HAT1, we have generated a conditional mouse knockout model of this enzyme. We previously reported that HAT1 is required for viability and important for mammalian development and genome stability. In this study, we show that haploinsufficiency of HAT1 results in a significant decrease in lifespan. Defects observed in HAT1+/- mice are consistent with an early-onset aging phenotype. These include lordokyphosis (hunchback), muscle atrophy, minor growth retardation, reduced subcutaneous fat, cancer, and paralysis. In addition, the expression of HAT1 is linked to the normal aging process as HAT1 mRNA and protein becomes undetectable in many tissues in old mice. At the cellular level, fibroblasts from HAT1 haploinsufficient embryos undergo early senescence and accumulate high levels of p21. HAT1+/- mouse embryonic fibroblasts (MEFs) display modest increases in endogenous DNA damage but have significantly higher levels of reactive oxygen species (ROS). Consistently, further studies show that HAT1-/- MEFs exhibit mitochondrial defects suggesting a critical role for HAT1 in mitochondrial function. Taken together, these data show that loss of HAT1 induces multiple hallmarks of early-onset aging.
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Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly
Nucleic acids research, 2017Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Michael E. Hoover, Pei Zhang, Michael A. Freitas, Mark R. ParthunAbstract:Histone acetyltransferase 1 (HAT1) catalyzes the acetylation of newly synthesized histone H4 at lysines 5 and 12 that accompanies replication-coupled chromatin assembly. The acetylation of newly synthesized H4 occurs in the cytoplasm and the function of this acetylation is typically ascribed to roles in either histone nuclear import or deposition. Using cell lines from HAT1+/+ and HAT1-/- mouse embryos, we demonstrate that HAT1 is not required for either histone nuclear import or deposition. We employed quantitative proteomics to characterize HAT1-dependent changes in the composition of nascent chromatin structure. Among the proteins depleted from nascent chromatin isolated from HAT1-/- cells are several bromodomain-containing proteins, including Brg1, Baz1A and Brd3. Analysis of the binding specificity of their bromodomains suggests that HAT1-dependent acetylation of H4 is directly involved in their recruitment. HAT1-/- nascent chromatin is enriched for topoisomerase 2α and 2β. The enrichment of topoisomerase 2 is functionally relevant as HAT1-/- cells are hyper-sensitive to topoisomerase 2 inhibition suggesting that HAT1 is required for proper chromatin topology. In addition, our results indicate that HAT1 is transiently recruited to sites of chromatin assembly, dissociating prior to the maturation of chromatin structure.
Anthony T. Annunziato - One of the best experts on this subject based on the ideXlab platform.
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Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4
PLoS genetics, 2013Co-Authors: Prabakaran Nagarajan, Anthony T. Annunziato, Paula A. Agudelo Garcia, Bianca M. Sirbu, Cheryl Doughty, Michaela Schlederer, David Cortez, Lukas Kenner, Mark R. ParthunAbstract: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.
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Schizosaccharomyces pombe HAT1 (Kat1) Is Associated with Mis16 and Is Required for Telomeric Silencing
Eukaryotic cell, 2012Co-Authors: Kevin Tong, Thomas Keller, Charles S. Hoffman, Anthony T. AnnunziatoAbstract:The HAT1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the HAT1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. HAT1 is found associated with the RbAp46 homologue Mis16, an essential protein. The HAT1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of HAT1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae HAT1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of HAT1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting HAT1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between HAT1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast.
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Properties of the type B histone acetyltransferase HAT1: H4 tail interaction, site preference, and involvement in DNA repair.
The Journal of biological chemistry, 2006Co-Authors: Laura J. Benson, Charles S. Hoffman, Jane A. Phillips, Mark R. Parthun, Anthony T. AnnunziatoAbstract:The HAT1 histone acetyltransferase catalyzes the acetylation of H4 at lysines 5 and 12, the same sites that are acetylated in newly synthesized histone H4. By performing histone acetyltransferase (HAT) assays on various synthetic H4 N-terminal peptides, we have examined the interactions between HAT1 and the H4 tail domain. It was found that acetylation requires the presence of positively charged amino acids at positions 8 and 16 of H4, positions that are normally occupied by lysine; however, lysine per se is not essential and can be replaced by arginine. In contrast, replacing Lys-8 and -16 of H4 with glutamines reduces acetylation to background levels. Similarly, phosphorylation of Ser-1 of the H4 tail depresses acetylation by both yeast HAT1p and the human HAT-B complex. These results strongly support the model proposed by Ramakrishnan and colleagues for the interaction between HAT1 and the H4 tail (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427-438) and may have implications for the genetic analysis of histone acetylation. It was also found that Lys-12 of H4 is preferentially acetylated by human HAT-B, in further agreement with the proposed model of H4 tail binding. Finally, we have demonstrated that deletion of the HAT1 gene from the fission yeast Schizosaccharomyces pombe causes increased sensitivity to the DNA-damaging agent methyl methanesulfonate in the absence of any additional mutations. This is in contrast to results obtained with a Saccharomyces cerevisiae HAT1Delta strain, which must also carry mutations of the acetylatable lysines of H3 for heightened methyl methanesulfonate sensitivity to be observed. Thus, although the role of HAT1 in DNA damage repair is evolutionarily conserved, the ability of H3 acetylation to compensate for HAT1 deletion appears to be more variable.
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Effects of acetylation of histone H4 at lysines 8 and 16 on activity of the HAT1 histone acetyltransferase.
The Journal of biological chemistry, 2001Co-Authors: Anita M. Makowski, Robert N. Dutnall, Anthony T. AnnunziatoAbstract:Abstract During nucleosome assembly in vivo, newly synthesized histone H4 is specifically diacetylated on lysines 5 and 12 within the H4 NH2-terminal tail domain. The highly conserved “K5/K12” deposition pattern of acetylation is thought to be generated by the HAT1 histone acetyltransferase, which in vivo is found in the HAT-B complex. In the following report, the activity and substrate specificity of the human HAT-B complex and of recombinant yeast HAT1p have been examined, using synthetic H4 NH2-terminal peptides as substrates. As expected, the unacetylated H4 peptide was a good substrate for acetylation by yeast HAT1p and human HAT-B, while the K5/K12-diacetylated peptide was not significantly acetylated. Notably, an H4 peptide previously diacetylated on lysines 8 and 16 was a very poor substrate for acetylation by either yeast HAT1p or human HAT-B. Treating the K8/K16-diacetylated peptide with histone deacetylase prior to the HAT-B reaction raised acetylation at K5/K12 to 70–80% of control levels. These results present strong support for the model of H4-HAT1p interaction proposed by Dutnall et al. (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998)Cell 94, 427–438) and provide evidence for the first time that site-specific acetylation of histones can regulate the acetylation of other substrate sites.
Prabakaran Nagarajan - One of the best experts on this subject based on the ideXlab platform.
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Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability
The Journal of biological chemistry, 2020Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Liudmila V. Popova, Michael A. Freitas, Callie M Lovejoy, Dongju Park, Mark R. ParthunAbstract:The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.
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HAT1-Dependent Lysine Acetylation Targets Diverse Cellular Functions
2019Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Mark R. ParthunAbstract: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.
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Early-onset aging and mitochondrial defects associated with loss of histone acetyltransferase 1 (HAT1).
Aging cell, 2019Co-Authors: Prabakaran Nagarajan, Paula A. Agudelo Garcia, Chitra C. Iyer, Liudmila V. Popova, William Arnold, Mark R. ParthunAbstract:Histone acetyltransferase 1 (HAT1) is responsible for the acetylation of newly synthesized histone H4 on lysines 5 and 12 during the process of chromatin assembly. To understand the broader biological role of HAT1, we have generated a conditional mouse knockout model of this enzyme. We previously reported that HAT1 is required for viability and important for mammalian development and genome stability. In this study, we show that haploinsufficiency of HAT1 results in a significant decrease in lifespan. Defects observed in HAT1+/- mice are consistent with an early-onset aging phenotype. These include lordokyphosis (hunchback), muscle atrophy, minor growth retardation, reduced subcutaneous fat, cancer, and paralysis. In addition, the expression of HAT1 is linked to the normal aging process as HAT1 mRNA and protein becomes undetectable in many tissues in old mice. At the cellular level, fibroblasts from HAT1 haploinsufficient embryos undergo early senescence and accumulate high levels of p21. HAT1+/- mouse embryonic fibroblasts (MEFs) display modest increases in endogenous DNA damage but have significantly higher levels of reactive oxygen species (ROS). Consistently, further studies show that HAT1-/- MEFs exhibit mitochondrial defects suggesting a critical role for HAT1 in mitochondrial function. Taken together, these data show that loss of HAT1 induces multiple hallmarks of early-onset aging.
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Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly
Nucleic acids research, 2017Co-Authors: Paula A. Agudelo Garcia, Prabakaran Nagarajan, Michael E. Hoover, Pei Zhang, Michael A. Freitas, Mark R. ParthunAbstract:Histone acetyltransferase 1 (HAT1) catalyzes the acetylation of newly synthesized histone H4 at lysines 5 and 12 that accompanies replication-coupled chromatin assembly. The acetylation of newly synthesized H4 occurs in the cytoplasm and the function of this acetylation is typically ascribed to roles in either histone nuclear import or deposition. Using cell lines from HAT1+/+ and HAT1-/- mouse embryos, we demonstrate that HAT1 is not required for either histone nuclear import or deposition. We employed quantitative proteomics to characterize HAT1-dependent changes in the composition of nascent chromatin structure. Among the proteins depleted from nascent chromatin isolated from HAT1-/- cells are several bromodomain-containing proteins, including Brg1, Baz1A and Brd3. Analysis of the binding specificity of their bromodomains suggests that HAT1-dependent acetylation of H4 is directly involved in their recruitment. HAT1-/- nascent chromatin is enriched for topoisomerase 2α and 2β. The enrichment of topoisomerase 2 is functionally relevant as HAT1-/- cells are hyper-sensitive to topoisomerase 2 inhibition suggesting that HAT1 is required for proper chromatin topology. In addition, our results indicate that HAT1 is transiently recruited to sites of chromatin assembly, dissociating prior to the maturation of chromatin structure.
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Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4
PLoS genetics, 2013Co-Authors: Prabakaran Nagarajan, Anthony T. Annunziato, Paula A. Agudelo Garcia, Bianca M. Sirbu, Cheryl Doughty, Michaela Schlederer, David Cortez, Lukas Kenner, Mark R. ParthunAbstract: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.
Christophe Thiriet - One of the best experts on this subject based on the ideXlab platform.
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Histones H3 and H4 require their relevant amino-tails for efficient nuclear import and replication-coupled chromatin assembly in vivo
Scientific Reports, 2017Co-Authors: Aïda Ejlassi, Vanessa Menil-philippot, Angélique Galvani, Christophe ThirietAbstract:Concomitant chromatin assembly and DNA duplication is essential for cell survival and genome integrity, and requires newly synthesized histones. Although the N-terminal domains of newly synthesized H3 and H4 present critical functions, their requirement for replication-coupled chromatin assembly is controversial. Using the unique capability of the spontaneous internalization of exogenous proteins in Physarum, we showed that H3 and H4 N-tails present critical functions in nuclear import during the S-phase, but are dispensable for assembly into nucleosomes. However, our data revealed that chromatin assembly in the S-phase of complexes presenting ectopic N-terminal domains occurs by a replication-independent mechanism. We found that replication-dependent chromatin assembly requires an H3/H4 complex with the relevant N-tail domains, suggesting a concomitant recognition of the two histone domains by histone chaperones. In eukaryotes, genomic DNA is associated with proteins to form chromatin. The basic sub-unit of chroma-tin is the nucleosome composed of a central tetramer of H3/H4, flanked by two heterodimers of H2A/H2B, and the octamer is wrapped by about two superhelical turns of DNA 1. The core histones are composed of two distinct domains; the fold domain involved in the histone-histone interaction with the nucleosome and the amino-tail domain that extends outside the nucleosome 2, 3. The histone tail domains have been shown to be post-translationally modified and these modifications are generally believed to be involved in chromatin activity regulation 4. During the S-phase of the cell cycle, the genome replicates and, in conjunction with DNA synthesis, chro-matin is assembled 5. The doubling of the genetic material associated with replication requires parental histone dilution and the synthesis of new histones to compact DNA within the nucleus. Using a pulse labeling strategy for studying newly synthesized histones revealed a conserved di-acetylation on lysines 5 and 12 of histone H4 (corresponding to 4 and 11 in Tetrahymena) related to chromatin deposition 6. This high conservation of the deposition-related di-acetylation of H4 suggested that histone acetyltransferase (HAT) is also highly conserved. In contrast, the newly synthesized H3 acetylation pattern in relation to replication presented a weaker degree of conservation. In the protozoan Tetrahymena, the partitioning of chromatin activities between two distinct nuclei, i.e. a macronucleus that transcribes and replicates and a micronucleus that only replicates, enabled HAT activities to be distinguished 7. Preparation of extracts from micronuclei and cytoplasm revealed that the deposition-related di-acetylation of H4 was catalyzed by a type B histone acetyltransferase, which does not acetylate histone in the chromatin form 8. This type B HAT was first isolated from yeast cytoplasmic extracts and corresponds to a two-subunit holoenzyme with HAT1p as the catalytic subunit and Hat2p 9. Consistently with the conservation of the deposition-related di-acetylation of H4, Human HAT1 has been identified and presents a high degree of conservation 10. Like the yeast enzyme, Human HAT1 is composed of two sub-units, HAT1 and RbA-p46. However, the enzyme was shown to have a nuclear localization in the vicinity of replication forks during the S-phase 10, 11. Furthermore, the chromatin assembly complex, which is composed of the three subunits of CAF-1 (p150, p60 and RbA-p48) and H3/H4 and which promotes replication-dependent chromatin assembly, exhibited acetylation of
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H4 replication-dependent diacetylation and HAT1 promote S-phase chromatin assembly in vivo.
Molecular biology of the cell, 2010Co-Authors: Aïda Ejlassi-lassallette, Eloïse Mocquard, Marie-claire Arnaud, Christophe ThirietAbstract:While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of HAT1, as coimmunoprecipitation experiments showed HAT1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks.
