Agudelo Garcia, P. A. | Search bioRxiv

bioRxiv2020

Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

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.

biochemistry↗

bioRxiv2019

Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions

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.

biochemistry↗