To advance our understanding of the histone acylation regulators and the response to DNA replication stress, we employ a variety of genetic screening techniques together with follow-up experiments... Show moreTo advance our understanding of the histone acylation regulators and the response to DNA replication stress, we employ a variety of genetic screening techniques together with follow-up experiments in yeast. By employing Epi-ID, we interrogated a common chromatin locus in the yeast deletion and DaMP collection. This was successful in identifying new histone acetylation and acylation regulators and we describe the crotonyltransferase activities of the ADA and NuA4 complexes. Increased crotonylation levels stimulated gene expression dependent on the activity of the HATs Gcn5 and Esa1, suggesting a role for these enzymes in the crotonylation-dependent transcriptional regulation. Additionally, extensive antibody validation studies revealed that non-acetyl pan-lysine specific acylation antibodies had a severe lack of specificity.To identify new regulators of replication stress, we developed a novel approach called Replication-Identifier (Repli-ID), which we used to investigate the accumulation of replicative polymerase ε near a barcoded origin of replication in mutant yeast collections. We demonstrate that this method is effective for studying DNA polymerase occupancy directly on chromatin and we identify new regulators of DNA replication fork progression/stability.Finally, we identify and characterize the Mediator complex in the replication stress response. We show how Mediator reduces replication stress by preventing R-loop formation. Show less
Kollenstart, L.; Horst, S.C. van der; Vreeken, K.; Janssen, G.M.C.; Martino, F.; Vlaming, H.; ... ; Attikum, H. van 2021
The collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and... Show moreThe collection of known posttranslational modifications (PTMs) has expanded rapidly with the identification of various non-acetyl histone lysine acylations, such as crotonylation, succinylation and butyrylation, yet their regulation is still not fully understood. Through an unbiased chromatin immunoprecipitation (ChIP)-based approach called Epigenetics-IDentifier (Epi-ID), we aimed to identify regulators of crotonylation, succinylation and butyrylation in thousands of yeast mutants simultaneously. However, highly correlative results led us to further investigate the specificity of the pan-K-acyl antibodies used in our Epi-ID studies. This revealed cross-reactivity and lack of specificity of pan-K-acyl antibodies in various assays. Our findings suggest that the antibodies might recognize histone acetylation in vivo, in addition to histone acylation, due to the vast overabundance of acetylation compared to other acylation modifications in cells. Consequently, our Epi-ID screen mostly identified factors affecting histone acetylation, including known (e.g. GCN5, HDA1, and HDA2) and unanticipated (MET7, MTF1, CLB3, and RAD26) factors, expanding the repertoire of acetylation regulators. Antibody-independent follow-up experiments on the Gcn5-Ada2-Ada3 (ADA) complex revealed that, in addition to acetylation and crotonylation, ADA has the ability to butyrylate histones. Thus, our Epi-ID screens revealed limits of using pan-K-acyl antibodies in epigenetics research, expanded the repertoire of regulators of histone acetylation, and attributed butyrylation activity to the ADA complex. Show less
Kollenstart, L.; Groot, A.J.L. de; Janssen, G.M.C.; Cheng, X.; Vreeken, K.; Martino, F.; ... ; Attikum, H. van 2019
Histone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the nonacetyl histone lysine acylation modifications such as... Show moreHistone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the nonacetyl histone lysine acylation modifications such as crotonylation, butyrylation, and succinylation. However, the biological relevance of these PTMs is not fully understood because their regulation is largely unknown. Here, we set out to investigate whether the main histone acetyltransferases in budding yeast, Gcn5 and Esa1, possess crotonyltransferase activity. In vitro studies revealed that the Gcn5-Ada2-Ada3 (ADA) and Esa1-Yng2-Epl1 (Piccolo NuA4) histone acetyltransferase complexes have the capacity to crotonylate histones. Mass spectrometry analysis revealed that ADA and Piccolo NuA4 crotonylate lysines in the N-terminal tails of histone H3 and H4, respectively. Functionally, we show that crotonylation selectively affects gene transcription in vivo in a manner dependent on Gcn5 and Esa1. Thus, we identify the Gcn5- and Esa1-containing ADA and Piccolo NuA4 complexes as bona fide crotonyltransferases that promote crotonylation-dependent transcription. Show less
