CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands.

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polymerase Delta (Polδ). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand these processes may be coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

A Network of Conserved Synthetic Lethal Interactions for Exploration of Precision Cancer Therapy.

An emerging therapeutic strategy for cancer is to induce selective lethality in a tumor by exploiting interactions between its driving mutations and specific drug targets. Here we use a multi-species approach to develop a resource of synthetic lethal interactions relevant to cancer therapy. First, we screen in yeast ∼169,000 potential interactions among orthologs of human tumor suppressor genes (TSG) and genes encoding drug targets across multiple genotoxic environments. Guided by the strongest signal, we evaluate thousands of TSG-drug combinations in HeLa cells, resulting in networks of conserved synthetic lethal interactions. Analysis of these networks reveals that interaction stability across environments and shared gene function increase the likelihood of observing an interaction in human cancer cells. Using these rules, we prioritize ∼10(5) human TSG-drug combinations for future follow-up. We validate interactions based on cell and/or patient survival, including topoisomerases with RAD17 and checkpoint kinases with BLM.

Gcn5 and Esa1 function as histone crotonyltransferases to regulate crotonylation-dependent transcription.

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 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.

Epigenetics Identifier screens reveal regulators of chromatin acylation and limited specificity of acylation antibodies.

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 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.