Abstract
Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD–H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases.
Highlights
Bromodomains (BD) are highly conserved epigenetic reader modules that recognize acetyl-lysine (Kac) on histones and other proteins [1, 2]. (Note: for clarity, histone residues will be referred to in one-letter code, and BD residues will be referred to in three-letter code.) In the nearly 30 years since BDs were first identified, the chromatin field has accumulated a wealth of biophysical, structural, and biochemical data on BDs and other epigenetic readers [3, 4]
While BD mutations that abolish acetyl-lysine binding in vitro only modestly impact C. elegans viability, we found that a combination of SMARCA4 BD binding mutants with genetic inactivation of the pbrm-1 gene, which encodes an accessory Switch/Sucrose Nonfermenting (SWI/SNF) subunit, resulted in enhanced embryonic lethality and fertility defects
To assess whether SMARCA4 BD–H3K14 binding is conserved between mammals and C. elegans, we screened a recombinantly expressed and purified Glutathione S– Transferase (GST)–bromodomain fusion protein against a microarrayed library of 300+ biotinylated histone peptides [34]
Summary
Bromodomains (BD) are highly conserved epigenetic reader modules that recognize acetyl-lysine (Kac) on histones and other proteins [1, 2]. (Note: for clarity, histone residues will be referred to in one-letter code, and BD residues will be referred to in three-letter code.) In the nearly 30 years since BDs were first identified, the chromatin field has accumulated a wealth of biophysical, structural, and biochemical data on BDs and other epigenetic readers [3, 4]. Nor have researchers begun to mine the vast structural and sequence data already available to elucidate potential global patterns of specificity and plasticity common among BD subfamilies targeting the same marks This paper addresses these gaps in studies of chromatin regulation by epigenetic readers. SMARCA4 can remodel nucleosomal substrates by itself in vitro [16, 17] and is functionally and structurally conserved among eukaryotes [14, 18] This remodeling enzyme possesses intrinsic ATPase and helicase activity and has a C-terminal BD motif capable of recognizing lysine 14 acetylation on histone H3 (H3K14ac) [19] (Fig. 1A). Based on the premise that binding specificity is evolutionarily conserved within each BD ortholog,
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