Regulation of the Dot1 histone H3K79 methyltransferase by histone H4K16 acetylation

Abstract

Cross-talk between histone modifications Histone modifications play pivotal roles within the intricate protein networks that underlie transcription and gene silencing in eukaryotic genomes. The enzymes that deposit them undergo spatiotemporal fine-tuning of their catalytic activity; one example is trans-histone cross-talk, in which one histone modification activates an enzyme responsible for another histone modification. Valencia-Sánchez et al. show that histone H4 lysine 16 acetylation (H4K16ac), a hallmark of decondensed, transcriptionally permissive chromatin, directly stimulates the Dot1 histone H3 lysine 79 methyltransferase. Structural, biochemical, and cellular data explain Dot1 s regulation by H4K16ac and show how it coordinates with a second positive regulator of Dot1, histone H2B ubiquitination. Science, this issue p. eabc6663 Mechanisms that regulate gene transcription and gene silencing to ensure optimal maintenance and propagation of an epigenetic state are elucidated. INTRODUCTION Nucleosomes—the primary, repeating unit of chromatin—package and protect the genome while also transmitting various regulatory signals through, in part, posttranslational modifications of histones. These histone modifications (for example, acetylation, ubiquitination, or methylation) affect critical processes such as transcription, replication, recombination, and repair, often forming complicated networks to ensure finely tuned signaling for chromatin enzymes. One such enzyme—evolutionarily conserved disruptor of telomeric silencing (Dot1)—catalyzes mono-, di-, and trimethylation of histone H3 lysine 79 (H3K79). H3K79 methylation by Dot1 is a prominent example of trans-histone cross-talk, a process in which one histone and its modification affects the modification of another histone. In mammals, the human homolog Dot1L plays critical roles in embryogenesis and hematopoiesis. Although recent advances have provided insights into Dot1L stimulation through histone H2B ubiquitination, how other modifications mechanistically regulate Dot1 activity is not known. We were particularly interested in how histone lysine acetylation contributes to the regulation of chromatin enzymes. Lysine acetylation plays pivotal roles in chromatin decondensation, transcriptional activation, and maintenance of euchromatin, serving as a general antisilencing mark in eukaryotes. Here, we present mechanistic studies that show how histone acetylation regulates the activity of Dot1. RATIONALE Histone H4 acetylation, histone H2B ubiquitination, and H3 methylation are conserved cotranscriptional histone modifications that work together to ensure the appropriate regulation of chromatin structure during transcription. The mechanisms of cross-talk between these modifications and enzymes that deposit them are crucial for understanding transcription. Advances in cryo–electron microscopy (cryo-EM), the ability to make specifically acetylated and ubiquitinated nucleosomes, and established in vitro and in vivo assays allowed us to determine the detailed mechanisms of yeast Dot1 regulation through histone acetylation and ubiquitination. RESULTS We tested nucleosomes with different acetylation states of histone H4 in vitro, and we show that Dot1 is allosterically stimulated by acetylation of H4 and that this effect is specific to lysine 16 (H4K16ac). The other known acetylation targets on histone H4 (H4K5ac, H4K8ac, and H4K12ac) do not stimulate the activity of Dot1, which highlights the distinctive role of H4K16 acetylation in regulating chromatin structure. We also show that the effect of H4K16 acetylation is direct and further enhanced by H2B ubiquitination (H2BUb), resulting in an optimal catalytic rate for Dot1. To gain mechanistic insights into stimulation by H4K16ac and its coordination with H2BUb, we determined two cryo-EM structures: one of Dot1 in complex, with nucleosomes bearing both H4K16ac and H2Bub, and the second of Dot1, with a nucleosome bearing only H2BUb. Upon examining our cryo-EM dataset of Dot1 bound to the nucleosome containing unacetylated H4, the particles classified into two main three-dimensional (3D) classes. In the first class, Dot1 is bound to the nucleosome in a catalytic conformation. In the second class, it is bound in a noncatalytic conformation. This is different from the dataset in which Dot1 is bound to an H4K16 acetylated nucleosome. When H4K16ac is present, the cryo-EM data is more homogeneous, and Dot1 is bound to the nucleosome predominantly in a catalytic conformation. This suggests a model in which acetylation of the H4 tail restricts the sampling space of Dot1, resulting in an active conformation leading to increased activity. We therefore propose that H2BUb partially restricts the conformation of yeast Dot1 on the nucleosome and that H4K16ac further restricts and stabilizes the active conformation. Comparing both of these cryo-EM structures allowed us to identify residues that are critical for Dot1 stimulation by H4K16ac and H2BUb. Site-directed mutagenesis of Dot1 coupled with enzymatic assays on nucleosomes revealed the details of these interfaces. These results show that the allosteric stimulation of Dot1 by H4K16ac and H2BUb plays a crucial role in H3K79 di- and trimethylation. CONCLUSION This work demonstrates how Dot1 is regulated by histone acetylation and how H4K16ac coordinates with H2BUb to regulate Dot1. H4K16ac plays a critical role in opening chromatin structure by counteracting the binding of silencing proteins, while simultaneously stimulating an enzyme that is important for transcription. We provide an example in which the activity of the fundamental methyltransferase Dot1 is modulated through cross-talk between distinct histone modifications to ensure optimal maintenance and propagation of an epigenetic state. Cross-talk such as this may represent a general property of chromatin enzymes. H3K79 methylation by Dot1 is allosterically stimulated by H4K16 acetylation and by H2BK123 ubiquitination. Cross-talk between different histone posttranslational modifications can orchestrate distinct chromatin states to regulate transcription and gene silencing. Dot1 (disruptor of telomeric silencing-1), the histone H3 lysine 79 (H3K79) methyltransferase, is conserved throughout evolution, and its deregulation is found in human leukemias. Here, we provide evidence that acetylation of histone H4 allosterically stimulates yeast Dot1 in a manner distinct from but coordinating with histone H2B ubiquitination (H2BUb). We further demonstrate that this stimulatory effect is specific to acetylation of lysine 16 (H4K16ac), a modification central to chromatin structure. We provide a mechanism of this histone cross-talk and show that H4K16ac and H2BUb play crucial roles in H3K79 di- and trimethylation in vitro and in vivo. These data reveal mechanisms that control H3K79 methylation and demonstrate how H4K16ac, H3K79me, and H2BUb function together to regulate gene transcription and gene silencing to ensure optimal maintenance and propagation of an epigenetic state.