Principles of nucleosome recognition by chromatin factors and enzymes

The recent torrent of structures of chromatin complexes determined by cryoelectron microscopy provides an opportunity to discern general principles for how chromatin factors and enzymes interact with their nucleosome substrate. We find that many chromatin proteins use a strikingly similar arginine anchor and variant arginine interactions to bind to the nucleosome acidic patch. We also observe that many chromatin proteins target the H3 and H2B histone fold α1-loop1 elbows and the H2B C-terminal helix on the nucleosomal histone face. These interactions with the histones can be complemented with interactions with and distortions of nucleosomal DNA

Recognition of the nucleosome by chromatin factors and enzymes

Dynamic expression of the genome requires coordinated binding of chromatin factors and enzymes that carry out genome-templated processes. Until recently, the molecular mechanisms governing how these factors and enzymes recognize and act on the fundamental unit of chromatin, the nucleosome core particle, have remained a mystery. A small, yet growing set of structures of the nucleosome in complex with chromatin factors and enzymes highlights the importance of multivalency in defining nucleosome binding and specificity. Many such interactions include an arginine anchor motif, which targets a unique acidic patch on the nucleosome surface. These emerging paradigms for chromatin recognition will be discussed, focusing on several recent structural breakthroughs

Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome

The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in many eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys 119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the human Ring1B-Bmi1-UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with several nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome

Multivalent interactions by the set8 histone methyltransferase with its nucleosome substrate

Set8 is the only mammalian monomethyltransferase responsible for H4K20me1, a methyl mark critical for genomic integrity of eukaryotic cells. We present here a structural model for how Set8 uses multivalent interactions to bind and methylate the nucleosome based on crystallographic and solution studies of the Set8/nucleosome complex. Our studies indicate that Set8 employs its i-SET and c-SET domains to engage nucleosomal DNA 1 to 1.5 turns from the nucleosomal dyad and in doing so, it positions the SET domain for catalysis with H4 Lys20. Surprisingly, we find that a basic N-terminal extension to the SET domain plays an even more prominent role in nucleosome binding, possibly by making an arginine anchor interaction with the nucleosome H2A/H2B acidic patch. We further show that proliferating cell nuclear antigen and the nucleosome compete for binding to Set8 through this basic extension, suggesting a mechanism for how nucleosome binding protects Set8 from proliferating cell nuclear antigen-dependent degradation during the cell cycle

A semisynthetic strategy to generate phosphorylated and acetylated histone H2B

Proteins are subject to numerous post-translational modifications (PTMs) that can alter the chemical structure, and hence function, of the molecule. The astonishing diversity of PTMs possible on proteins is exemplified by histones, nuclear proteins that form the protein core of the nucleosome particle. Histones can be modified in a variety of ways including acetylation, phosphorylation, methylation, ADP-ribosylation and ubiquitylation. Moreover, many, if not all, of these modifications can occur in combination. Indeed, there is growing evidence that functional cross-talk between histone PTMs is essential for the regulation of gene expression and ultimately cell fate and identity. Biochemical studies into the role of histone PTMs are often confounded by the difficulty associated with obtaining large quantities of homogeneously modified proteins. For this reason chemical approaches to obtaining post-translationally modified histones have received considerable attention in recent years. Among the available strategies, the protein ligation approach, expressed protein ligation (EPL), offers the most flexibility in terms of the number and type of PTMs that can be incorporated. To date, EPL has been used to generate phosphorylated, acetylated, and methylated forms of histone H3, acetylated H4, and ubiquitylated H2B. Nonetheless, many modified histones have yet to be accessed using semi-synthesis. A notable case in point is the N-terminal region of H2B, which has been described to possess several PTMs, including (poly)lysine acetylation and serine 14 phosphorylation, which have been implicated in transcription and apoptotic chromatin compaction, respectively. Differentially modified semi-synthetic H2B analogs would be useful to assess the affect of acetylation on both antibody recognition as well as on the efficiency of phosphorylation. In this report, we describe a general semi-synthetic route to H2B that allows the installation of PTMs into an otherwise native polypeptide background

Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation

Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome

Crosstalk among Set1 complex subunits involved in H2B ubiquitylation-dependent H3K4 methylation

H2B ubiquitylation (H2Bub)-dependent H3K4 methylation is mediated by the multisubunit Set1 complex (Set1C) in yeast, but precisely how Set1C subunits contribute to this histone modification remains unclear. Here, using reconstituted Set1Cs and recombinant H2Bub chromatin, we identified Set1C subunits and domains involved in the H2Bub-dependent H3K4 methylation process, showing that the Spp1 PHDL domain, in conjunction with the Set1 n-SET domain, interacts with Swd1/Swd3 and that this interaction is essential for H2Bub-dependent H3K4 methylation. Importantly, Set1C containing an Spp1-Swd1 fusion bypasses the requirement for H2Bub for H3K4 methylation, suggesting that the role of H2Bub is to induce allosteric rearrangements of the subunit-interaction network within the active site of Set1C that are necessary for methylation activity. Moreover, the interaction between the Set1 N-terminal region and Swd1 renders the Spp1-lacking Set1C competent for H2Bub-dependent H3K4 methylation. Collectively, our results suggest that H2Bub induces conformational changes in Set1C that support H3K4 methylation activity

Selective disruption of Tcf7l2 in the pancreatic β cell impairs secretory function and lowers β cell mass.

Type 2 diabetes (T2D) is characterized by β cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired β cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the β cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in β cells from the earliest expression of the Ins1 gene (∼E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (∼20%) and Glp1r (∼40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual β cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ∼30% decrease in β cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of β cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed

Structural basis of nucleosome-dependent cGAS inhibition

Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) recognizes cytosolic foreign or damaged DNA to activate the innate immune response to infection, inflammatory diseases, and cancer. By contrast, cGAS reactivity against self-DNA in the nucleus is suppressed by chromatin tethering. We report a 3.3-angstrom-resolution cryo-electron microscopy structure of cGAS in complex with the nucleosome core particle. The structure reveals that cGAS uses two conserved arginines to anchor to the nucleosome acidic patch. The nucleosome-binding interface exclusively occupies the strong double-stranded DNA (dsDNA)-binding surface on cGAS and sterically prevents cGAS from oligomerizing into the functionally active 2:2 cGAS-dsDNA state. These findings provide a structural basis for how cGAS maintains an inhibited state in the nucleus and further exemplify the role of the nucleosome in regulating diverse nuclear protein functions

Histone monoubiquitylation position determines specificity and direction of enzymatic cross-talk with histone methyltransferases Dot1L and PRC2

It is well established that chromatin is a destination for signal transduction, affecting many DNA-templated processes. Histone proteins in particular are extensively post-translationally modified. We are interested in how the complex repertoire of histone modifications is coordinately regulated to generate meaningful combinations of "marks" at physiologically relevant genomic locations. One important mechanism is "cross-talk" between pre-existing histone post-translational modifications and enzymes that subsequently add or remove modifications on chromatin. Here, we use chemically defined "designer" nucleosomes to investigate novel enzymatic cross-talk relationships between the most abundant histone ubiquitylation sites, H2AK119ub and H2BK120ub, and two important histone methyltransferases, Dot1L and PRC2. Although the presence of H2Bub in nucleosomes greatly stimulated Dot1L methylation of H3K79, we found that H2Aub did not influence Dot1L activity. In contrast, we show that H2Aub inhibited PRC2 methylation of H3K27, but H2Bub did not influence PRC2 activity. Taken together, these results highlight how the position of nucleosome monoubiquitylation affects the specificity and direction of cross-talk with enzymatic activities on chromatin