Molecular glue compounds induce protein-protein interactions that, inthe context of a ubiquitin ligase, lead to protein degradation. 1 Unlike traditional enzyme inhibitors, such molecular glue degraders act sub-stoichiometrically to catalyse rapid depletion of previously inaccessible targets. 2 They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Through systematic mining of databases for correlations between the cytotoxicity of 4,518 clinical and pre-clinical small molecules and E3 ligase expression levels across hundreds of human cancer cell lines, 3-5 we identified CR8, a cyclin-dependent kinase (CDK) inhibitor, 6 as a compound that acts as a molecular glue degrader. A solvent-exposed pyridyl moiety of CR8, in its CDKbound form, induces CDK12-cyclin K complex formation with DDB1, the CUL4 adaptor protein, bypassing the requirement for a substrate receptor and presenting cyclin K (cycK) for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy to turn target binders into molecular glues.

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Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo–electron microscopy at two preferred positions. Depending on motif location, OCT4 and SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from histone H2A and histone H3; however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA-binding domains to engage DNA in both structures, reading out a partial motif. These findings explain site-specific nucleosome engagement by the pluripotency factors OCT4 and SOX2, and they reveal how TFs distort nucleosomes to access chromatinized motifs.

histones in situ, which were partially lost upon aclarubicin treatment (Extended Data Fig. 1c, d). Thus, histones appear to dynamically engage cGAS in the nucleus.Consistent with prior work [13], functional analysis of cGAS in vitro enzymatic activity revealed that mononucleosomes (hereafter nucleosomes) inhibited DNA-induced cGAMP synthesis (Extended Data Fig. 1e). Likewise, compact chromatin fibres (12-mer nucleosome arrays) suppressed cGAS activity (Extended Data Fig. 1e). H2A-H2B dimers also had an inhibitory effect, but neither H2A or H2B monomers nor H3 or H4 monomers, respectively (Extended Data Fig. 1f, g). Thus, H2A-H2B dimers on their own can suppress cGAS (Extended Data Fig. 1h), albeit with weaker overall potency compared to fullassembled nucleosomes with additional features of nucleosomes in chromatin being necessary to exert maximal inhibition. Overall structure of the cGAS-NCP complexTo determine how cGAS interacts with nucleosomes, we pursued structural studies. A 1.5:1 molar mixture of human cGAS (residues 155 to 522) with a 147 bp 601 DNA nucleosome core particle (NCP) resulted in heterogenous particle distributions (Extended Data Fig. 2ad). To select for and stabilize more homogenous cGAS-NCP complexes, we combined gradient centrifugation with chemical crosslinking (GraFix) [15]. Both WT cGAS and cGAS K394E, a mutant impaired in dsDNA-mediated cGAS dimerisation [16], were used for structure determination. For the cGAS K394E mutant, we obtained a 4.1 Å reconstruction revealing two NCPs organized in a NCP 1 -cGAS 1 -cGAS 2 -NCP 2 sandwich arrangement with an expected molecular weight around 560 kDa, consistent with the most prominent peak fraction in multi-angle light scattering (MALS) (Fig. 1a, b, Extended Data Fig. 3, Supplementary Video 1, 2, and Extended Data Table 1a). The two individual nucleosomes are held together by two cGAS protomers. While the first cGAS protomer and its corresponding NCP (designated cGAS 1 and NCP 1 ) are well-resolved, the second nucleosome/cGAS pair (NCP 2 and cGAS 2 ) is less ordered (Extended Data Fig. 3e). In the dimeric NCP 1 -cGAS 1 -cGAS 2 -NCP 2 arrangement, each cGAS protomer interacts with the histone octamer of one NCP through histones H2A and H2B and the nucleosomal DNA (e.g. cGAS 1 and NCP 1 ), while contacting the second nucleosome (e.g. cGAS 1 and NCP 2 ) primarily through interactions with the nucleosomal DNA (Fig. 1a, b). In the WT cGAS structure, we observed a similar overall structural arrangement, with the NCP 1 -cGAS 1 -cGAS 2 -NCP 2 complex at 5.1Å and the focused NCP 1 -cGAS 1 structure at 4.7Å resolution (Extended Data

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The genomic binding sites of the transcription factor (TF) and tumour suppressor p53 are unusually diverse in regards to their chromatin features, including histone modifications, opening the possibility that chromatin provides context-dependence for p53 regulation. Here, we show that the ability of p53 to open chromatin and activate its target genes is indeed locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated lysine 4 of histone H3, thereby preferentially locating to those p53 sites that reside in closed chromatin, while it is deterred from accessible chromatin by lysine 4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to impact gene expression as a function of the local chromatin state. These findings link histone methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors which locally modulate TF function.

The genomic binding sites of the transcription factor (TF) and tumor suppressor p53 are unusually diverse with regard to their chromatin features, including histone modifications, raising the possibility that the local chromatin environment can contextualize p53 regulation. Here, we show that epigenetic characteristics of closed chromatin, such as DNA methylation, do not influence the binding of p53 across the genome. Instead, the ability of p53 to open chromatin and activate its target genes is locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated histone 3 lysine 4 (H3K4), thereby preferentially localizing to those p53 sites that reside in closed chromatin, whereas it is deterred from accessible chromatin by H3K4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to affect gene expression as a function of the local chromatin state. These findings link H3K4 methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors that locally modulate TF function.