Biology subjects

Waterbury, A. L.

Publications and source records attributed to Waterbury, A. L..

3 recordsLinked to original sources

bioRxiv2024

Asymmetric Engagement of Dimeric CRL3KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal1, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

molecular biology↗

bioRxiv2023

DNA methylation insulates genic regions from CTCF loops near nuclear speckles

The insulator protein CTCF is essential for mediating chromatin loops and regulating gene expression. While it is established that DNA methylation hinders CTCF binding, the impacts of this methylation-sensitive CTCF binding on chromatin architecture and transcription are poorly defined. Here, we used a selective DNMT1 inhibitor (DNMT1i) to investigate the characteristics and functions of DNMT1i-specific CTCF peaks resulting from global DNA demethylation. We found that DNMT1i-specific peaks preferentially form chromatin loops on gene bodies and interact with highly-looping partner peaks located in regions of active chromatin. Notably, both DNMT1i-specific CTCF peaks and their highly-looping partners are enriched near nuclear speckles, condensate bodies implicated in transcription and splicing. Utilizing targeted protein degradation, we specifically depleted CTCF and nuclear speckles to elucidate their functional interplay. By degrading CTCF upon DNMT1 inhibition, we revealed that CTCF is important for DNMT1i-dependent interactions between chromatin and speckle proteins. Moreover, we found that CTCF promotes the activation of genes near speckles upon DNMT1 inhibition. Conversely, acute depletion of nuclear speckles revealed that they influence RNA abundance but do not maintain CTCF binding or looping. Collectively, our study suggests a model wherein DNA methylation prevents spurious CTCF occupancy and interactions with regulatory elements near nuclear speckles, yet CTCF looping is robust towards the loss of speckles.

genomics↗

bioRxiv2022

Drug addiction mutations unveil a methylation ceiling in EZH2-mutant lymphoma

Cancer mutations in Polycomb Repressive Complex 2 (PRC2) drive aberrant epigenetic states. Although therapies inhibiting the PRC2 enzymatic component EZH2 are FDA-approved, oncogene-specific dependencies remain to be discovered. Here, we identify mutations that confer both resistance and drug addiction to PRC2 inhibitors in EZH2-mutant lymphoma, resulting in cancer cells that paradoxically depend on drug for survival. Drug addiction is mediated by hypermorphic mutations in the CXC domain of EZH2, which maintain H3K27me3 levels even in the presence of PRC2 inhibitors. Drug removal leads to overspreading of H3K27me3, surpassing a repressive methylation ceiling compatible with lymphoma cell survival. Activating EZH2 cancer mutations establish an epigenetic state precariously close to this ceiling, which we show can be breached by inhibition of SETD2, a PRC2 antagonist, to block lymphoma growth. More broadly, we highlight how approaches to identify drug addiction mutations can be leveraged to discover cancer vulnerabilities.

cancer biology↗