Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function. As a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma, the most common embryonal brain tumour in children. These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST.

UM171 glues asymmetric CRL3-HDAC1/2 assembly to degrade CoREST corepressors.

UM171 is a potent agonist of ex vivo human haematopoietic stem cell self-renewal. By co-opting KBTBD4, a substrate receptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, UM171 promotes the degradation of the LSD1-CoREST corepressor complex, thereby limiting haematopoietic stem cell attrition. However, the direct target and mechanism of action of UM171 remain unclear.

An autoinhibitory switch of the LSD1 disordered region controls enhancer silencing.

Transcriptional coregulators and transcription factors (TFs) contain intrinsically disordered regions (IDRs) that are critical for their association and function in gene regulation. More recently, IDRs have been shown to promote multivalent protein-protein interactions between coregulators and TFs to drive their association into condensates. By contrast, here we demonstrate how the IDR of the corepressor LSD1 excludes TF association, acting as a dynamic conformational switch that tunes repression of active cis-regulatory elements.

Asymmetric Engagement of Dimeric CRL3 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-renewal, 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 attrition. However, the direct target and mechanism of action of UM171 remain unclear.

KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes.

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function . As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB) , the most common embryonal brain tumor in children, and pineoblastoma . These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST .

Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery.

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification remains highly challenging.

CRISPR-Suppressor Scanning for Systematic Discovery of Drug-Resistance Mutations.

CRISPR-Cas9 genome editing technologies have enabled complex genetic manipulations in situ, including large-scale, pooled screening approaches to probe and uncover mechanistic insights across various biological processes. The RNA-programmable nature of CRISPR-Cas9 greatly empowers tiling mutagenesis approaches to elucidate molecular details of protein function, in particular the interrogation of mechanisms of resistance to small molecules, an approach termed CRISPR-suppressor scanning. In a typical CRISPR-suppressor scanning experiment, a pooled library of single-guide RNAs is designed to target across the coding sequence(s) of one or more genes, enabling the Cas9 nuclease to systematically mutate the targeted proteins and generate large numbers of diverse protein variants in situ.

Base editor scanning charts the DNMT3A activity landscape.

DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation of this process is known to cause aberrant development and oncogenesis, yet how DNMT3A is regulated holistically by its three domains remains challenging to study. Here, we integrate base editing with a DNA methylation reporter to perform in situ mutational scanning of DNMT3A in cells.