Biology subjects

Liau, B. B.

Publications and source records attributed to Liau, B. B..

11 recordsLinked to original sources

bioRxiv2025

Genetic landscape of DNMT3A R882H reveals mechanism of aberrant oligomerization

DNA methyltransferase 3A (DNMT3A) is a de novo DNA methyltransferase that is recurrently mutated in hematological malignancies and developmental disorders. The most prevalent mutation, R882H, compromises DNMT3A activity in a dominant-negative manner, but its precise biochemical mechanism has been debated. Here, we use paired deep mutational scanning of the wild-type and R882H-mutant proteins to systematically identify mutations on a massively parallel scale that modify DNMT3A activity by suppressing, phenocopying, or selectively rescuing the dominant-negative effect of R882H. By leveraging the mutational depth and unbiased nature of the paired genetic landscapes, we uncover two distinct mechanisms that can rescue DNMT3AR882H activity, providing novel insights into the function of the R882 hotspot. First, by analyzing the effects of combinatorial mutations in the target recognition domain (TRD), we reveal that its crosstalk with the ADD regulatory domain modulates DNMT3A DNA binding and enzymatic activity, partially compensating for R882H-induced loss-of-function. Second, pairwise analysis of variant effects across the two genetic backgrounds supports the notion that R882H promotes aberrant macro-oligomerization of DNMT3A via its central dimerization interface, which accounts for its dominant-negative effect. Critically, we show that the R882 position exhibits a distinct dominant-negative signature in the genetic landscape, where positively charged residues at this position safeguard against aberrant macro-oligomerization. By performing hydrogen-deuterium exchange mass spectrometry (HDX-MS) on DNMT3A mutants, we show that R882H dramatically alters the protein dynamics of DNMT3A, rigidifying its central dimerization interface to promote oligomerization. Our data support a new model in which R882H removes the critical function of R882, where the arginine attenuates the pre-organization of the interface and subsequent oligomerization at a supramolecular assembly hotspot1. Altogether, we map the genetic landscape underpinning the DNMT3AR882H hotspot mutation, illuminating the unexpected molecular mechanism of macro-oligomerization that drives its dominant-negative effect.

biochemistry↗

bioRxiv2025

A chemical-genetic interaction between PAF1 and ENL/AF9 YEATS inhibition

Transcriptional regulatory proteins are frequent drivers of oncogenesis and common targets for drug discovery. The transcriptional co-activator, ENL, which is localized to chromatin through its acetyllysine-binding YEATS domain, is preferentially required for the survival and pathogenesis of acute leukemia. Small molecules that inhibit the ENL YEATS domain show anti-leukemia effects in preclinical models, which is thought to be caused by the downregulation of pro-leukemic ENL target genes. However, the transcriptional effects of ENL YEATS domain inhibitors have not been studied in models of intrinsic or acquired resistance and, therefore, the connection between proximal transcriptional effects and downstream anti-proliferative response is poorly understood. To address this, we identified models of intrinsic and acquired resistance and used them to study the effects of ENL YEATS domain inhibitors. We first discovered that ENL YEATS domain inhibition produces similar transcriptional responses in naive models of sensitive and resistant leukemia. We then performed a CRISPR/Cas9-based genetic modifier screen and identified in-frame deletions of the essential transcriptional regulator, PAF1, that confer resistance to ENL YEATS domain inhibitors. Using these drug-resistance alleles of PAF1 to construct isogenic models, we again found that the downregulation of ENL target genes is shared in both sensitive and resistant leukemia. Altogether, these data support the conclusion that the suppression of ENL target genes is not sufficient to explain the anti-leukemia effects of ENL antagonists.

biochemistry↗

bioRxiv2025

Phosphorylation-inducing chimera rewires oncogenic kinase to trigger apoptosis

The active sites electric field is integral to enzymatic catalysis (e.g., substrate recognition) and nature employs charge-altering post-translational modifications (e.g., phosphorylation) to perturb this electric field and regulate enzymes. A chromosomal translocation converts Abelson kinase (ABL) to BCR-ABL, whose hyperactivity drives several cancers. Here, we developed a small molecule, BRD8833, that induces BCR-ABL phosphorylation, which perturbs its active sites electric field with loss of hyperactivity. Unlike "occupancy-driven" inhibitors that require stoichiometric concentrations, BRD8833 operates through an event-driven, substoichiometric mechanism by inducing proximity between two BCR-ABL molecules to trigger the inhibitory phosphorylation and selective apoptosis of BCR-ABL-dependent cancer cells. Furthermore, BRD8833 is effective against other oncogenic ABL fusions or clinically observed resistance mutations, including those to occupancy-driven drugs with the same binding site as BRD8833, suggesting differences in their resistance mechanisms. These studies lay the foundation for electric-field and "event-driven" modalities to control hyperactive enzymes with orthogonal resistance mechanisms to occupancy-driven drugs. GRAPHICAL ABSTRACT (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/659082v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1607a40org.highwire.dtl.DTLVardef@fa10deorg.highwire.dtl.DTLVardef@1698232org.highwire.dtl.DTLVardef@1e2e0f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Coupling CRISPR Scanning with Targeted Chromatin Accessibility Profiling using a Double-Stranded DNA Deaminase

Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms and the development of gene therapies. However, these approaches cannot be combined with direct scalable readouts of chromatin structure and accessibility across long single-molecule chromatin fibers. Here we leverage a double-stranded DNA cytosine deaminase to profile chromatin accessibility at high depth and resolution at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). Powered by high sequence coverage at targeted loci of interest, TDAC-seq can be uniquely integrated with CRISPR perturbations to enable the functional dissection of cis-regulatory elements, where genetic perturbations and their effects on chromatin accessibility are superimposed on the same single chromatin fiber and resolved at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34+ hematopoietic stem and progenitor cells during erythroid differentiation as well as in pooled CRISPR and base editing screens tiling an enhancer controlling the globin locus. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.

molecular biology↗

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↗

bioRxiv2024

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

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function1. As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB)2, the most common embryonal brain tumor in children, and pineoblastoma3. These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST4. However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.

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

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 their fundamental importance, systematic identification of allosteric sites remains highly challenging. Here we perform CRISPR scanning on the essential maintenance methylation machinery--DNMT1 and its partner UHRF1--with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function mutations that increase DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our findings highlight the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, even beyond the direct drug target.

genomics↗

bioRxiv2022

Polycomb-lamina antagonism partitions heterochromatin at the nuclear periphery

The genome can be divided into two spatially segregated compartments, A and B,1,2 which broadly partition active and inactive chromatin states, respectively. Constitutive heterochromatin is predominantly located within the B compartment and comprises chromatin that is in close contact with the nuclear lamina.3-5 By contrast, facultative heterochromatin marked by H3K27me3 can span both compartments.2-5 How epigenetic modifications, A/B compartmentalization, and lamina association collectively maintain heterochromatin architecture and function remains unclear.6,7 Here we developed an approach termed Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) that jointly measures chromosome conformation, DNA methylation, and nuclear lamina positioning. Through this approach, we identified topologically distinct A/B sub-compartments characterized by high levels of H3K27me3 and differing degrees of lamina association. To study the regulation of these sub-compartments, we inhibited Polycomb repressive complex 2 (PRC2), revealing that H3K27me3 is an essential factor in sub-compartment segregation. Unexpectedly, PRC2 inhibition also elicited broad gains in lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with repositioning to the lamina, genes originally marked with H3K27me3 in the B compartment, but not in the A compartment, remained largely repressed, suggesting that constitutive heterochromatin spreading can compensate for loss of H3K27me3 at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with nuclear lamina association are fundamental organizational features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how dynamic changes in compartmentalization and nuclear lamina association represent distinct but interdependent modes of heterochromatin regulation.

genomics↗

bioRxiv2022

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. We identify mutations throughout the protein that perturb function, including ones at an interdomain interface that block allosteric activation. Unexpectedly, we also find mutations in the PWWP domain, a histone reader, that modulate enzyme activity despite preserving histone recognition and protein stability. These effects arise from altered PWWP domain DNA affinity, which we show is a noncanonical function required for full activity in cells. Our findings highlight mechanisms of interdomain crosstalk and demonstrate a generalizable strategy to probe sequence-activity relationships of nonessential chromatin regulators.

biochemistry↗

bioRxiv2021

Profiling the Landscape of Drug Resistance Mutations in Neosubstrates to Molecular Glue Degraders

Targeted protein degradation (TPD) holds immense promise for drug discovery but mechanisms of acquired resistance to degraders remain to be fully identified. Here we used CRISPR-suppressor scanning to identify mechanistic classes of drug resistance mutations to molecular glue degraders in GSPT1 and RBM39, neosubstrates targeted by E3 ligase substrate receptors cereblon and DCAF15, respectively. While many mutations directly alter the ternary complex heterodimerization surface, distal resistance sites were also identified. Several distal mutations in RBM39 led to modest decreases in degradation yet can enable cell survival, underscoring how small differences in degradation can lead to resistance. Integrative analysis of resistance sites across GSPT1 and RBM39 revealed varying levels of sequence conservation and mutational constraint that control the emergence of different resistance mechanisms, highlighting that many regions co-opted by TPD are inessential. Altogether, our study identifies common resistance mechanisms for molecular glue degraders and outlines a general approach to survey neosubstrate requirements necessary for effective degradation.

molecular biology↗