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The demethylase activity of KDM5A is allosterically enhanced by binding of histone H3 to its PHD1 reader domain, through an unknown mechanism. Here the authors show that the PHD1 domain drives ligand-induced allosteric stimulation by stabilizing the binding of substrate to the catalytic domain.

Summary: Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH recapitulates previous GJC structures in lipid bilayers. At pH 6.4, we identify two conformational states, one resembling the open physiological-pH structure and a closed conformation that displays six threads of density, that join to form a pore-occluding density. Crosslinking and hydrogen-deuterium exchange mass spectrometry reveal closer association between the N-terminal (NT) domains and the cytoplasmic loops (CL) at acidic pH. Previous electrophysiologic studies suggest an association between NT residue N14 and H100 near M2, which may trigger the observed movement of M2 toward M1 in our cryo-EM maps, thereby accounting for additional NT-CL crosslinks at acidic pH. We propose that these pH-induced interactions and conformational changes result in extension, ordering, and association of the acetylated NT domains to form a hexameric “ball-and-chain” gating particle. : During tissue injury, acidic pH blocks gap junction channel intercellular communication. Khan et al. use cryo-EM and mass spectrometry to show that acidification causes extension, ordering, and association of the N-terminal domains to form a hexameric gating particle that sterically occludes the human Cx26 gap junction channel pore. Keywords: connexins, gap junction channels, pH gating, cryo-EM, mass spectrometry, hydrogen deuterium exchange, crosslinking, membrane proteins, protein structure, conformational changes

Smoothened receptors (SMO) play a key role in the Hedgehog signalling pathway. Here the authors present the structure of a multi-domain human SMO with a rationally designed stabilizing ligand bound in the transmembrane domain of the receptor, and propose a model for SMO activation.

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ERα)) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER +breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We found that BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show BZA’s selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations.

Somatic mutations in the estrogen receptor alpha (ERα) gene (ESR1), especially Y537S and D538G, have been linked to acquired resistance to endocrine therapies. Cell-based studies demonstrated that these mutants confer ERα constitutive activity and antiestrogen resistance and suggest that ligand-binding domain dysfunction leads to endocrine therapy resistance. Here, we integrate biophysical and structural biology data to reveal how these mutations lead to a constitutively active and antiestrogen-resistant ERα. We show that these mutant ERs recruit coactivator in the absence of hormone while their affinities for estrogen agonist (estradiol) and antagonist (4-hydroxytamoxifen) are reduced. Further, they confer antiestrogen resistance by altering the conformational dynamics of the loop connecting Helix 11 and Helix 12 in the ligand-binding domain of ERα, which leads to a stabilized agonist state and an altered antagonist state that resists inhibition.

Cartilage plays a crucial role in skeletal development and function, and abnormal development contributes to genetic and age-related skeletal disease. To better understand how human cartilage develops in vivo , we jointly profiled the transcriptome and open chromatin regions in individual nuclei recovered from distal femurs at 2 fetal timepoints. We used these multiomic data to identify transcription factors expressed in distinct chondrocyte subtypes, link accessible regulatory elements with gene expression, and predict transcription factor-based regulatory networks that are important for growth plate or epiphyseal chondrocyte differentiation. We developed a human pluripotent stem cell platform for interrogating the function of predicted transcription factors during chondrocyte differentiation and used it to test NFATC2 . We expect new regulatory networks we uncovered using multiomic data to be important for promoting cartilage health and treating disease, and our platform to be a useful tool for studying cartilage development in vitro ., Statement of Significance: The identity and integrity of the articular cartilage lining our joints are crucial to pain-free activities of daily living. Here we identified a gene regulatory landscape of human chondrogenesis at single cell resolution, which is expected to open new avenues of research aimed at mitigating cartilage diseases that affect hundreds of millions of individuals world-wide.

To address large gaps in our understanding of the molecular regulation of articular and growth plate cartilage development in humans, we used our directed differentiation approach to generate these distinct cartilage tissues from human embryonic stem cells. The resulting transcriptomic profiles of hESC-derived articular and growth plate chondrocytes were similar to fetal epiphyseal and growth plate chondrocytes, with respect to genes both known and previously unknown to cartilage biology. With the goal to characterize the regulatory landscapes accompanying these respective transcriptomes, we mapped chromatin accessibility in hESC-derived chondrocyte lineages, and mouse embryonic chondrocytes, using ATAC-sequencing. Integration of the expression dataset with the differentially accessible genomic regions revealed lineage-specific gene regulatory networks. We validated functional interactions of two transcription factors (TFs) (RUNX2 in growth plate chondrocytes and RELA in articular chondrocytes) with their predicted genomic targets. The maps we provide thus represent a framework for probing regulatory interactions governing chondrocyte differentiation. This work constitutes a substantial step towards comprehensive and comparative molecular characterizations of distinct chondrogenic lineages and sheds new light on human cartilage development and biology., Competing Interests: DR, SP, DV, RR, PM, ZL, TC, AC No competing interests declared, (© 2023, Richard, Pregizer et al.)

As pluripotent human embryonic stem cells progress toward one germ layer fate, they lose the ability to adopt alternative fates. Using a low-dimensional reaction coordinate to monitor progression toward ectoderm, we show that a differentiating stem cell's probability of adopting a mesendodermal fate given appropriate signals falls sharply at a point along the ectoderm trajectory. We use this reaction coordinate to prospectively isolate and profile differentiating cells based on their mesendoderm competence and analyze their RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) profiles to identify transcription factors that control the cell's mesendoderm competence. By modulating these key transcription factors, we can expand or contract the window of competence to adopt the mesendodermal fate along the ectodermal differentiation trajectory. The ability of the underlying gene regulatory network to modulate competence is essential for understanding human development and controlling the fate choices of stem cells in vitro., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)