Sexual Misconduct by Board Certified Family Physicians.

Purpose: Sexual misconduct by physicians is a consequential violation of patient trust. The purpose of this study was to determine the frequency and patterns of sexual misconduct by physicians certified by the American Board of Family Medicine (ABFM).

Methods: We described a cohort of current or formerly ABFM certified physicians ("Diplomates") disciplined for sexual misconduct in 2016 to 2022.

CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters.

CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches.

The H3K36me2 writer-reader dependency in H3K27M-DIPG.

Histone H3K27M is a driving mutation in diffuse intrinsic pontine glioma (DIPG), a deadly pediatric brain tumor. H3K27M reshapes the epigenome through a global inhibition of PRC2 catalytic activity and displacement of H3K27me2/3, promoting oncogenesis of DIPG. As a consequence, a histone modification H3K36me2, antagonistic to H3K27me2/3, is aberrantly elevated.

An Esrrb and Nanog Cell Fate Regulatory Module Controlled by Feed Forward Loop Interactions.

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory "dimensions" coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator.

RNA-Mediated Feedback Control of Transcriptional Condensates.

Regulation of biological processes typically incorporates mechanisms that initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively, but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism where low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates.

MeCP2 links heterochromatin condensates and neurodevelopmental disease.

Methyl CpG binding protein 2 (MeCP2) is a key component of constitutive heterochromatin, which is crucial for chromosome maintenance and transcriptional silencing. Mutations in the MECP2 gene cause the progressive neurodevelopmental disorder Rett syndrome, which is associated with severe mental disability and autism-like symptoms that affect girls during early childhood. Although previously thought to be a dense and relatively static structure, heterochromatin is now understood to exhibit properties consistent with a liquid-like condensate.

LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells.

FACT (facilitates chromatin transcription) is a protein complex that allows RNA polymerase II (RNAPII) to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation.

Automethylation of PRC2 promotes H3K27 methylation and is impaired in H3K27M pediatric glioma.

The histone methyltransferase activity of PRC2 is central to the formation of H3K27me3-decorated facultative heterochromatin and gene silencing. In addition, PRC2 has been shown to automethylate its core subunits, EZH1/EZH2 and SUZ12. Here, we identify the lysine residues at which EZH1/EZH2 are automethylated with EZH2-K510 and EZH2-K514 being the major such sites in vivo.

Capturing the Onset of PRC2-Mediated Repressive Domain Formation.

Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus.

Allosteric Activation Dictates PRC2 Activity Independent of Its Recruitment to Chromatin.

PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin.

Low-Grade Astrocytoma Mutations in IDH1, P53, and ATRX Cooperate to Block Differentiation of Human Neural Stem Cells via Repression of SOX2.

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs.

Identification of Nidogen 1 as a lung metastasis protein through secretome analysis.

Secreted proteins play crucial roles in mediating tumor-stroma interactions during metastasis of cancer to different target organs. To comprehensively profile secreted proteins involved in lung metastasis, we applied quantitative mass spectrometry-based proteomics and identified 392 breast cancer-derived and 302 melanoma-derived proteins secreted from highly lung metastatic cells. The cancer-specific lung metastasis secretome signatures (LMSSs) displayed significant prognostic value in multiple cancer clinical data sets.

Chromatin Starts to Come Clean.

In an effort to identify a chromatin-associated pluripotent network, Rafiee et al., (2016) developed a powerful ChIP-MS technique and discovered a novel protein, TRIM24, enriched on OCT4-, SOX2-, and NANOG-associated chromatin, paving the way for future proteomic studies on chromatin.

Multi-faceted quantitative proteomics analysis of histone H2B isoforms and their modifications.

Background: Histone isoforms and their post-translational modifications (PTMs) play an important role in the control of many chromatin-related processes including transcription and DNA damage. Variants of histones H2A and H3 have been studied in depth and have been found to have distinct functions. Although 13 somatic histone H2B isoforms have been identified by various biochemical and mass spectrometric (MS) approaches, the distinct roles of these isoforms within human cells are as yet unknown.

Proteogenomics analysis reveals specific genomic orientations of distal regulatory regions composed by non-canonical histone variants.

Background: Histone variants play further important roles in DNA packaging and controlling gene expression. However, our understanding about their composition and their functions is limited.

Results: Integrating proteomic and genomic approaches, we performed a comprehensive analysis of the epigenetic landscapes containing the four histone variants H3.

BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones.

Small-molecule BET inhibitors interfere with the epigenetic interactions between acetylated histones and the bromodomains of the BET family proteins, including BRD4, and they potently inhibit growth of malignant cells by targeting cancer-promoting genes. BRD4 interacts with the pause-release factor P-TEFb and has been proposed to release RNA polymerase II (Pol II) from promoter-proximal pausing. We show that BRD4 occupies widespread genomic regions in mouse cells and directly stimulates elongation of both protein-coding transcripts and noncoding enhancer RNAs (eRNAs), in a manner dependent on bromodomain function.

H3R42me2a is a histone modification with positive transcriptional effects.

Histone posttranslational modification leads to downstream effects indirectly by allowing or preventing docking of effector molecules, or directly by changing the intrinsic biophysical properties of local chromatin. To date, little has been done to study posttranslational modifications that lie outside of the unstructured tail domains of histones. Core residues, and in particular arginines in H3 and H4, mediate key interactions between the histone octamer and DNA in forming the nucleosomal particle.

A quantitative atlas of histone modification signatures from human cancer cells.

Background: An integral component of cancer biology is the understanding of molecular properties uniquely distinguishing one cancer type from another. One class of such properties is histone post-translational modifications (PTMs). Many histone PTMs are linked to the same diverse nuclear functions implicated in cancer development, including transcriptional activation and epigenetic regulation, which are often indirectly assayed with standard genomic technologies.

Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation.

How oncogenic signalling coordinates glycolysis and anabolic biosynthesis in cancer cells remains unclear. We recently reported that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate and product 2-phosphoglycerate. Here we report a novel mechanism in which Y26 phosphorylation enhances PGAM1 activation through release of inhibitory E19 that blocks the active site, stabilising cofactor 2,3-bisphosphoglycerate binding and H11 phosphorylation.

MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency.

The chromatin template imposes an epigenetic barrier during the process of somatic cell reprogramming. Using fibroblasts derived from macroH2A double knockout (dKO) mice, here we show that these histone variants act cooperatively as a barrier to induced pluripotency. Through manipulation of macroH2A isoforms, we further demonstrate that macroH2A2 is the predominant barrier to reprogramming.

SETD6 monomethylates H2AZ on lysine 7 and is required for the maintenance of embryonic stem cell self-renewal.

The histone H2A variant H2AZ is an essential chromatin signaling factor. Herein, we report that H2AZ is monomethylated at lysine 7 (H2AZK7me1) by the lysine methyltransferase SETD6. We observed that methylation of H2AZ increased noticeably upon cellular differentiation of mouse embryonic stem cells (mESCs).

Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila.

X-chromosome dosage compensation by the MSL (male-specific lethal) complex is required in Drosophila melanogaster to increase gene expression from the single male X to equal that of both female X chromosomes. Instead of focusing solely on protein complexes released from DNA, here we used chromatin-interacting protein MS (ChIP-MS) to identify MSL interactions on cross-linked chromatin. We identified MSL-enriched histone modifications, including histone H4 Lys16 acetylation and histone H3 Lys36 methylation, and CG4747, a putative Lys36-trimethylated histone H3 (H3K36me3)-binding protein.

Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth.

It is unclear how cancer cells coordinate glycolysis and biosynthesis to support rapidly growing tumors. We found that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, contributes to biosynthesis regulation in part by controlling intracellular levels of its substrate, 3-phosphoglycerate (3-PG), and product, 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels.