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Bone remodeling consists of resorption by osteoclasts followed by formation by osteoblasts, and osteoclasts are a source of bone formation-stimulating factors. Here we utilize osteoclast ablation by denosumab (DMAb) and RNA-sequencing of bone biopsies from postmenopausal women to identify osteoclast-secreted factors suppressed by DMAb. Based on these analyses, LIF, CREG2, CST3, CCBE1 , and DPP4 are likely osteoclast-derived coupling factors in humans. Given the role of Dipeptidyl Peptidase-4 (DPP4) in glucose homeostasis, we further demonstrate that DMAb-treated participants have a significant reduction in circulating DPP4 and increase in Glucagon-like peptide (GLP)-1 levels as compared to the placebo-treated group, and also that type 2 diabetic patients treated with DMAb show significant reductions in HbA1c as compared to patients treated either with bisphosphonates or calcium and vitamin D. Thus, our results identify several coupling factors in humans and uncover osteoclast-derived DPP4 as a potential link between bone remodeling and energy metabolism. Anti-resorptive bone therapies also inhibit bone formation, as osteoclasts secrete factors that stimulate bone formation by osteoblasts. Here, the authors identify osteoclast-secreted factors that couple bone resorption to bone formation in healthy subjects, and show that osteoclast-derived DPP4 may be a factor coupling bone resorption to energy metabolism.

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin’s favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities. Alteration of the epigenetic landscape has been implicated in several disease processes, where targeting histone modifiers may have therapeutic applications. Here the authors report a bifunctional small molecule inhibitor that simultaneously targets the deacetylase (HDAC1) and demethylase (LSD1) activities of the CoREST complex.

The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitination of the DEUBAD is a general feature of ASXLs and Asx. BAP1 promotes DEUBAD monoubiquitination resulting in an increased stability of ASXL2, which in turn stimulates BAP1 DUB activity. ASXL2 monoubiquitination is directly catalyzed by UBE2E family of Ubiquitin-conjugating enzymes and regulates mammalian cell proliferation. Remarkably, Calypso also regulates Asx monoubiquitination and transgenic flies expressing monoubiquitination-defective Asx mutant exhibit developmental defects. Finally, the protein levels of ASXL2, BAP1 and UBE2E enzymes are highly correlated in mesothelioma tumors suggesting the importance of this signaling axis for tumor suppression. We propose that monoubiquitination orchestrates a molecular symbiosis relationship between ASXLs and BAP1. Additional sex combs-like (ASXLs) stimulate BAP1 deubiquitinase activity to induce tumor suppression, but how these complexes work in coordination in vivo is unclear. Here, the authors show the mutually reinforcing roles of BAP1 and ASXLs such that BAP1 promotes DEUBAD monoubiquitination of ASXL2, which in turn stimulates BAP1 DUB activity.

Stem cell proviral silencing Endogenous retroviruses are widely dispersed in mammalian genomes, and are silenced in somatic cells by DNA methylation. Here, an endogenous retroviruses silencing pathway independent of DNA methylation is shown to operate in embryonic stem cells. The pathway involves the histone H3K9 methyltransferase ESET/SETDB1 and might be important for endogenous retrovirus silencing during the stages in embryogenesis when DNA methylation is reprogrammed. Endogenous retroviruses (ERVs) are widely dispersed in mammalian genomes, and are silenced in somatic cells by DNA methylation. Here, an ERV silencing pathway independent of DNA methylation is shown to operate in embryonic stem cells. The pathway involves the histone H3K9 methyltransferase ESET and might be important for ERV silencing during the stages in embryogenesis when DNA methylation is reprogrammed. Endogenous retroviruses (ERVs), retrovirus-like elements with long terminal repeats, are widely dispersed in the euchromatic compartment in mammalian cells, comprising ∼10% of the mouse genome 1 . These parasitic elements are responsible for >10% of spontaneous mutations 2 . Whereas DNA methylation has an important role in proviral silencing in somatic and germ-lineage cells 3 , 4 , 5 , an additional DNA-methylation-independent pathway also functions in embryonal carcinoma and embryonic stem (ES) cells to inhibit transcription of the exogenous gammaretrovirus murine leukaemia virus (MLV) 6 , 7 , 8 . Notably, a recent genome-wide study revealed that ERVs are also marked by histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3 in ES cells but not in mouse embryonic fibroblasts 9 . However, the role that these marks have in proviral silencing remains unexplored. Here we show that the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and the Krüppel-associated box (KRAB)-associated protein 1 (KAP1, also called TRIM28) 10 , 11 are required for H3K9me3 and silencing of endogenous and introduced retroviruses specifically in mouse ES cells. Furthermore, whereas ESET enzymatic activity is crucial for HP1 binding and efficient proviral silencing, the H4K20 methyltransferases Suv420h1 and Suv420h2 are dispensable for silencing. Notably, in DNA methyltransferase triple knockout ( Dnmt1 -/- Dnmt3a -/- Dnmt3b -/- ) mouse ES cells, ESET and KAP1 binding and ESET-mediated H3K9me3 are maintained and ERVs are minimally derepressed. We propose that a DNA-methylation-independent pathway involving KAP1 and ESET/ESET-mediated H3K9me3 is required for proviral silencing during the period early in embryogenesis when DNA methylation is dynamically reprogrammed.

Driver genes with a mutually exclusive mutation pattern across tumor genomes are thought to have overlapping roles in tumorigenesis. In contrast, we show here that mutually exclusive prostate cancer driver alterations involving the ERG transcription factor and the ubiquitin ligase adaptor SPOP are synthetic sick. At the molecular level, the incompatible cancer pathways are driven by opposing functions in SPOP. ERG upregulates wild type SPOP to dampen androgen receptor (AR) signaling and sustain ERG activity through degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG-function and enable oncogenic androgen receptor signaling. This dichotomy regulates the response to therapeutic interventions in the AR pathway. While mutant SPOP renders tumor cells susceptible to androgen deprivation therapies, ERG promotes sensitivity to high-dose androgen therapy and pharmacological inhibition of wild type SPOP. More generally, these results define a distinct class of antagonistic cancer drivers and a blueprint toward their therapeutic exploitation. Gene fusions involving the ERG transcription factor and point mutations in the ubiquitin ligase adaptor SPOP are two truncal mutations that are mutually exclusively present in prostate cancer. Here, the authors show that mutations in SPOP render prostate tumor cells sensitive to antiandrogen therapy and that the presence of ERG promotes sensitivity to high dose of androgen and SPOP inhibition.

NR4A1 is shown to be an endogenous inhibitor of TGF-β-induced fibrosis and represents a therapeutic target for this condition. Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific binding of a lac repressor, labeled with a fluorescent protein, to a chromosomal lac operator. Using single-molecule detection techniques, we measured the kinetics of binding and dissociation of the repressor in response to metabolic signals. Furthermore, we characterized the nonspecific binding to DNA, one-dimensional (1D) diffusion along DNA segments, and 3D translocation among segments through cytoplasm at the single-molecule level. In searching for the operator, a lac repressor spends ~90% of time nonspecifically bound to and diffusing along DNA with a residence time of <5 milliseconds. The methods and findings can be generalized to other nucleic acid binding proteins.

Leaf size and shape are mainly determined by coordinated cell division and differentiation in lamina. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors are key regulators of leaf development. However, the mechanisms that control TCP activities during leaf development are largely unknown. We identified the TCP Interactor containing EAR motif protein1 (TIE1), a novel transcriptional repressor, as a major modulator of TCP activities during leaf development. Overexpression of TIE1 leads to hyponastic and serrated leaves, whereas disruption of TIE1 causes epinastic leaves. TIE1 is expressed in young leaves and encodes a transcriptional repressor containing a C-terminal EAR motif, which mediates interactions with the TOPLESS (TPL)/TOPLESS-RELATED (TPR) corepressors. In addition, TIE1 physically interacts with CIN-like TCPs. We propose that TIE1 regulates leaf size and morphology by inhibiting the activities of TCPs through recruiting the TPL/TPR corepressors to form a tertiary complex at early stages of leaf development.

Dysregulation of the transcriptional repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor is important in a broad range of diseases, including cancer, diabetes, and heart disease. The role of REST-dependent epigenetic modifications in neurodegeneration is less clear. Here, we show that neuronal insults trigger activation of REST and CoREST in a clinically relevant model of ischemic stroke and that REST binds a subset of \"transcriptionally responsive\" genes (gria2, grin1, chrnb2, nefh, nfκb2, trpv1, chrm4, and syt6), of which the AMPA receptor subunit GluA2 is a top hit. Genes with enriched REST exhibited decreased mRNA and protein. We further show that REST assembles with CoREST, mSin3A, histone deacetylases 1 and 2, histone methyl-transferase G9a, and methyl CpG binding protein 2 at the promoters of target genes, where it orchestrates epigenetic remodeling and gene silencing. RNAi-mediated depletion of REST or administration of dominant-negative REST delivered directly into the hippocampus in vivo prevents epigenetic modifications, restores gene expression, and rescues hippocampal neurons. These findings document a causal role for REST-dependent epigenetic remodeling in the neurodegeneration associated with ischemic stroke and identify unique therapeutic targets for the amelioration of hippocampal injury and cognitive deficits.

Key messageSCL3 inhibits transcriptional activity of IDD-DELLA complex by acting as a co-repressor and repression activity is enhanced in the presence of GAF1 in a TOPLESS-independent manner. GRAS [GIBBERELLIN-INSENSITIVE (GAI), REPRESSOR OF ga1-3 (RGA) and SCARECROW (SCR)] proteins are a family of plant-specific transcriptional regulators that play diverse roles in development and signaling. GRAS family DELLA proteins act as growth repressors by inhibiting gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also act as co-activators of transcription factor GAI-ASSOCIATED FACTOR1 (GAF1)/INDETERMINATE DOMAIN2 (IDD2), the GAF1-DELLA complex activating transcription of GAF1 target genes. GAF1 also interacts with TOPLESS (TPL), a transcriptional co-repressor, in the absence of DELLA, the GAF1-TPL complex repressing transcription of the target genes. SCARECROW-LIKE3 (SCL3), another member of the GRAS family, is thought to inhibit transcriptional activity of the IDD-DELLA complex through competitive interaction with IDD. Here, we also revealed that SCL3 inhibits transcriptional activation by the GAF1-DELLA complex via repression activity rather than via competitive inhibition of the GAF1-DELLA interaction. Moreover, the repression activity of SCL3 was enhanced by GAF1 in a TPL-independent manner. While the GRAS domain of DELLA has transcriptional activation activity, that of SCL3 has repression activity. SCL3 also inhibited transcriptional activity of GAF1-RGA fusion proteins. Results from the co-immunoprecipitation assays and the yeast three-hybrid assay suggested the possibility that SCL3 forms a ternary complex with GAF1 and DELLA. These findings provide important information on DELLA-regulated GA signaling and new insight into the transcriptional repression mechanism.