A cytoplasmic COMPASS is necessary for cell survival and triple-negative breast cancer pathogenesis by regulating metabolism.

Mutations and translocations within the COMPASS (complex of proteins associated with Set1) family of histone lysine methyltransferases are associated with a large number of human diseases, including cancer. Here we report that SET1B/COMPASS, which is essential for cell survival, surprisingly has a cytoplasmic variant. SET1B, but not its SET domain, is critical for maintaining cell viability, indicating a novel catalytic-independent role of SET1B/COMPASS. Loss of SET1B or its unique cytoplasmic-interacting protein, BOD1, leads to up-regulation of expression of numerous genes modulating fatty acid metabolism, including ADIPOR1 (adiponectin receptor 1), COX7C, SDC4, and COQ7 Our detailed molecular studies identify ADIPOR1 signaling, which is inactivated in both obesity and human cancers, as a key target of SET1B/COMPASS. Collectively, our study reveals a cytoplasmic function for a member of the COMPASS family, which could be harnessed for therapeutic regulation of signaling in human diseases, including cancer.

A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation.

Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. Here we identify an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4-RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which we named the CryptoTudor domain. Using CRISPR-Cas9 genetic knockouts, we demonstrate that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, we demonstrate that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family.

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome.

Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A, and this occurred through suppression of IFN-responsive transcription factor activity. Moreover, we discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. Kinase inhibition altered splicing in pathway-specific ways and selectively affected IFN-responsive gene splicing in T21 cells. To further probe Mediator kinase function, we completed cytokine screens and untargeted metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways, including broad up-regulation of anti-inflammatory lipid mediators. Elevated levels of lipid mediators persisted at least 24hr after Mediator kinase inhibition, and many identified lipids serve as ligands for nuclear receptors (e.g. PPAR, LXR) or G-protein coupled receptors (GPCRs; e.g. FFAR4). Notably, ligand-dependent activation of these GPCRs or nuclear receptors will propagate anti-inflammatory signaling pathways and gene expression programs, and this mechanistic link suggests that metabolic changes caused by CDK8/CDK19 inhibition can durably and independently suppress pro-inflammatory IFN responses. Collectively, our results establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

An isothermal shift assay for proteome scale drug-target identification.

Most small molecule drugs act on living systems by physically interacting with specific proteins and modulating target function. Identification of drug binding targets, within the complex milieu of the human proteome, remains a challenging task of paramount importance in drug discovery. Existing approaches for target identification employ complex workflows with limited throughput. Here, we present the isothermal shift assay (iTSA), a mass spectrometry method for proteome-wide identification of drug targets within lysates or living cells. Compared with prevailing methods, iTSA uses a simplified experimental design with increased statistical power to detect thermal stability shifts that are induced by small molecule binding. Using a pan-kinase inhibitor, staurosporine, we demonstrate improved performance over commonly used thermal proteome profiling methods, identifying known targets in cell lysates and living cells. We also demonstrate the identification of both known targets and additional candidate targets for the kinase inhibitor harmine in cell and tissue lysates.

Resetting the epigenetic balance of Polycomb and COMPASS function at enhancers for cancer therapy.

The lysine methyltransferase KMT2C (also known as MLL3), a subunit of the COMPASS complex, implements monomethylation of Lys4 on histone H3 (H3K4) at gene enhancers. KMT2C (hereafter referred to as MLL3) frequently incurs point mutations across a range of human tumor types, but precisely how these lesions alter MLL3 function and contribute to oncogenesis is unclear. Here we report a cancer mutational hotspot in MLL3 within the region encoding its plant homeodomain (PHD) repeats and demonstrate that this domain mediates association of MLL3 with the histone H2A deubiquitinase and tumor suppressor BAP1. Cancer-associated mutations in the sequence encoding the MLL3 PHD repeats disrupt the interaction between MLL3 and BAP1 and correlate with poor patient survival. Cancer cells that had PHD-associated MLL3 mutations or lacked BAP1 showed reduced recruitment of MLL3 and the H3K27 demethylase KDM6A (also known as UTX) to gene enhancers. As a result, inhibition of the H3K27 methyltransferase activity of the Polycomb repressive complex 2 (PRC2) in tumor cells harboring BAP1 or MLL3 mutations restored normal gene expression patterns and impaired cell proliferation in vivo. This study provides mechanistic insight into the oncogenic effects of PHD-associated mutations in MLL3 and suggests that restoration of a balanced state of Polycomb-COMPASS activity may have therapeutic efficacy in tumors that bear mutations in the genes encoding these epigenetic factors.

Amyloid Accumulation Drives Proteome-wide Alterations in Mouse Models of Alzheimer's Disease-like Pathology.

Amyloid beta (Aß) peptides impair multiple cellular pathways and play a causative role in Alzheimer's disease (AD) pathology, but how the brain proteome is remodeled by this process is unknown. To identify protein networks associated with AD-like pathology, we performed global quantitative proteomic analysis in three mouse models at young and old ages. Our analysis revealed a robust increase in Apolipoprotein E (ApoE) levels in nearly all brain regions with increased Aß levels. Taken together with prior findings on ApoE driving Aß accumulation, this analysis points to a pathological dysregulation of the ApoE-Aß axis. We also found dysregulation of protein networks involved in excitatory synaptic transmission. Analysis of the AMPA receptor (AMPAR) complex revealed specific loss of TARPγ-2, a key AMPAR-trafficking protein. Expression of TARPγ-2 in hAPP transgenic mice restored AMPA currents. This proteomic database represents a resource for the identification of protein alterations responsible for AD.

Structural and Developmental Disparity in the Tentacles of the Moon Jellyfish Aurelia sp.1.

Tentacles armed with stinging cells (cnidocytes) are a defining trait of the cnidarians, a phylum that includes sea anemones, corals, jellyfish, and hydras. While cnidarian tentacles are generally characterized as structures evolved for feeding and defense, significant variation exists between the tentacles of different species, and within the same species across different life stages and/or body regions. Such diversity suggests cryptic distinctions exist in tentacle function. In this paper, we use confocal and transmission electron microscopy to contrast the structure and development of tentacles in the moon jellyfish, Aurelia species 1. We show that polyp oral tentacles and medusa marginal tentacles display markedly different cellular and muscular architecture, as well as distinct patterns of cellular proliferation during growth. Many structural differences between these tentacle types may reflect biomechanical solutions to different feeding strategies, although further work would be required for a precise mechanistic understanding. However, differences in cell proliferation dynamics suggests that the two tentacle forms lack a conserved mechanism of development, challenging the textbook-notion that cnidarian tentacles can be homologized into a conserved bauplan.