Braveheart, a long noncoding RNA required for cardiovascular lineage commitment.

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.

H2A.Z acidic patch couples chromatin dynamics to regulation of gene expression programs during ESC differentiation.

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z(AP3)) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z(AP3) interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z(AP3) was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z(AP3) ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z(AP3) ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z(AP3) displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z(AP3) mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment.

Phosphate dysregulation via the XPR1-KIDINS220 protein complex is a therapeutic vulnerability in ovarian cancer.

Despite advances in precision medicine, the clinical prospects for patients with ovarian and uterine cancers have not substantially improved. Here, we analyzed genome-scale CRISPR-Cas9 loss-of-function screens across 851 human cancer cell lines and found that frequent overexpression of SLC34A2-encoding a phosphate importer-is correlated with sensitivity to loss of the phosphate exporter XPR1, both in vitro and in vivo. In patient-derived tumor samples, we observed frequent PAX8-dependent overexpression of SLC34A2, XPR1 copy number amplifications and XPR1 messenger RNA overexpression. Mechanistically, in SLC34A2-high cancer cell lines, genetic or pharmacologic inhibition of XPR1-dependent phosphate efflux leads to the toxic accumulation of intracellular phosphate. Finally, we show that XPR1 requires the novel partner protein KIDINS220 for proper cellular localization and activity, and that disruption of this protein complex results in acidic "vacuolar" structures preceding cell death. These data point to the XPR1-KIDINS220 complex and phosphate dysregulation as a therapeutic vulnerability in ovarian cancer.

ATRAID regulates the action of nitrogen-containing bisphosphonates on bone.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.

The sensitivity of yeast mutants to oleic acid implicates the peroxisome and other processes in membrane function.

The peroxisome, sole site of beta-oxidation in Saccharomyces cerevisiae, is known to be required for optimal growth in the presence of fatty acid. Screening of the haploid yeast deletion collection identified approximately 130 genes, 23 encoding peroxisomal proteins, necessary for normal growth on oleic acid. Oleate slightly enhances growth of wild-type yeast and inhibits growth of all strains identified by the screen. Nonperoxisomal processes, among them chromatin modification by H2AZ, Pol II mediator function, and cell-wall-associated activities, also prevent oleate toxicity. The most oleate-inhibited strains lack Sap190, a putative adaptor for the PP2A-type protein phosphatase Sit4 (which is also required for normal growth on oleate) and Ilm1, a protein of unknown function. Palmitoleate, the other main unsaturated fatty acid of Saccharomyces, fails to inhibit growth of the sap190delta, sit4delta, and ilm1delta strains. Data that suggest that oleate inhibition of the growth of a peroxisomal mutant is due to an increase in plasma membrane porosity are presented. We propose that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids.

Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates.

Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here, we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

H2A.Z.1 Monoubiquitylation Antagonizes BRD2 to Maintain Poised Chromatin in ESCs.

Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in embryonic stem cells (ESCs) to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 monoubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1(K3R3)). We show that H2A.Z.1(K3R3) is properly incorporated at target promoters in murine ESCs (mESCs), but loss of monoubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member BRD2, are enriched in H2A.Z.1 chromatin. We further show that BRD2 is gained at de-repressed promoters in H2A.Z.1(K3R3) mESCs, whereas BRD2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and BRD2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs.

Polycomb group proteins set the stage for early lineage commitment.

Precise control of gene expression patterns is critical for the specification of cellular diversity during metazoan development. Polycomb group (PcG) proteins comprise a class of transcriptional modifiers that have dynamic and essential roles in regulating a number of key processes including lineage commitment. How this is accomplished during mammalian development is incompletely understood. Here, we discuss recent studies in embryonic stem cells (ESCs) that provide critical new insights into how PcG proteins may be targeted to genomic sites as well as the mechanisms by which these regulators influence gene expression and multilineage differentiation in mammals.