Runx1-R188Q germ line mutation induces inflammation and predisposition to hematologic malignancies in mice.

Germ line mutations in the RUNX1 gene cause familial platelet disorder (FPD), an inherited disease associated with lifetime risk to hematopoietic malignancies (HM). Patients with FPD frequently show clonal expansion of premalignant cells preceding HM onset. Despite the extensive studies on the role of RUNX1 in hematopoiesis, its function in the premalignant bone marrow (BM) is not well-understood. Here, we characterized the hematopoietic progenitor compartments using a mouse strain carrying an FPD-associated mutation, Runx1R188Q. Immunophenotypic analysis showed an increase in the number of hematopoietic stem and progenitor cells (HSPCs) in the Runx1R188Q/+ mice. However, the comparison of Sca-1 and CD86 markers suggested that Sca-1 expression may result from systemic inflammation. Cytokine profiling confirmed the dysregulation of interferon-response cytokines in the BM. Furthermore, the expression of CD48, another inflammation-response protein, was also increased in Runx1R188Q/+ HSPCs. The DNA-damage response activity of Runx1R188Q/+ hematopoietic progenitor cells was defective in vitro, suggesting that Runx1R188Q may promote genomic instability. The differentiation of long-term repopulating HSCs was reduced in Runx1R188Q/+ recipient mice. Furthermore, we found that Runx1R188Q/+ HSPCs outcompete their wild-type counterparts in bidirectional repopulation assays, and that the genetic makeup of recipient mice did not significantly affect the clonal dynamics under this setting. Finally, we demonstrate that Runx1R188Q predisposes to HM in cooperation with somatic mutations found in FPDHM, using 3 mouse models. These studies establish a novel murine FPDHM model and demonstrate that germ line Runx1 mutations induce a premalignant phenotype marked by BM inflammation, selective expansion capacity, defective DNA-damage response, and predisposition to HM.

Replication Gaps Underlie BRCA Deficiency and Therapy Response.

Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes BRCA1 and BRCA2 (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. SIGNIFICANCE: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype "BRCAness," yielding promising biomarkers, targets, and opportunities to resensitize refractory disease.See related commentary by Canman, p. 1214.

Lats1/2 Sustain Intestinal Stem Cells and Wnt Activation through TEAD-Dependent and Independent Transcription.

Intestinal homeostasis is tightly regulated by complex yet poorly understood signaling networks. Here, we demonstrate that Lats1/2, the core Hippo kinases, are essential to maintain Wnt pathway activity and intestinal stem cells. Lats1/2 deletion leads to loss of intestinal stem cells but drives Wnt-uncoupled crypt expansion. To explore the function of downstream transcriptional enhanced associate domain (TEAD) transcription factors, we identified a selective small-molecule reversible inhibitor of TEAD auto-palmitoylation that directly occupies its lipid-binding site and inhibits TEAD-mediated transcription in vivo. Combining this chemical tool with genetic and proteomics approaches, we show that intestinal Wnt inhibition by Lats deletion is Yes-associated protein (YAP)/transcriptional activator with PDZ-binding domain (TAZ) dependent but TEAD independent. Mechanistically, nuclear YAP/TAZ interact with Groucho/Transducin-Like Enhancer of Split (TLE) to block Wnt/T-cell factor (TCF)-mediated transcription, and dual inhibition of TEAD and Lats suppresses Wnt-uncoupled Myc upregulation and epithelial over-proliferation in Adenomatous polyposis coli (APC)-mutated intestine. Our studies highlight a pharmacological approach to inhibit TEAD palmitoylation and have important implications for targeting Wnt and Hippo signaling in human malignancies.

YAP/TAZ Activation Drives Uveal Melanoma Initiation and Progression.

Uveal melanoma (UM), the most common ocular malignancy, is characterized by GNAQ/11 mutations. Hippo/YAP and Ras/mitogen-activated protein kinase (MAPK) emerge as two important signaling pathways downstream of G protein alpha subunits of the Q class (GαQ/11)-mediated transformation, although whether and how they contribute to UM genesis in vivo remain unclear. Here, we adapt an adeno-associated virus (AAV)-based ocular injection method to directly deliver Cre recombinase into the mouse uveal tract and demonstrate that Lats1/2 kinases suppress UM formation specifically in uveal melanocytes. We find that genetic activation of YAP, but not Kras, is sufficient to initiate UM. We show that YAP/TAZ activation induced by Lats1/2 deletion cooperates with Kras to promote UM progression via downstream transcriptional reinforcement. Furthermore, dual inhibition of YAP/TAZ and Ras/MAPK synergizes to suppress oncogenic growth of human UM cells. Our data highlight the functional significance of Lats-YAP/TAZ in UM initiation and progression in vivo and suggest combination inhibition of YAP/TAZ and Ras/MAPK as a new therapeutic strategy for UM.

IMP3 Stabilization of WNT5B mRNA Facilitates TAZ Activation in Breast Cancer.

Insulin-like growth factor-2 mRNA-binding protein 3 (IMP3) is an oncofetal protein associated with many aggressive cancers and implicated in the function of breast cancer stem cells (CSCs). The mechanisms involved, however, are poorly understood. We observed that IMP3 facilitates the activation of TAZ, a transcriptional co-activator of Hippo signaling that is necessary for the function of breast CSCs. The mechanism by which IMP3 activates TAZ involves both mRNA stability and transcriptional regulation. IMP3 stabilizes the mRNA of an alternative WNT ligand (WNT5B) indirectly by repressing miR145-5p, which targets WNT5B, resulting in TAZ activation by alternative WNT signaling. IMP3 also facilitates the transcription of SLUG, which is necessary for TAZ nuclear localization and activation, by a mechanism that is also mediated by WNT5B. These results demonstrate that TAZ can be regulated by an mRNA-binding protein and that this regulation involves the integration of Hippo and alternative WNT-signaling pathways.

Cbx4 Sumoylates Prdm16 to Regulate Adipose Tissue Thermogenesis.

Transcriptional co-activator Prdm16 controls brown fat development and white fat browning, but how this thermogenic function is modulated post-translationally is poorly understood. Here, we report that Cbx4, a Polycomb group protein, is a SUMO E3 ligase for Prdm16 and that Cbx4-mediated sumoylation of Prdm16 is required for thermogenic gene expression. Cbx4 expression is enriched in brown fat and is induced in adipose tissue by acute cold exposure. Sumoylation of Prdm16 at lysine 917 by Cbx4 blocks its ubiquitination-mediated degradation, thereby augmenting its stability and thermogenic function. Moreover, this sumoylation event primes Prdm16 to be further stabilized by methyltransferase Ehmt1. Heterozygous Cbx4-knockout mice develop metabolic phenotypes resembling those of Prdm16-knockout mice. Furthermore, fat-specific Cbx4 knockdown and overexpression produce remarkable, opposite effects on white fat remodeling. Our results identify a modifying enzyme for Prdm16, and they demonstrate a central role of Cbx4 in the control of Prdm16 stability and white fat browning.

MELK Promotes Melanoma Growth by Stimulating the NF-κB Pathway.

Melanoma accounts for more than 80% of skin cancer-related deaths, and current therapies provide only short-term benefit to patients. Here, we show in melanoma cells that maternal embryonic leucine zipper kinase (MELK) is transcriptionally upregulated by the MAPK pathway via transcription factor E2F1. MELK knockdown or pharmacological inhibition blocked melanoma growth and enhanced the effectiveness of BRAFV600E inhibitor against melanoma cells. To identify mediators of MELK function, we performed stable isotope labeling with amino acids in cell culture (SILAC) and identified 469 proteins that had downregulated phosphorylation after MELK inhibition. Of these proteins, 139 were previously reported as substrates of BRAF or MEK, demonstrating that MELK is an important downstream mediator of the MAPK pathway. Furthermore, we show that MELK promotes melanoma growth by activating NF-κB pathway activity via Sequestosome 1 (SQSTM1/p62). Altogether, these results underpin an important role for MELK in melanoma growth downstream of the MAPK pathway.

Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs.

During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

YAP/TAZ and Hedgehog Coordinate Growth and Patterning in Gastrointestinal Mesenchyme.

YAP/TAZ are the major mediators of mammalian Hippo signaling; however, their precise function in the gastrointestinal tract remains poorly understood. Here we dissect the distinct roles of YAP/TAZ in endodermal epithelium and mesenchyme and find that, although dispensable for gastrointestinal epithelial development and homeostasis, YAP/TAZ function as the critical molecular switch to coordinate growth and patterning in gut mesenchyme. Our genetic analyses reveal that Lats1/2 kinases suppress expansion of the primitive mesenchymal progenitors, where YAP activation also prevents induction of the smooth muscle lineage through transcriptional repression of Myocardin. During later development, zone-restricted downregulation of YAP/TAZ provides the positional cue and allows smooth muscle cell differentiation induced by Hedgehog signaling. Taken together, our studies identify the mesenchymal requirement of YAP/TAZ in the gastrointestinal tract and highlight the functional interplays between Hippo and Hedgehog signaling underlying temporal and spatial control of tissue growth and specification in developing gut.

Transcription factor Hlx controls a systematic switch from white to brown fat through Prdm16-mediated co-activation.

Browning of subcutaneous white fat (iWAT) involves several reprograming events, but the underlying mechanisms are incompletely understood. Here we show that the transcription factor Hlx is selectively expressed in brown adipose tissue (BAT) and iWAT, and is translationally upregulated by ß3-adrenergic signaling-mediated suppression of the translational inhibitor 4E-BP1. Hlx interacts with and is co-activated by Prdm16 to control BAT-selective gene expression and mitochondrial biogenesis. Hlx heterozygous knockout mice have defects in brown-like adipocyte formation in iWAT, and develop glucose intolerance and high fat-induced hepatic steatosis. Conversely, transgenic expression of Hlx at a physiological level drives a full program of thermogenesis and converts iWAT to brown-like fat, which improves glucose homeostasis and prevents obesity and hepatic steatosis. The adipose remodeling phenotypes are recapitulated by fat-specific injection of Hlx knockdown and overexpression viruses, respectively. Our studies establish Hlx as a powerful regulator for systematic white adipose tissue browning and offer molecular insights into the underlying transcriptional mechanism.The transcriptional co-activator Prdm16 regulates browning of white adipose tissue (WAT). Here, the authors show that Prdm16 interacts with the transcription factor Hlx, which is stabilized in response to ß3-adrenergic signaling, to increase thermogenic gene expression and mitochondrial biogenesis in subcutaneous WAT.

DNA methylation directs genomic localization of Mbd2 and Mbd3 in embryonic stem cells.

Cytosine methylation is an epigenetic and regulatory mark that functions in part through recruitment of chromatin remodeling complexes containing methyl-CpG binding domain (MBD) proteins. Two MBD proteins, Mbd2 and Mbd3, were previously shown to bind methylated or hydroxymethylated DNA, respectively; however, both of these findings have been disputed. Here, we investigated this controversy using experimental approaches and re-analysis of published data and find no evidence for methylation-independent functions of Mbd2 or Mbd3. We show that chromatin localization of Mbd2 and Mbd3 is highly overlapping and, unexpectedly, we find Mbd2 and Mbd3 are interdependent for chromatin association. Further investigation reveals that both proteins are required for normal levels of cytosine methylation and hydroxymethylation in murine embryonic stem cells. Furthermore, Mbd2 and Mbd3 regulate overlapping sets of genes that are also regulated by DNA methylation/hydroxymethylation factors. These findings reveal an interdependent regulatory mechanism mediated by the DNA methylation machinery and its readers.

Regulation of X-linked gene expression during early mouse development by Rlim.

Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.

Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors.

Vascular endothelial growth factor C (Vegfc) activates its receptor, Flt4, to induce lymphatic development. However, the signals that act downstream of Flt4 in this context in vivo remain unclear. To understand Flt4 signaling better, we generated zebrafish bearing a deletion in the Flt4 cytoplasmic domain that eliminates tyrosines Y1226 and 1227. Embryos bearing this deletion failed to initiate sprouting or differentiation of trunk lymphatic vessels and did not form a thoracic duct. Deletion of Y1226/7 prevented ERK phosphorylation in lymphatic progenitors, and ERK inhibition blocked trunk lymphatic sprouting and differentiation. Conversely, endothelial autonomous ERK activation rescued lymphatic sprouting and differentiation in flt4 mutants. Interestingly, embryos bearing the Y1226/7 deletion formed a functional facial lymphatic network enabling them to develop normally to adulthood. By contrast, flt4 null larvae displayed hypoplastic facial lymphatics and severe lymphedema. Thus, facial lymphatic vessels appear to be the first functional lymphatic network in the zebrafish, whereas the thoracic duct is initially dispensable for lymphatic function. Moreover, distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations.

Vegfa signals through ERK to promote angiogenesis, but not artery differentiation.

Vascular endothelial growth factor a (Vegfa) is essential for blood vessel formation and can induce activation of numerous signaling effectors in endothelial cells. However, it is unclear how and where these function in developmental contexts during vascular morphogenesis. To address this issue, we have visualized activation of presumptive Vegfa effectors at single-cell resolution in zebrafish blood vessels. From these studies, we find that phosphorylation of the serine/threonine kinase ERK (pERK) preferentially occurs in endothelial cells undergoing angiogenesis, but not in committed arterial endothelial cells. pERK in endothelial cells was ectopically induced by Vegfa and lost in Vegfa signaling mutants. Both chemical and endothelial autonomous inhibition of ERK prevented endothelial sprouting, but did not prevent initial artery differentiation. Timed chemical inhibition during angiogenesis caused a loss of genes implicated in coordinating tip/stalk cell behaviors, including flt4 and, at later stages, dll4 ERK inhibition also blocked excessive angiogenesis and ectopic flt4 expression in Notch-deficient blood vessels. Together, these studies implicate ERK as a specific effector of Vegfa signaling in the induction of angiogenic genes during sprouting.

Tead and AP1 Coordinate Transcription and Motility.

The Tead family transcription factors are the major intracellular mediators of the Hippo-Yap pathway. Despite the importance of Hippo signaling in tumorigenesis, Tead-dependent downstream oncogenic programs and target genes in cancer cells remain poorly understood. Here, we characterize Tead4-mediated transcriptional networks in a diverse range of cancer cells, including neuroblastoma, colorectal, lung, and endometrial carcinomas. By intersecting genome-wide chromatin occupancy analyses of Tead4, JunD, and Fra1/2, we find that Tead4 cooperates with AP1 transcription factors to coordinate target gene transcription. We find that Tead-AP1 interaction is JNK independent but engages the SRC1-3 co-activators to promote downstream transcription. Furthermore, we show that Tead-AP1 cooperation regulates the activity of the Dock-Rac/CDC42 module and drives the expression of a unique core set of target genes, thereby directing cell migration and invasion. Together, our data unveil a critical regulatory mechanism underlying Tead- and AP1-controlled transcriptional and functional outputs in cancer cells.

Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

Jmjd3-Mediated H3K27me3 Dynamics Orchestrate Brown Fat Development and Regulate White Fat Plasticity.

Progression from brown preadipocytes to adipocytes engages two transcriptional programs: the expression of adipogenic genes common to both brown fat (BAT) and white fat (WAT), and the expression of BAT-selective genes. However, the dynamics of chromatin states and epigenetic enzymes involved remain poorly understood. Here we show that BAT development is selectively marked and guided by repressive H3K27me3 and is executed by its demethylase Jmjd3. We find that a significant subset of BAT-selective genes, but not common fat genes or WAT-selective genes, are demarcated by H3K27me3 in both brown and white preadipocytes. Jmjd3-catalyzed removal of H3K27me3, in part through Rreb1-mediated recruitment, is required for expression of BAT-selective genes and for development of beige adipocytes both in vitro and in vivo. Moreover, gain- and loss-of-function Jmjd3 transgenic mice show age-dependent body weight reduction and cold intolerance, respectively. Together, we identify an epigenetic mechanism governing BAT fate determination and WAT plasticity.

IRE1 prevents endoplasmic reticulum membrane permeabilization and cell death under pathological conditions.

The endoplasmic reticulum (ER) has emerged as a critical regulator of cell survival. IRE1 is a transmembrane protein with kinase and RNase activities that is localized to the ER and that promotes resistance to ER stress. We showed a mechanism by which IRE1 conferred protection against ER stress-mediated cell death. IRE1 signaling prevented ER membrane permeabilization mediated by Bax and Bak and cell death in cells experiencing ER stress. Suppression of IRE1 signaling triggered by its kinase activity led to the accumulation of the BH3 domain-containing protein Bnip3, which in turn triggered the oligomerization of Bax and Bak in the ER membrane and ER membrane permeabilization. Consequently, in response to ER stress, cells deficient in IRE1 were susceptible to leakage of ER contents, which was associated with the accumulation of calcium in mitochondria, oxidative stress in the cytosol, and ultimately cell death. Our results reveal a role for IRE1 in preventing a cell death-initializing step that emanates from the ER and provide a potential target for treating diseases characterized by ER stress, including diabetes and Wolfram syndrome.

Global Promotion of Alternative Internal Exon Usage by mRNA 3' End Formation Factors.

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.