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Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, includingIER5, which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes.IER5is epistatic toPPP2R2A, a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation.

The bromodomain and extraterminal (BET) family comprises epigenetic reader proteins that are important regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of proinflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLN(R9C)) — a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, we treated PLN(R9C) or age-matched WT mice longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLN(R9C) mice by inhibiting expression of proinflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on proinflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NF-κB–mediated proinflammatory gene expression in cardiac fibroblasts. Suppressing proinflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.

Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in long- range enhancers. Among the direct Notch target genes are multiple DNA damage response genes, includingIER5, which is regulated by Notch through several enhancer elements. We show thatIER5is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic toPPP2R2A, which encodes the B55αsubunit of PP2A, and IER5 interacts with B55αin cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers.ImpactOur findings highlight context-specific crosstalk between Notch, DNA damage response genes, and PP2A, and provide a roadmap for understanding how Notch induces the differentiation of squamous cells.

Canonical roles for macrophages in mediating the fibrotic response after a heart attack include extracellular matrix turnover and activation of cardiac fibroblasts to initiate collagen deposition. Here we reveal that macrophages directly contribute collagen to the forming post-injury scar. Unbiased transcriptomics shows an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. Adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors, enhances scar formation via cell autonomous production of collagen. In zebrafish, the majority of tagged collagen localises proximal to the injury, within the overlying epicardial region, suggesting a possible distinction between macrophage-deposited collagen and that predominantly laid-down by myofibroblasts. Macrophage-specific targeting of col4a3bpa and cognate col4a1 in zebrafish significantly reduces scarring in cryoinjured hosts. Our findings contrast with the current model of scarring, whereby collagen deposition is exclusively attributed to myofibroblasts, and implicate macrophages as direct contributors to fibrosis during heart repair., Macrophages mediate the fibrotic response after a heart attack by extracellular matrix turnover and cardiac fibroblasts activation. Here the authors identify an evolutionarily-conserved function of macrophages that contributes directly to the forming post-injury scar through cell-autonomous deposition of collagen.

Recent studies have identified a genetic variant rs641738 near two genes encoding membrane bound O-acyltransferase domain-containing 7 (MBOAT7) and transmembrane channel-like 4 (TMC4) that associate with increased risk of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcohol-related cirrhosis, and liver fibrosis in those infected with viral hepatitis (Buch et al., 2015; Mancina et al., 2016; Luukkonen et al., 2016; Thabet et al., 2016; Viitasalo et al., 2016; Krawczyk et al., 2017; Thabet et al., 2017). Based on hepatic expression quantitative trait loci analysis, it has been suggested that MBOAT7 loss of function promotes liver disease progression (Buch et al., 2015; Mancina et al., 2016; Luukkonen et al., 2016; Thabet et al., 2016; Viitasalo et al., 2016; Krawczyk et al., 2017; Thabet et al., 2017), but this has never been formally tested. Here we show that Mboat7 loss, but not Tmc4, in mice is sufficient to promote the progression of NAFLD in the setting of high fat diet. Mboat7 loss of function is associated with accumulation of its substrate lysophosphatidylinositol (LPI) lipids, and direct administration of LPI promotes hepatic inflammatory and fibrotic transcriptional changes in an Mboat7-dependent manner. These studies reveal a novel role for MBOAT7-driven acylation of LPI lipids in suppressing the progression of NAFLD., eLife digest Non-alcoholic fatty liver disease, or NAFLD for short, is a medical condition that develops when the liver accumulates excess fat. It can lead to complications such as diabetes and liver scarring. In humans, mutations that inactivate a protein called MBOAT7 increase the risk of fat accumulating in the liver. Genetic studies suggest that low levels of MBOAT7 in a human’s liver cells increase the severity of NAFLD. Yet the links between MBOAT7, NAFLD and obesity are not well understood. Helsley et al. used data from humans and from obese mice that had been fed a high-fat diet to investigate the relationship between NAFLD and MBOAT7. This revealed that people who are obese have lower levels of MBOAT7 in their livers. Next, obese mice were genetically manipulated to produce less MBOAT7, which led them to develop more severe NAFLD. Helsley et al. then grew human liver cells in the laboratory and lowered their levels of MBOAT7, which led to excess fat accumulating in the cells. This increase in fat accumulation was, at least in part, due to how these cells metabolize fats when MBOAT7 is reduced: they start making more new fats and consume fewer lipids to produce energy. These findings provide a link between obesity and liver damage in both humans and mice, and show how a decrease in MBOAT7 levels causes changes in fat metabolism that could lead to NAFLD. The results could drive new approaches to treating liver damage in patients with mutations in the gene that codes for MBOAT7.

Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1's importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1's functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sor(tm1(Cre/ERT)Nat)/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1's role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation.

The authors would like to clarify that the controls previously depicted in Figs ​Figs4E4E and ​and7A7A were for different experiments and were included in error. Fig 4 UCP2 is Up-Regulated in Sirt1 Knockdown Cells and in Sirt1 KO Mice. Fig 7 UCP2 mRNA or Protein Levels in Fed or Starved Wild-Type Mice. The correct control for Fig 4E was located and used to prepare a corrected figure. The correct control for the original Fig 7A could not be located; this panel has therefore been removed after a careful assessment and investigation determined that the result for which original Fig 7A was cited is supported elsewhere in this article, and that removal of this panel does not affect the conclusions of the paper. We have also taken this opportunity to provide new versions of several figures (Figs ​(Figs4,4, ​,5,5, ​,6,6, ​,7)7) in which gel/blot splices and a non-linear level adjustment were made but were not previously indicated or declared, or to replace incorrectly spliced gels/blots with the un-spliced originals. We also take the opportunity to correct two errors in the legend to Fig 6, first to remove a redundant and incorrect sentence, and second to address incorrect description of p values. Fig 5 Sirt1 Binds at the UCP2 Promoter and Represses the Gene. Fig 6 Knockdown of UCP2 in Sirt1 Knockdown Cells Restores Glucose-Induced Insulin Secretion. The text in the Results section titled “UCP2 Levels Increase in Food-Deprived Mice” has been edited to accommodate the removal of the original Fig 7A and the relabeling of Fig 7B, 7C and 7D as Fig 7A, 7B and 7C, respectively. The corrected text and Figs ​Figs4,4, ​,5,5, ​,66 and ​and77 are provided here.

Fusion of the EWS gene to FLI1 produces a fusion oncoprotein that drives an aberrant gene expression program responsible for the development of Ewing sarcoma. We used a homogenous proximity assay to screen for compounds that disrupt the binding of EWS-FLI1 to its cognate DNA targets. A number of DNA-binding chemotherapeutic agents were found to non-specifically disrupt protein binding to DNA. In contrast, actinomycin D was found to preferentially disrupt EWS-FLI1 binding by comparison to p53 binding to their respective cognate DNA targets in vitro. In cell-based assays, low concentrations of actinomycin D preferentially blocked EWS-FLI1 binding to chromatin, and disrupted EWS-FLI1-mediated gene expression. Higher concentrations of actinomycin D globally repressed transcription. These results demonstrate that actinomycin D preferentially disrupts EWS-FLI1 binding to DNA at selected concentrations. Although the window between this preferential effect and global suppression is too narrow to exploit in a therapeutic manner, these results suggest that base-preferences may be exploited to find DNA-binding compounds that preferentially disrupt subclasses of transcription factors.

Global expression analysis of fetal liver hematopoietic stem cells (FL HSCs) revealed the presence of unspliced pre-mRNA for a number of genes in normal FL HSCs. In a subset of these genes, Crebbp+/- FL HSCs had less unprocessed pre-mRNA without a corresponding reduction in total mRNA levels. Among the genes thus identified were the key regulators of HSC function Itga4, Msi2 and Tcf4. A similar but much weaker effect was apparent in Ep300+/- FL HSCs, indicating that, in this context as in others, the two paralogs are not interchangeable. As a group, the down-regulated intronic probe sets could discriminate adult HSCs from more mature cell types, suggesting that the underlying mechanism is regulated with differentiation stage and is active in both fetal and adult hematopoiesis. Consistent with increased myelopoiesis in Crebbp hemizygous mice, targeted reduction of CREBBP abundance by shRNA in the multipotent EML cell line triggered spontaneous myeloid differentiation in the absence of the normally required inductive signals. In addition, differences in protein levels between phenotypically distinct EML subpopulations were better predicted by taking into account not only the total mRNA signal but also the amount of unspliced message present. CREBBP thus appears to selectively influence the timing and degree of pre-mRNA processing of genes essential for HSC regulation and thereby has the potential to alter subsequent cell fate decisions in HSCs.

NUT midline carcinoma (NMC) is a lethal pediatric tumor defined by the presence of BRD-NUT fusion proteins that arrest differentiation. Here we explore the mechanisms underlying the ability of BRD4-NUT to prevent squamous differentiation. In both gain-of and loss-of-expression assays, we find that expression of BRD4-NUT is associated with globally decreased histone acetylation and transcriptional repression. Bulk chromatin acetylation can be restored by treatment of NMC cells with histone deacetylase inhibitors (HDACi), engaging a program of squamous differentiation and arrested growth in vitro that closely mimics the effects of siRNA-mediated attenuation of BRD4-NUT expression. The potential therapeutic utility of HDACi differentiation therapy was established in three different NMC xenograft models, where it produced significant growth inhibition and a survival benefit. Based on these results and translational studies performed with patient-derived primary tumor cells, a child with NMC was treated with the FDA-approved HDAC inhibitor, vorinostat. An objective response was obtained after five weeks of therapy, as determined by positron emission tomography. These findings provide preclinical support for trials of HDACi in patients with NMC. Cancer Res; 71(7); 2686–96. ©2011 AACR.

Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM.

In PLoS Biology, volume 4, issue 2: DOI: 10.1371/journal.pbio.0040031 In the first paragraph of the Materials and Methods subsection “Retroviral infection of INS1 and MIN6 cells,” “pSUPERretro SiRNA-T1 (5′-GCTGCATCCAAGGGCCATG-3′)” should be “pSUPERretro SiRNA-T1 (5′-gatgaagttgacctcctca-3′)”.

The NAD-dependent deacetylase SIR2 and the forkhead transcription factor DAF-16 regulate lifespan in model organisms, such as yeast and C. elegans. Here we show that the mammalian SIR2 ortholog SIRT1 deacetylates and represses the activity of the forkhead transcription factor Foxo3a and other mammalian forkhead factors. This regulation appears to be in the opposite direction from the genetic interaction of SIR2 with forkhead in C. elegans. By restraining mammalian forkhead proteins, SIRT1 also reduces forkhead-dependent apoptosis. The inhibition of forkhead activity by SIRT1 parallels the effect of this deacetylase on the tumor suppressor p53. We speculate how downregulating these two classes of damage-responsive mammalian factors may favor long lifespan under certain environmental conditions, such as calorie restriction.