A small molecule that blocks fat synthesis by inhibiting the activation of SREBP

Sterol regulatory element binding proteins (SREBPs) are transcription factors that activate transcription of the genes involved in cholesterol and fatty acid biosynthesis. In the present study, we show that a small synthetic molecule we previously discovered to block adipogenesis is an inhibitor of the SREBP activation. The diarylthiazole derivative, now called fatostatin, impairs the activation process of SREBPs, thereby decreasing the transcription of lipogenic genes in cells. Our analysis suggests that fatostatin inhibits the ER-Golgi translocation of SREBPs through binding to their escort protein, the SREBP cleavage-activating protein (SCAP), at a distinct site from the sterol-binding domain. Fatostatin blocked increases in body weight, blood glucose, and hepatic fat accumulation in obese ob/ob mice, even under uncontrolled food intake. Fatostatin may serve as a tool for gaining further insights into the regulation of SREBP

ERAD components Derlin-1 and Derlin-2 are essential for postnatal brain development and motor function

Derlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function

Novel repressor regulates insulin sensitivity through interaction with Foxo1

This study characterizes a novel Foxo1 CoRepressor (FCoR) that regulates insulin sensitivity and energy metabolism as revealed by whole-body knockout. As target of PKA phosphorylation, FCoR modulates Foxo's acetylation known to control Foxo's biological activity

Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.

Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells

PPAR beta/delta activation of CD300a controls intestinal immunity

Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPAR beta/delta (peroxisome proliferator-activated receptor beta/delta) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a(-/-) mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a(-/-) macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis

Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation

Genome defence in hypomethylated developmental contexts

Retrotransposons constitute around 40% of the mammalian genome and their aberrant activation can have wide ranging detrimental consequences, both throughout development and into somatic lineages. DNA methylation is one of the major epigenetic mechanisms in mammals, and is essential in repressing retrotransposons throughout mammalian development. Yet during normal mouse embryonic development some cell lineages become extensively DNA hypomethylated and it is not clear how these cells maintain retrotransposon silencing in a globally hypomethylated genomic context. In this thesis I determine that hypomethylation in multiple contexts results in the consistent activation of only one gene in the mouse genome - Tex19.1. Thus if a generic compensatory mechanism for loss of DNA methylation exists in mice, it must function through this gene. Tex19.1-/- mice de-repress retrotransposons in the hypomethylated component of the placenta and in the mouse germline, and have developmental defects in these tissues. In this thesis I examine the mechanism of TEX19.1 mediated genome defence and the developmental consequences upon its removal. I show that TEX19.1 functions in repressing retrotransposons, at least in part, through physically interacting with the transcriptional co-repressor, KAP1. Tex19.1-/- ES cells have reduced levels of KAP1 bound retrotransposon chromatin and reduced levels of the repressive H3K9me3 modification at these loci. Furthermore, these subsets of retrotransposon loci are de-repressed in Tex19.1-/- placentas. Thus, my data indicates that mouse cells respond to hypomethylation by activating expression of Tex19.1, which in turn augments compensatory, repressive histone modifications at retrotransposon sequences, thereby helping developmentally hypomethylated cells to maintain genome stability. I next aimed to further elucidate the role of Tex19.1 in the developing hypomethylated placenta. I determine that Tex19.1-/- placental defects precede intrauterine growth restriction of the embryo and that alterations in mRNA abundance in E12.5 Tex19.1-/- placentas is likely in part due to genic transcriptional changes. De-repression of LINE- 1 is evident in these placentas and elements of the de-repressed subfamily are associated with significantly downregulated genes. If retrotransposon de-repression is contributing to developmental defects by interfering with gene expression remains to be determined, however I identify a further possible mechanism leading to placental developmental defects. I determine that Tex19.1-/- placentas have an increased innate immune response and I propose that this is contributing to the developmental defects observed. Developmental defects and retrotransposon de-repression are also observed in spermatogenesis in Tex19.1-/- testes, the molecular basis for which is unclear. I therefore investigate the possibility that the TEX19.1 interacting partners, the E3 ubiquitin ligase proteins, may be contributing to the phenotypes observed in Tex19.1- /- testes. I show that repression of MMERVK10C in the testes is dependent on UBR2, alongside TEX19.1. Furthermore, I have identified a novel role for the TEX19.1 interacting partner, UBR5, in spermatogenesis, whose roles are distinct from those of TEX19.1. The work carried out during the course of this thesis provides mechanistic insights into TEX19.1 mediated genome defence and highlights the importance of protecting the genome from aberrant retrotransposon expression

Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition.

Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions