17-409 Sigma-AldrichEZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit

Lipotoxicity caused by saturated fatty acids (SFAs) induces tissue damage and inflammation in metabolic disorders. SCD1 (stearoyl-coenzyme A desaturase 1) converts SFAs to mono-unsaturated fatty acids (MUFAs) that are incorporated into triglycerides and stored in lipid droplets. SCD1 thus helps protect hepatocytes from lipotoxicity and its reduced expression is associated with increased lipotoxic injury in cultured hepatic cells and mouse models. To further understand the role of SCD1 in lipotoxicity, we examined the regulation of Scd1 in hepatic cells treated with palmitate, and found that NR1H/LXR (nuclear receptor subfamily 1 group H) ligand, GW3965, induced Scd1 expression and lipid droplet formation to improve cell survival. Surprisingly, ULK1/ATG1 (unc-51 like kinase) played a critical role in protecting hepatic cells from SFA-induced lipotoxicity via a novel mechanism that did not involve macroautophagy/autophagy. Specific loss of Ulk1 blocked the induction of Scd1 gene transcription by GW3965, decreased lipid droplet formation, and increased apoptosis in hepatic cells exposed to palmitate. Knockdown of ULK1 increased RPS6KB1 (ribosomal protein S6 kinase, polypeptide 1) signaling that, in turn, induced NCOR1 (nuclear receptor co-repressor 1) nuclear uptake, interaction with NR1H/LXR, and recruitment to the Scd1 promoter. These events abrogated the stimulation of Scd1 gene expression by GW3965, and increased lipotoxicity in hepatic cells. In summary, we have identified a novel autophagy-independent role of ULK1 that regulates NR1H/LXR signaling, Scd1 expression, and intracellular lipid homeostasis in hepatic cells exposed to a lipotoxic environment.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in chronic HBV infection and replication, and is an important factor for HBV surface antigen loss indicating the endpoint of HBV treatment. However, there is a known problem that current anti-HBV drugs, including interferons and nucleos(t)ide analogues, reduce HBV replication but have a little or no effect on reducing cccDNA. Therefore, the development of new therapeutic agents is necessary to eradicate cccDNA. In this study, we identified pre-mRNA processing factor 31 (PRPF31) by siRNA screening as a factor associated with cccDNA. PRPF31 knockdown by siRNA decreased cccDNA formation without serious cytotoxicity. In rescue experiments, expression of siRNA-resistant PRPF31 recovered cccDNA formation. PRPF31 knockdown did not affect HBV core protein and HBV core DNA levels in HBV-replicating cells. Chromatin immunoprecipitation and immunoprecipitation assays revealed an association between PRPF31 and cccDNA. Furthermore, co-overexpression of PRPF31 and HBx enhanced cccDNA formation in HepAD38 cells. Taken together, the present findings suggest that the interaction between PRPF31 and HBx may be a novel target for anti-HBV treatment.

p53 activation by cellular stresses induces the transcription of hundreds of its target genes. To elucidate the entire picture of its downstream pathway, we screened a cDNA microarray dataset of adriamycin-treated HCT116 p53-/- or p53+/+ cells and identified EPSIN 3 as a novel p53 target.Potential p53 binding sequences in the EPSIN 3 locus were evaluated by reporter and CHIP assays. To investigate the role of EPSIN 3 in the p53 downstream pathway, we assessed DNA damage-induced apoptosis in EPSIN 3-knockdown HCT116 cells or Epsin 3-deficient mice. In addition, we evaluated EPSIN 3 expression levels in various tissues, including gastric adenocarcinoma, human gastric mucosa with or without Helicobacter pylori infection, and mouse acute gastritis tissues induced by indomethacin.In response to DNA damage, p53 induced the expression of EPSIN 3 through the p53 binding elements in the EPSIN 3 promoter and the first intron. Knockdown of EPSIN 3 resulted in resistance to DNA damage-induced apoptosis both in vitro and in vivo. EPSIN 3 expression was down-regulated in gastric cancer tissues compared with normal tissues. In addition, Helicobacter pylori infection and indomethacin-induced acute gastritis repressed EPSIN 3 expression in gastric mucosa.EPSIN 3 is a novel p53 target and a key mediator of apoptosis. Chronic or acute mucosal inflammation as well as p53 inactivation induced down-regulation of EPSIN 3 and subsequently caused apoptosis resistance, which is a hallmark of cancer cells.

Conventional therapeutic processes in patient with OSCC are associated with several unfavorable effects leading to patients with poor survival rate. Metformin has been shown to protect against a variety of specific diseases, including cancer. However, the precise roles and mechanisms underlying the therapeutic effects of metformin on OSCC remain elusive. In the current study, in vitro and xenograft model experiments revealed that metformin inhibited growth and metastasis of oral cancer cells. Importantly, metformin-restrained tumorigenesis of oral cancer was accompanied with strong decrease of both Aurora-A and Late SV40 Factor (LSF) expressions. Furthermore, LSF contributed to Aurora-A-elicited malignancy behaviors of oral cancer via binding to the promoter region of Aurora-A. A significant correlation was observed between LSF and Aurora-A levels in a cohort of specimens of oral cancer. These findings showed that a novel LSF/Aurora-A-signaling inhibition supports the rationale of using metformin as potential OSCC therapeutics.

p53, one of the most frequently mutated genes in colon cancer, suppresses cancer development through transactivation of its targets. Herein, we conducted a comprehensive analysis of the p53 downstream pathway in colorectal cancer by using multi-omics analysis. Mass spectrometric analysis of HCT116 p53+/+ and HCT116 p53-/- cells treated with adriamycin identified 124 proteins increased by DNA damage in a p53-dependent manner. Further screening using a cDNA microarray and the TCGA database revealed MICALL1 as a novel p53 target, and we identified functional p53 binding motifs located approximately 3000 base pairs upstream of the MICALL1 gene. MICALL1 expression was significantly decreased in colorectal cancer tissues with p53 mutation compared with those without p53 mutation. In response to DNA damage, MICALL1 co-localized with RAB8A and CD2AP at tubular recycling endosomes, whereas these proteins hardly localized at tubular recycling endosomes when p53 or MICALL1 expression was inhibited by siRNA. Our findings show that p53 regulates tubular recycling endosome biogenesis via transcriptional regulation of MICALL1, whose expression is frequently suppressed in colorectal cancer tissues.

Endothelial cell injury can promote the development of various cardiovascular diseases, thus, fully understanding the mechanisms underlying the maintenance of vascular endothelial cell homoeostasis may help prevent and treat cardiovascular disease. Kaiso, a zinc finger and BTB domain containing transcription factor, is key to embryonic development and cancer, but how Kaiso interacts with vascular endothelium is not fully understood. We report that Kaiso has an anti-apoptotic function in human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMEC-1s). Overexpression of Kaiso significantly increased cell viability and inhibited hydrogen peroxide-induced apoptosis. Furthermore, Kaiso increased expression of B-cell CLL/lymphoma 2 (BCL2) and reduced expression of BCL2-associated X protein (BAX) and BCL2-interacting killer (BIK) by differentially regulating gene promoter activity. Methylated DNA and specific Kaiso binding site (KBS) contributed to gene regulatory activity of Kaiso. In addition, p120ctn functioned cooperatively in Kaiso-mediated transcriptional regulation.

On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation, and contains the DNA-binding homeodomain but is devoid of transactivation domain. It is predominantly expressed in crypt cells, whereas the CDX2 protein is present in crypt cells but also in differentiated villous cells. Functional studies revealed a dominant-negative effect exerted by miniCDX2 on the transcriptional activity of CDX2, and conversely similar effects regarding several transcription-independent functions of CDX2. In addition, a regulatory role played by the CDX2 and miniCDX2 homeoproteins on their pre-mRNA splicing is displayed, through interactions with splicing factors. Overexpression of miniCDX2 in the duodenal Brunner glands leads to the expansion of the territory of these glands and ultimately to brunneroma. As a whole, this study characterized a new and original variant of the CDX2 homeobox gene. The production of this variant represents not only a novel level of regulation of this gene, but also a novel way to fine-tune its biological activity through the versatile functions exerted by the truncated variant compared to the full-length homeoprotein. This study highlights the relevance of generating protein diversity through alternative splicing in the gut and its diseases.

The downregulation of tight junction protein CLDN6 promotes breast cancer cell migration and invasion; however, the exact mechanism underlying CLDN6 downregulation remains unclear. CLDN6 silence is associated with DNA methyltransferase 1 (DNMT1) mediated DNA methylation, and DNMT1 is regulated by the transforming growth factor beta (TGFβ)/SMAD pathway. Therefore, we hypothesized that TGFβ/SMAD pathway, specifically SMAD2, may play a critical role for CLDN6 downregulation through DNA methyltransferase 1 (DNMT1) mediated DNA methylation. To test this hypothesis, we blocked the SMAD2 pathway with SB431542 in two human breast cancer cell lines (MCF-7 and SKBR-3). Our results showed that treatment with SB431542 led to a decrease of DNMT1 expression and the binding activity for CLDN6 promoter. The methylation level of CLDN6 promoter was decreased, and simultaneously CLDN6 protein expression increased. Upregulation of CLDN6 inhibited epithelial to mesenchymal transition (EMT) and reduced the migration and invasion ability of both MCF-7 and SKBR-3 cells. Furthermore, knocked down of CLDN6 abolished SB431542 effects on suppression of EMT associated gene expression and inhibition of migration and invasion. Thus, we demonstrated that the downregulation of CLDN6 is regulated through promoter methylation by DNMT1, which depends on the SMAD2 pathway, and that CLDN6 is a key regulator in the SMAD2/DNMT1/CLDN6 pathway to inhibit EMT, migration and invasion of breast cancer cells.

p53 encodes a transcription factor that transactivates downstream target genes involved in tumour suppression. Although osteosarcoma frequently has p53 mutations, the role of p53 in osteosarcomagenesis is not fully understood. To explore p53-target genes comprehensively in calvarial bone and find out novel druggable p53 target genes for osteosarcoma, we performed RNA sequencing using the calvarial bone and 23 other tissues from p53 +/+ and p53 -/- mice after radiation exposure. Of 23,813 genes, 69 genes were induced more than two-fold in irradiated p53 +/+ calvarial bone, and 127 genes were repressed. Pathway analysis of the p53-induced genes showed that genes associated with cytokine-cytokine receptor interactions were enriched. Three genes, CD137L, CDC42 binding protein kinase gamma and Follistatin, were identified as novel direct p53 target genes that exhibited growth-suppressive effects on osteosarcoma cell lines. Of the three genes, costimulatory molecule Cd137l was induced only in calvarial bone among the 24 tissues tested. CD137L-expressing cells exhibited growth-suppressive effects in vivo. In addition, recombinant Fc-fusion Cd137l protein activated the immune response in vitro and suppressed osteosarcoma cell growth in vivo. We clarified the role of CD137L in osteosarcomagenesis and its potential therapeutic application. Our transcriptome analysis also indicated the regulation of the immune response through p53.

p53 mutation is a marker of poor prognosis in breast cancers. To identify downstream targets of p53, we screened two transcriptome datasets, including cDNA microarrays of MCF10A breast epithelial cells with wild-type p53 or p53-null background, and RNA sequence analysis of breast invasive carcinoma. Here, we unveil ten novel p53 target candidates that are up-regulated after the induction of p53 in wild-type cells. Their expressions are also high in breast invasive carcinoma tissues with wild-type p53. The GO analysis identified epidermis development and ectoderm development, which COL17A1 participates, as significantly up-regulated by wild-type p53. The COL17A1 expressions increased in a p53-dependent manner in human breast cells and mouse mammary tissues. Reporter assay and ChIP assay identified intronic p53-binding sequences in the COL17A1 gene. The MDA-MB-231 cells that genetically over-express COL17A1 gene product exhibited reduced migration and invasion in vitro. Similarly, COL17A1 expression was decreased in metastatic tumors compared to primary tumors and normal tissues, even from the same patients. Moreover, high COL17A1 expression was associated with longer survival of patients with invasive breast carcinoma. In conclusion, we revealed that COL17A1 is a novel p53 transcriptional target in breast tissues that inhibits cell migration and invasion and is associated with better prognosis.