Identification of putative target genes of the transcription factor RUNX2
Ontology highlight
ABSTRACT: We overexpress RUNX2 in ten human cell lines and identify genes that are affected by RUNX2 expression. These target genes provide a valuable resource into pathways regulated by RUNX2
Project description:We found frequent epigenetic silencing of microRNA-34b/c in human colorectal cancer. Introduction of miR-34b/c into a colorectal cancer cell line induced significant changes in gene expression profile. We also found overlap between the genes downregulated by miR-34b/c and those downregulated by DAC. Keywords: dose response A colorecal cancer cell line HCT116 was transfected with miR-34b or -c precursor or negative control. Also, HCT116 was treated with 5-aza-2'-deoxycytidine (DAC) or mock. Genes up- or downregulated by miR-34b/c and those by DAC was compared.
Project description:Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcription program essential for bone formation through both genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation: proliferation, matrix deposition and mineralization, we identified Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) over the course of these stages, we discovered close to 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibited distinct patterns during osteogenesis, and were associated with proximal promoters as well as a large percentage of Runx2 occupancy in non-promoter regions: upstream, introns, exons, transcription termination site (TTS) regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identified novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of extracellular matrix. We demonstrated by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis. MC3T3-E1 cells were treated with scramble or Runx2 shRNA, then harvested at proliferating stage (day 0) and differentiating stage (day 9). Total RNAs recovered from these cells were hybridization on Affymetrix microarrays. We sought to find new target genes or pathways regulated by Runx2 during osteoblast differentiation. When combined with genome-wide occupancy of Runx2, we expect to gain new insights on how Runx2 controls a transcriptional program essential for osteoblast differentiation.
Project description:Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcription program essential for bone formation through both genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation: proliferation, matrix deposition and mineralization, we identified Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) over the course of these stages, we discovered close to 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibited distinct patterns during osteogenesis, and were associated with proximal promoters as well as a large percentage of Runx2 occupancy in non-promoter regions: upstream, introns, exons, transcription termination site (TTS) regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identified novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of extracellular matrix. We demonstrated by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. Our data establish that Runx2 interactions withM-BM- chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis. To identiy the genome-wide occupancy of Runx2, DNA bound by Runx2 at the prolieration, matrix deposition, and mineralization stages were recovered by Runx2 ChIP. Libraries of purified DNA were generated using Illumina SR adapters (Illumina) following manufacturerM-bM-^@M-^Ys manual, and were selected for the inserted fragments of 200 M-BM-1 50 bp, and sequenced 36 bases on an Illumina Genome Analyzer II. Base calls and sequence reads were generated by Illumina CASAVA software (version 1.6, Illumina). Two independent biological repeats of Runx2 ChIP-Seq libraries were prepared for each time point, and two input libraries were prepared with sonicated DNA from day 9 MC3T3-E1 cells. We pooled the reads from two biological replicates for peaking calling using MACS (version 1.4.1) with a stringent p value threshold (p < 1e-10) in contrast to input control, and used these peaks for further bioinformatic analyses. Each sample deposited here contains three files: the sequence file with short reads combined from two biological replicates, a Bed file with peaks called from the pooled short reads, and a Wig file with peak signals.
Project description:Cells of MDA-MB-231 breast cancer cell-line were transfected with siRNA against Runx2, CBF-beta or non-specific siRNA used as control. Runx2 is a member of the Runx transcription factor family and possesses a Runt domain capable of binding to the consensus DNA sequence ACC(A/G)CA. This domain also interacts with the co-activator protein core binding factor beta (CBF-beta), which enhances its binding to DNA. Runx2, primarily identified as a master regulator of bone development, but was also found expressed in the epithelium of the nascent mammary gland in mice. In contrast with its normal role in breast, it has been shown that Runx2 is over-expressed in breast cancer cell lines.
Project description:LH-indced RUNX2 expression is important for luteal gene expression. Granulosa cells were treated with Runx2 siRNA and forskolin to knockdown agonist-induced Runx2 expression. Two separate cultures were performed (n=2) and universal siRNA was used for negative control. Total RNAs were isolated from cultured granulosa cells and used for DNA microarray.
Project description:We used microarrays to identify genes differentially expressed between mouse RUNX2 -/- and wt embryonic humeri at stage E14.5 Experiment Overall Design: To minimize the effects of random biological variation, humeri from different preparations were pooled (approximately 75 wildtype and 60 Runx2-/- for each of the two biological replicates).
Project description:Previously, we have identified the RUNX2 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from epithelial ovarian cancer (EOC) patients, when compared to primary cultures derived from matched primary (prior to CT) tumors. However, we found no differences in the RUNX2 methylation in primary EOC tumors and EOC omental metastases, suggesting that DNA methylation-based epigenetic mechanisms have no impact on RUNX2 expression in advanced (metastatic) stage of the disease. Moreover, RUNX2 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. Knockdown of the RUNX2 expression in EOC cells led to sharp decrease of cell proliferation and significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as various genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX2 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the RUNX2 gene in EOC progression and suggest that RUNX2 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX2 and other members of the RUNX gene family in ovarian tumorigenesis. To better understand the molecular mechanisms of RUNX2 gene action in ovarian cancer cells, we employed the Agilent Whole Human Genome microarrays, containing ~ 44,000 genes to identify global gene expression changes upon RUNX2 suppression in SKOV3 cells. We compared the gene expression of the previously selected clone shRNA- RUNX2-knockdown clone 3 (cl3) against the corresponding control clone. The microarray experiments were performed in duplicates, as two hybridizations were carried out for the RUNX2-suppressing cell clone against the corresponding control, using a fluorescent dye reversal (dye-swap) technique.
Project description:The nuclear receptor PPAR gamma is required for adipocyte differentiation, but its role in mature adipocytes is less clear. Here we report that knockdown of PPAR gamma expression in 3T3-L1 adipocytes returned the expression of most adipocyte genes towards preadipocyte levels. Consistently, down regulated but not up regulated genes showed strong enrichment of PPAR gamma binding. Surprisingly, not all adipocyte genes were reversed and the adipocyte morphology was maintained for an extended period after PPAR gamma depletion. To explain this, we focused on transcriptional regulators whose adipogenic regulation was not reversed upon PPAR gamma depletion. We identified GATA2, a transcription factor whose down-regulation early in adipogenesis is required for preadipocyte differentiation, remaining low after PPAR gamma knockdown. Forced expression of GATA2 in mature adipocytes complemented PPAR gamma depletion and impaired adipocyte functionality with a more preadipocyte- like gene expression profile. Ectopic expression of GATA2 in adipose tissue in vivo had similar effect on adipogenic gene expression. These results suggest that PPAR gamma-independent down regulation of GATA2 prevents reversion of mature adipocytes after PPAR gamma depletion. Experiment Overall Design: This dataset consists of three sample groups: preadipocytes, control siRNA treated adipocytes, and PPAR gamma siRNA treated adipocytes. Each sample group consists of three replicates samples. Each sample was hybridized to a separate array for a total of nine arrays. Experiment Overall Design: Technical replicates: Pread 1, Pread 2, Pread 3 Experiment Overall Design: Technical replicates: Cont siRNA 1, Cont siRNA 2, Cont siRNA 3 Experiment Overall Design: Technical replicates: PPAR gamma siRNA 1, PPAR gamma siRNA 2, PPAR gamma siRNA 3
Project description:RUNX2 is a transcription factor that is first expressed in early osteoblast-lineage cells and represents a primary determinant of osteoblastogenesis. While numerous target genes are regulated by RUNX2, little is known of sites on the genome occupied by RUNX2 or of the gene networks that are controlled by these sites. To explore this, we conducted a genome-wide analysis of the RUNX2 cistrome in both pre-osteoblastic MC3T3-E1 cells (POB) and their mature osteoblast progeny (OB), characterized the two cistromes and assessed their relationship to changes in gene expression. We found that although RUNX2 was widely bound to the genome in POB cells, this binding profile was reduced upon differentiation to OBs. Numerous sites were lost upon differentiation, new sites were also gained; many sites remained common to both cell states. Additional features were identified as well including location relative to potential target genes, abundance with respect to single genes, the frequent presence of a consensus TGTGGT RUNX2 binding motif, co-occupancy by C/EBPβ and the presence of a typical epigenetic histone enhancer signature. This signature was changed quantitatively following differentiation. While RUNX2 binding sites were associated extensively with adjacent genes, the distal nature of the majority of these sites prevented assessment of whether they represented direct targets of RUNX2 action. Changes in gene expression, however, revealed an abundance of genes that contained RUNX2 binding sites and were regulated in concert. These studies establish a basis for further analysis of the role of RUNX2 activity and its function during osteoblast lineage maturation. 2 transcription factors and 5 histone modifications were examined in undifferentiated MC3T3-E1 cells as well as post 15 day osteogenic differentiation MC3T3-E1 cells. The samples were completed in biological replicate and examined separately.
Project description:Expression profiling of transcription factors.<br>Two further processed data files, Sox2-Chd7-probesets.txt and Sox2-Chd7-genes.txt are available in the E-MEXP-2743.additional.zip archive on the FTP site for this experiment.