Project description:Runx2 is a metastatic transcription factor (TF) increasingly expressed during prostate cancer (PCa) progression. Using PCa cells conditionally expressing Runx2, we previously identified Runx2-regulated genes with known roles in epithelial-mesenchymal transition, invasiveness, angiogenesis, extracellular matrix proteolysis and osteolysis. To map Runx2-occupied regions (R2ORs) in PCa cells, we first analyzed regions predicted to bind Runx2 based on the expression data, and found that recruitment to sites upstream of the KLK2 and CSF2 genes was cyclical over time. Genome-wide ChIP-seq analysis at a time of maximum occupancy at these sites revealed 1,603 high-confidence R2ORs, enriched with cognate motifs for RUNX, GATA and ETS TFs. The R2ORs were distributed with little regard to annotated transcription start sites (TSSs), mainly in introns and intergenic regions. Runx2-upregulated genes, however, displayed enrichment for R2ORs within 40 kb of their TSSs. The main annotated functions enriched in 98 Runx2-upregulated genes with nearby R2ORs were related to invasiveness and membrane trafficking/secretion. Indeed, using SDS-PAGE, mass spectrometry and western analyses, we show that Runx2 enhances secretion of several proteins, including fatty acid synthase and metastasis-associated laminins. Thus, combined analysis of Runx2’s transcriptome and genomic occupancy in PCa cells lead to defining its novel role in regulating protein secretion. Examination of Runx2 in Prostate Cancer cells
Project description:Runx2 is a metastatic transcription factor (TF) increasingly expressed during prostate cancer (PCa) progression. Using PCa cells conditionally expressing Runx2, we previously identified Runx2-regulated genes with known roles in epithelial-mesenchymal transition, invasiveness, angiogenesis, extracellular matrix proteolysis and osteolysis. To map Runx2-occupied regions (R2ORs) in PCa cells, we first analyzed regions predicted to bind Runx2 based on the expression data, and found that recruitment to sites upstream of the KLK2 and CSF2 genes was cyclical over time. Genome-wide ChIP-seq analysis at a time of maximum occupancy at these sites revealed 1,603 high-confidence R2ORs, enriched with cognate motifs for RUNX, GATA and ETS TFs. The R2ORs were distributed with little regard to annotated transcription start sites (TSSs), mainly in introns and intergenic regions. Runx2-upregulated genes, however, displayed enrichment for R2ORs within 40 kb of their TSSs. The main annotated functions enriched in 98 Runx2-upregulated genes with nearby R2ORs were related to invasiveness and membrane trafficking/secretion. Indeed, using SDS-PAGE, mass spectrometry and western analyses, we show that Runx2 enhances secretion of several proteins, including fatty acid synthase and metastasis-associated laminins. Thus, combined analysis of Runx2’s transcriptome and genomic occupancy in PCa cells lead to defining its novel role in regulating protein secretion.
Project description:The androgen receptor (AR) has a pivotal role in regulating gene expression in the male reproductive system. Due to the involvement of AR in prostate cancer, its role is best studied in the prostate gland epithelium and prostate cancer cell lines. Here we investigate the transcriptional program of AR in normal human epididymis epithelial (HEE) cells. After AR stimulation of caput HEE cells with the synthetic androgen R1881, AR targets were revealed with RNA-sequencing. Next, AR occupancy genome-wide was determined in control or R1881-stimulated HEE cells by chromatin immunoprecipitation and deep sequencing (ChIP-seq). The results identify about 200 genes that are differentially expressed (DEGs) in HEE cells after AR activation. Some of these DEGs show occupancy of AR at their promoters or cis-regulatory elements suggesting direct regulation. However there is little overlap in AR-associated DEGs between HEE and prostate epithelial cells. Inspection of over-represented motifs in AR ChIP-seq peaks identified CAAT-enhancer binding protein beta (CEBPB) and Runt-related transcription factor 1 (RUNX1) as potential co-factors, with no evidence for FOXA1, which is an important co-factor in the prostate epithelium. CEBPB and RUNX1 ChIP-seq in HEE cells showed that both these factors often occupied AR-binding sites, though rarely simultaneously. Further analysis at a single AR-regulated locus (FK506-binding protein 5, FKPB5) suggests that RUNX1 may inhibit AR occupancy, while CEBP appears to be a co-activator. These data suggest a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium.
Project description:The androgen receptor (AR) has a pivotal role in regulating gene expression in the male reproductive system. Due to the involvement of AR in prostate cancer, its role is best studied in the prostate gland epithelium and prostate cancer cell lines. Here we investigate the transcriptional program of AR in normal human epididymis epithelial (HEE) cells. After AR stimulation of caput HEE cells with the synthetic androgen R1881, AR targets were revealed with RNA-sequencing. Next, AR occupancy genome-wide was determined in control or R1881-stimulated HEE cells by chromatin immunoprecipitation and deep sequencing (ChIP-seq). The results identify about 200 genes that are differentially expressed (DEGs) in HEE cells after AR activation. Some of these DEGs show occupancy of AR at their promoters or cis-regulatory elements suggesting direct regulation. However there is little overlap in AR-associated DEGs between HEE and prostate epithelial cells. Inspection of over-represented motifs in AR ChIP-seq peaks identified CAAT-enhancer binding protein beta (CEBPB) and Runt-related transcription factor 1 (RUNX1) as potential co-factors, with no evidence for FOXA1, which is an important co-factor in the prostate epithelium. CEBPB and RUNX1 ChIP-seq in HEE cells showed that both these factors often occupied AR-binding sites, though rarely simultaneously. Further analysis at a single AR-regulated locus (FK506-binding protein 5, FKPB5) suggests that RUNX1 may inhibit AR occupancy, while CEBP appears to be a co-activator. These data suggest a novel AR transcriptional network governs differentiated functions of the human epididymis epithelium.
Project description:We report the application of single cell transcriptome, bulk transcriptome, and chromatin accessibility analysis for investigating the role of Runx2 in regulating soft palate muscle development. By isolating single cells from soft palate tissue of wild type embryos at E13.5, E14.5 and E15.5, we describe the heterogeneity of soft palate mesenchyme during development by analyzing single cell transcriptome. Combined analysis of bulk and single cell transcriptome of soft palate from wild type and Runx2 mutant suggests Runx2 activate expression of perimysial markers. Finally, we show that Runx2 activates expression of perimysial markers probably by repressing Twist1 through chromatin accessibility analysis. This study provides the first single cell level heterogeneity analysis of developing soft palate and shows the important role of Runx2 in regulating soft palate muscle development.
Project description:Prostate carcinogenesis is associated with changes in androgen signaling from driving cellular differentiation to promoting oncogenic behaviors. RUNX2 binds the androgen receptor (AR), and ectopic expression of RUNX2 is linked to prostate cancer (PCa) progression. We therefore investigated genome-wide the influence of RUNX2 on androgen-induced gene expression and AR DNA binding in PCa cells. The predominant function of RUNX2 is to inhibit the androgen response, attributable in part to dissociation of AR from target genes such as the tumor suppressor NKX3-1. At a minority of AR target genes, however, AR activity persists in the presence of RUNX2. Some of these genes are co-operatively stimulated by androgen and RUNX2 signaling and are characterized by the presence of putative enhancers co-occupied by AR and RUNX2. Genes synergistically stimulated by AR and RUNX2 include the invasion-promoting transcription factor SNAI2. Indeed, co-activation of AR and RUNX2, but neither alone, stimulated PCa cell invasiveness, which was abolished by SNAI2 silencing. Accordingly, PCa biopsies most strongly stained for SNAI2 exhibit high nuclear expression of both RUNX2 and AR. The RUNX2-mediated locus-dependent modulation of AR activity in PCa opens a research avenue that may guide the development of novel diagnostic and therapeutic approaches to patient management. total RNA from C4-2B/Rx2dox cells was extracted in biological triplicates from four different conditions. Ethanol vehicle control, dox to induce RUNX2 expression, DHT to activate androgen receptor and DHT+dox combined.
Project description:Prostate carcinogenesis is associated with changes in androgen signaling from driving cellular differentiation to promoting oncogenic behaviors. RUNX2 binds the androgen receptor (AR), and ectopic expression of RUNX2 is linked to prostate cancer (PCa) progression. We therefore investigated genome-wide the influence of RUNX2 on androgen-induced gene expression and AR DNA binding in PCa cells. The predominant function of RUNX2 is to inhibit the androgen response, attributable in part to dissociation of AR from target genes such as the tumor suppressor NKX3-1. At a minority of AR target genes, however, AR activity persists in the presence of RUNX2. Some of these genes are co-operatively stimulated by androgen and RUNX2 signaling and are characterized by the presence of putative enhancers co-occupied by AR and RUNX2. Genes synergistically stimulated by AR and RUNX2 include the invasion-promoting transcription factor SNAI2. Indeed, co-activation of AR and RUNX2, but neither alone, stimulated PCa cell invasiveness, which was abolished by SNAI2 silencing. Accordingly, PCa biopsies most strongly stained for SNAI2 exhibit high nuclear expression of both RUNX2 and AR. The RUNX2-mediated locus-dependent modulation of AR activity in PCa opens a research avenue that may guide the development of novel diagnostic and therapeutic approaches to patient management.
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:Background: Prostate cancer (PCa) cells preferentially metastasize to bone at least in part by acquiring osteomimetic properties. Runx2, an osteoblast master transcription factor, is aberrantly expressed in PCa cells, and promotes their metastatic phenotype. The transcriptional programs regulated by Runx2 have been extensively studied during osteoblastogenesis, where it activates or represses target genes in a context-dependent manner. However, little is known about the gene regulatory networks influenced by Runx2 in PCa cells. We therefore investigated genome-wide mRNA expression changes in PCa cells in response to Runx2. Results: We engineered a C4-2B PCa sub-line called C4-2B/Rx2dox, in which doxycycline (Dox) treatment stimulates Runx2 expression from very low levels to levels observed in other PCa cells. Transcriptome profiling using whole genome expression array followed by in silico analysis indicated that Runx2 upregulated a multitude of genes with prominent cancer-associated functions. They included secreted factors (CSF2, SDF-1), proteolytic enzymes (MMP9, CST7), cytoskeleton modulators (SDC2, Twinfilin, SH3PXD2A), intracellular signaling molecules (DUSP1, SPHK1, RASD1) and transcription factors (Sox9, SNAI2, SMAD3) functioning in epithelium to mesenchyme transition (EMT), tissue invasion, as well as homing and attachment to bone. Consistent with the gene expression data, induction of Runx2 in C4-2B cells enhanced their invasiveness. It also promoted cellular quiescence by blocking the G1/S phase transition during cell cycle progression. Furthermore, the cell cycle block was reversed as Runx2 levels declined after Dox withdrawal. Conclusions: The effects of Runx2 in C4-2B/Rx2dox cells, as well as similar observations made by employing LNCaP, 22RV1 and PC3 cells, highlight multiple mechanisms by which Runx2 promotes the metastatic phenotype of PCa cells, including tissue invasion, homing to bone and induction of high bone turnover. Runx2 is therefore an attractive target for the development of novel diagnostic, prognostic and therapeutic approaches to PCa management. Targeting Runx2 may prove more effective than focusing on its individual downstream genes and pathways. C4-2B/Rx2dox cells were subjected to microarray gene expression analysis after one and two days of treatment with either Dox or vehicle in biological quadruplicates (a total of 16 samples).