Genome-wide changes in DNase-hypersensitivity during osteoblastogenesis reveal differential usage of DNA motifs and define novel cis-regulatory regions that control gene expression during differentiation
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Jane Lian
PROVIDER: E-GEOD-55047 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
The bone-specific Runx2-P1 promoter displays conserved three-dimensional chromatin structure with the syntenic Supt3h promoter.
Barutcu A Rasim AR Tai Phillip W L PW Wu Hai H Gordon Jonathan A R JA Whitfield Troy W TW Dobson Jason R JR Imbalzano Anthony N AN Lian Jane B JB van Wijnen André J AJ Stein Janet L JL Stein Gary S GS
Nucleic acids research 20140812 16
Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for differentiation of precursor cells into mature osteoblasts. Although organization and control of the bone-specific Runx2-P1 promoter have been studied extensively, long-range regulation has not been explored. In this study, we investigated higher-order ...[more]
Similar Datasets
Project description:Gene regulation during the process of osteoblastogenesis has been well-described, yet the discovery of novel regulatory regions has been limited by how we currently predict the locations of functional cis-regulatory modules. Historically, the de novo identification of sequences critical for the control of gene expression relied primarily on sequence conservation in promoters. Queries for binding motifs were based on position weight matrices of known transcription factors and the identification of disease-causing, non-coding mutations near critical genes. However, we now must consider that regulatory elements also rely on 3-dimensional chromosomal interactions between far-distal regions, epigenetic chromosomal modifications, and RNA:DNA interactions. Traditionally, DNaseI-hypersensitivity assays have been used for the identification of regulatory regions via preferential digestion at chromatin depleted or displaced of nucleosomes, as a result of transcription factor occupancy. We probed DNase hypersensitivity on a genome-wide scale to determine whether osteogenic differentiation and/or bone-related gene regulation is marked by the presence of commonly utilized DNA motifs within active cis-regulatory modules. We thus sought to evaluate the gain or loss of motif representation within hypersensitive regions during osteoblastogenesis, from day-0 (growth-phase) to day-28 (mineralizing) MC3T3 cultures. We find that differentiation is marked by an increased enrichment of NFkB-p65, MEF2, and bHLH/E-box motifs within hypersensitive regions, while CTCF, NF1, TEAD, and AP1 motifs decrease. Furthermore, grouping hypersensitive regions based on genomic positioning (promoters, introns, exons, and far-distal regions) reveals significant differences in motif abundance in first introns versus other genomic positions. This finding suggests that the regulation conferred within first intron sequences may be somewhat distinct. Interestingly, the majority of motifs that were enriched, regardless of genomic position or differentiation time-point, were not completely matched to currently known transcription factor motifs (curated in the JASPAR database). Taken together, the changes in DNase-hypersensitive regions during osteoblastogenesis and the enrichment of distinct motifs within these regions indicate that osteoblasts utilize unique sets of motif rules for transcription factor binding or that regulatory control operates through undiscovered factors. Genome-wide DNase hypersensitivity mapping of osteoblast cultures was performed by adapting the DNase-seq protocol from Song et al. (Song and Crawford, 2010) with slight modifications. Growth-phase (day 0), matrix-deposition stage (day 9), or mineralization stage (day 28) MC3T3-E1 clone-4 cultures were subjected to DNase-seq library preparation. Libraries of purified DNA were generated using custom adapters described in Song et al. High-throughput sequencing was performed by Illumina Genome Analyzer II with 36 base reads and on an Illumina Hiseq-1000 with 100 base reads. Base calls and sequence reads were generated by Illumina CASAVA software (version 1.6, Illumina). Two independent biological repeats of DNase-seq libraries were prepared for each time point. Each biological repeat is represented by two technical repeats.
2015-05-18 | E-GEOD-55046 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2014-02-13 | E-GEOD-54014 | biostudies-arrayexpress
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.
2014-02-13 | E-GEOD-54013 | biostudies-arrayexpress
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.
2014-02-13 | E-GEOD-53982 | biostudies-arrayexpress
Project description:Hemogenic endothelium (HE) is the source of HSCs in the developing embryo. In this study we have identified the hemogenic endothelial progenitors and their precursors originating from differentiated H1 cells on OP9 stromal cells. RNA-seq of hemogenic endothelial progenitors and their precursors originating from differentiated H1 cells on OP9 stromal cells.
2012-09-18 | E-GEOD-39661 | biostudies-arrayexpress
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. RNA was isolated and applied to gene expression microarrays in undifferentiated MC3T3-E1 cells as well as post 15 day osteogenic differentiation MC3T3-E1 cells. The samples were completed in biological triplicate.
2014-04-24 | E-GEOD-41954 | biostudies-arrayexpress
Project description:The biological effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast differentiation and function differ significantly depending upon the cellular state of maturation. To explore this phenomenon mechanistically, we examined the impact of 1,25(OH)2D3 on the transcriptomes of both pre-osteoblastic (POBs) and differentiated osteoblastic (OBs) MC3T3-E1 cells, and assessed localization of the vitamin D receptor (VDR) at sites of action on a genome-scale using ChIP-seq analysis. We observed that the 1,25(OH)2D3-induced transcriptomes of POBs and OBs were quantitatively and qualitatively different, supporting not only the altered biology observed but the potential for a change in VDR interaction at the genome as well. This idea was confirmed through discovery that VDR cistromes in POBs and OBs were also strikingly different. Depletion of VDR binding sites in OBs, due in part to reduced VDR expression, was the likely cause of the loss of VDR-target gene interaction. Continued novel regulation by 1,25(OH)2D3, however, suggested that factors in addition to the VDR might also be involved. Accordingly, we show that transcriptomic modifications are also accompanied by changes in genome binding of the master osteoblast regulator RUNX2 and the chromatin remodeler C/EBPβ. Importantly, genome occupancy was also highlighted by the presence of epigenetic enhancer signatures which were selectively changed in response to both differentiation and 1,25(OH)2D3. The impact of VDR, RUNX2, and C/EBPβ on osteoblast differentiation is exemplified by their actions at the Runx2 and Sp7 gene loci. We conclude that each of these mechanisms may contribute to the diverse actions of 1,25(OH)2D3 on differentiating osteoblasts. RNA was isolated and applied to gene expression microarrays in undifferentiated MC3T3-E1 cells as well as post 15 day osteogenic differentiation MC3T3-E1 cells, which were treated for 24 hours with 10-7M 1,25(OH)2D3. For the vehicle matched samples, please refer to study GSE41955. The samples were completed in biological triplicate.
2014-06-02 | E-GEOD-51514 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series:; GSE12264: Gene Expression Profile of Osteogenic Cells Derived from Human Bone Marrow and Trabecular Bone I; GSE12265: Gene Expression Profile of Osteogenic Cells Derived from Human Bone Marrow and Trabecular Bone II; GSE12266: Gene Expression Profile of Osteogenic Cells Derived from Human Bone Marrow and Trabecular Bone III Experiment Overall Design: Refer to individual Series
2008-10-21 | E-GEOD-12267 | biostudies-arrayexpress
Project description:One of the long-standing goals in the field has been to establish a culture system that would allow maintenance of HSC properties ex vivo. In the absence of such system, the ability to model human hematopoiesis in vitro has been limited, and there has been little progress in the expansion of human HSCs for clinical application. To that end, we defined a mesenchymal stem cell co-culture system based on a monoclonal OP9 stromal cell line (OP9M2), for expansion of clonally multipotent human HSPCs that were protected from apoptosis and immediate differentiation, and retained the HSPC phenotype. To identify the supportive mechanisms, we performed a genome-wide gene expression analysis of OP9M2 stromal cells and compared the expression to a non-supportive stomal line (BFC012). This co-culture system provides a new, well-defined platform for studying mechanisms involved in HSC-niche interactions and protection of critical HSC properties ex vivo. To determine the cellular identity and the supportive mechanism of the OP9M2 cells, we compared the OP9M2 cells with non-supportive BFC012 stromal cells using Affymetrix mouse microarrays.
2013-01-31 | E-GEOD-34973 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-01-31 | E-GEOD-34974 | biostudies-arrayexpress