Abstract
The ability to discover regulatory sequences that control bone-related genes during development has been greatly improved by massively parallel sequencing methodologies. To expand our understanding of cis-regulatory regions critical to the control of gene expression during osteoblastogenesis, we probed the presence of open chromatin states across the osteoblast genome using global DNase hypersensitivity (DHS) mapping. Our profiling of MC3T3 mouse pre-osteoblasts during differentiation has identified more than 224,000 unique DHS sites. Approximately 65% of these sites are dynamic during temporal stages of osteoblastogenesis, and a majority of them are located within non-promoter (intergenic and intronic) regions. Nearly half of all DHS sites (both constitutive and dynamic) overlap binding events of the bone-essential RUNX2 and/or the chromatin-related CTCF transcription factors. This finding reinforces the role of these regulatory proteins as essential components of the bone gene regulome. We observe a reduction in chromatin accessibility throughout the genome between pre-osteoblast and early osteoblasts. Our analysis also defined a class of differentially expressed genes that harbor DHS peaks centered within 1 kb downstream of transcriptional end sites (TES). These DHSs at the 3’-flanks of genes exhibit dynamic changes during differentiation that may impact regulation of the osteoblast genome. Taken together, the distribution of DHS regions within non-promoter locations harboring osteoblast and chromatin related transcription factor binding motifs, reflect novel cis-regulatory requirements to support temporal gene expression in differentiating osteoblasts.
Highlights
The process of osteoblast differentiation is controlled by an abundance of cellular signaling events that impact the regulation of gene transcription, and in turn, direct cellular identity and behavior
DNase hypersensitivity (DHS) peaks that were common among all three stages totaled 75,228, while more than 65% (80,857 peaks) are exclusive to an individual osteoblast stage (Fig 1B)
Prompted by the overrepresentation of RUNX and CCCTC-binding factor (CTCF) motifs within DHS sites of differentiating osteoblasts, we examined the extent to which regions of differential hypersensitivity coincided with RUNX2 and CTCF binding profiled by our previous ChIP-seq analyses [7]
Summary
Cell cultureThe MC3T3-E1 clone-4 pre-osteoblastic murine cell line [20] (American Type Culture Collection, Manassas, VA) was used in this study. Growth-phase cultures were maintained as reported previously [7, 23]. When cultures reached ~90% confluency, differentiation was initiated by the addition of 142 μM ascorbic acid (Sigma-Aldrich, St. Louis, MO) to 10% FBS (Hyclone, Thermo Fisher Scientific) in α-MEM. After 2 days, the ascorbic acid concentration was increased to 280 μM, and 5 mM β-Glycerophosphate (Sigma- Aldrich) was added [20]. Cultures were maintained at 37 ̊C at 5% CO2, with fresh media changes every 2 days. Phase contrast images of DNase-seq reveals a dynamic osteoblast regulome differentiation progression were captured on a Nikon Eclipse TS100 inverted microscope (Nikon Instruments Inc., Melville, NY), conjugated to a SPOT RT3 CCD camera using SPOT imaging software v5.0 (Diagnostic Instruments Inc., Sterling Heights, MI)
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