Analysis of Dnase I hypersensitive sites during OB differentiation.

DHS sites in cells shifted to 39°C in BM or OIM for 24 and 48hr (d1 and d2) were compared to control cells growing in BM at 34°C (BM 34). (A) Total number of DHS sites from 2-way comparison as indicated by colors on the right of the bar graph (blue: B39d1-B34, red: B39d2-B34, green: OIMd1-B34, purple: OIMd2-B34) was separated into common, unique and unique enhancers, designated as sites after removing the promoters. (B) Ratio of unique DHS sites separated into promoters, exons, introns, and distal up and downstream sites. The first 2 bars represent BM control cells compared to differentiating cells with no significant shift of fractions. The last 2 bars show the reverse comparison, using DHS unique for BM39 and OIM. (C) Aggregation plots showing average tag density relative to TSS from 3-way comparison with significant increase in MaxD from OIM treated cells. (D) An example of DHS tracing for WNT5A locus (UCSC browser, [27]) show changes in the proximal enhancer located upstream within 5kb of WNT5A TSS. Chromosomal coordinates and scale are indicated on the top. Arrows indicate sites changed between control and treated cells. Each condition is annotated on the left of each tracing. DHS for mature bone osteoblasts is added for comparison (from ENCODE database, Short Read Archive: GSM816654). d. (E) DHS tracing for DKK1 locus shows changes in the promoter. Designations are as above. Each tracing represents replicate concordant from 2–4 independent experiments. DHS for mature bone osteoblasts is added for comparison (from ENCODE database, Short Read Archive: GSM816654). (F) Expression changes in WNT5A and DKK1 showing log2 values from 3 biological replicas. Color labels are indicated on the right.