FIG. 7.

HMGN1 binds to Sox9 chromatin. (A and B) ChIP analysis (with anti HMGN1) of mouse MEFs and E10.5 limb bud cells. (A) Schematic diagram of Sox9 gene, with the positions of primer sets used to amplify the immunoprecipitated DNAs are indicated at the bottom. The arrow indicates transcription start site. (B) ChIP assay for HMGN1 in MEFs and limb bud cells. Enrichment of each DNA sequence in the HMGN1 immunoprecipitate relative to the input DNA was normalized and plotted as the position of the PCR primer pair within the Sox9 gene locus. Note that HMGN1 is associated with Sox9 chromatin in limb bud cells but not in MEFs. (C) Expression levels of Hmgn1 and Sox9 mRNA in MEFs and limb bud cells. Note that while HMGN1 is expressed in both cell types, Sox9 is expressed only in limb bud cells. (D) Western analysis of HMGN1 and Sox9 proteins in limb bud cells and MEFs. Coomassie blue staining of histones indicates equal loading of extracts from MEFs and limb bud cells. (E) Enhanced DNase I sensitivity of Sox9 chromatin in limb bud cells. Nuclei isolated from MEF or E10.5 limb bud cells were digested with the indicated concentrations of DNase I, the DNA from the digested nuclei purified and amplified with forward primer of primer pair 3 and reverse primer of primer pair 4. The level of amplification is inversely related to degree of digestion. Each point is an average from three experiments, with a new preparation of cells each time. (F) Similar rate of DNase I sensitivity of the beta-globin gene in chromatin of limb bud cells and MEFs. Primers used for amplification are described in Materials and Methods. LB, limb bud cells.