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. 2016 Dec 29;292(6):2237–2254. doi: 10.1074/jbc.M116.764233

FIGURE 5.

FIGURE 5.

HMGN2 affects STAT5 chromatin accessibility. A, HMGN2 knockdown results in decreased STAT5 binding at CISH. T47D cells expressing shCTL or shHMGN2 were treated with or without PRL for 45 min and analyzed by ChIP-qPCR. Nuclear lysates were precipitated with an antibody against STAT5, and normal IgG served as a control for nonspecific binding. Primers amplify the region of the CISH promoter from −81 to −9 bp, which includes STAT5 consensus elements. Recovered DNA is graphed as a percentage of input. Results are presented as the mean ± S.E. (error bars) of three independent experiments. Statistical significance was determined by two-way repeated measures ANOVA. *, p ≤ 0.05. B, STAT5 is probably not recruited to the promoter region of IER3 or PHLDA2. T47D cells were analyzed by ChIP-qPCR for STAT5 recruitment following 45 min of PRL treatment. The x axis labels indicate the location of each qPCR amplicon relative to the TSS of the indicated gene. All amplicons target STAT5 consensus elements, except IER3 −0.2 kb, IER3 TSS, and PHLDA2 +0.1 kb. CISH primers are described in A. Results are presented as the mean ± S.E. of three independent experiments. Statistical significance was determined by a two-sided t test assuming equal sample variance. ****, p ≤ 0.0001 comparing without and with PRL (No PRL and +PRL) at CISH. No other regions exhibited significant STAT5 enrichment. C, HMGN2 knockdown does not affect STAT5 activation, as indicated by STAT5 phosphorylation and nuclear translocation. T47D cells expressing shCTL or shHMGN2 were treated with or without PRL for 45 min. Nuclear lysates were isolated and analyzed by Western blotting using an antibody against phosphorylated (p-) STAT5. The membrane was stripped and reprobed with an antibody against total STAT5. Knockdown of HMGN2 was verified, and histone H4 was used as a loading control. D, HMGN2 knockdown does not affect global chromatin accessibility. Nuclei from T47D shCTL and shHMGN2 cells were permeabilized, and the chromatin was digested with MNase for the indicated times. Purified DNA was analyzed by agarose gel electrophoresis.