HMGD1 and H1 knockdown reciprocally mediate higher order chromatin structure leading to changes in distinct gene expression outcomes. (A) S2 cells were treated with the indicated siRNA for 48 hours and knockdown (KD) was validated by western blot. For the H1 KD, there was a ~40% reduction in H1 protein levels; for the HMGD1 KD, there was a ~90% reduction in HMGD1 protein levels. (B) Relative occupancy of HMGD1 and H1 as measured by ChIP-qPCR at promoters that were initially bound by H1 and HMGD1. Relative occupancy was computed by setting the IgG control to 1. (C) Gene expression changes measured by RT-qPCR following HMGD1 or H1 knockdown in S2 cells. Expression was normalized to β-actin levels and fold change in expression was calculated by setting the expression in the mock control to 1. In (B) and (C), error bars are mean ± SD from three independent experiments. Using a student t-test, the p values from all experiments were significant with values ranging from p = 1.35 × 10-4 to p = 2.87 × 10-8(D) Nucleosome repeat length changes caused by KD of H1 or HMGD1. S2 nuclei (5 × 106) were digested with 25 units of MNase for 1, 3, 5, 7 and 10mins. Purified DNA from these digests was run on a 3.3% Nusieve™ agarose gel. M indicates DNA ladders. Red stars indicate the nucleosome ladders. A decrease in H1 levels decreased the nucleosome repeat length by ~24 bp. Knockdown of HMGD1 increased the nucleosome repeat length by ~7 bp.