Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei
Figure 5
Chromatin structure in the presence of reduced histone H1.
A. Schematic of the T. brucei BF RYT3-H1 cell line in which micrococcal nuclease (MNase) sensitivity experiments were performed after the induction of histone H1 RNAi. The large blue box indicates the BF T. brucei cell, with a blasticidin (Bla) gene integrated in the active VSGT3 ES (T3), and the puromycin (Pur) resistance gene and eGFP integrated immediately behind the promoter of the silent VSG221 ES. The expression site (ES) promoters are indicated with white flags, and ES transcription with an arrow. A construct allowing tetracycline inducible transcription of histone H1 RNAi (H1) from opposing T7 promoters (facing arrows) has also been introduced into these cells using a hygromycin resistance gene (Hyg) transcribed from an rDNA promoter (black flag). B. Parental (Par) BF T. brucei RYT3BSR cells and RYT3-H1 cells in which histone H1 had been knocked down by the induction of RNAi for 48 h (H1+48 h) were permeabilized and incubated with increasing concentrations of MNase (Lane 1 = 0.0625 units MNase, lane 2 = 0.125 units, lane 3 = 0.25 units and lane 4 = 0.5 units. Isolated DNA was visualized on ethidium bromide-stained agarose gels, and a characteristic ladder pattern was observed. Products corresponding to DNA that had been bound to mono-, di, or trinucleosomes, as well as undigested DNA (undig.) are indicated. DNA sizes are indicated in kilobases (kb) on the left. C. Sucrose gradient fractionation of MNase-digested chromatin from BF T. brucei RYT3BSR cells (Par). Chromatin in permeabilized cells was subjected to MNase treatment and then loaded onto a 5–30% sucrose step gradient (input). After centrifugation, fractions were removed, and DNA isolated. Fractions 11–24 are shown with top to bottom of the gradient indicated with an arrow. Fractions 1–10 contained very little DNA and are not shown. Fractions containing mono-, di-, di-/tri-, tri-/tetra-, and >tetranucleosomes are indicated with white bars. These fractions were used to create five pools of DNA which were used as templates for qPCR. D. BF T. brucei cells in which histone H1 RNAi had been induced for 48 hours. Chromatin in permeabilized cells was subsequently subjected to MNase treatment and was further analysed as described in panel C. E. Induction of histone H1 RNAi for 48 hours affects the distribution of various T. brucei genomic regions in the mononucleosomal fractions containing open chromatin. Results show qPCR analysis of fractionated, MNase-treated DNA. The amount of each target detected in the mononucleosome pool is plotted as a percentage of the total amount of target detected in all pools. Genomic regions analysed are as indicated in Fig. 3 panel D. The mean of three independent experiments is shown with error bars indicating standard deviation. Histone H1 knock-down resulted in a statistically significant increased distribution of various regions in the mononucleosomal fraction, with asterisks indicating statistical significance (*, P<0.05; **, P<0.01; ***, P<0.001).