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Drosophila ISWI regulates the association of histone H1 with interphase chromosomes in vivo - PubMed

Drosophila ISWI regulates the association of histone H1 with interphase chromosomes in vivo

Giorgia Siriaco et al. Genetics. 2009 Jul.

Abstract

Although tremendous progress has been made toward identifying factors that regulate nucleosome structure and positioning, the mechanisms that regulate higher-order chromatin structure remain poorly understood. Recent studies suggest that the ISWI chromatin-remodeling factor plays a key role in this process by promoting the assembly of chromatin containing histone H1. To test this hypothesis, we investigated the function of H1 in Drosophila. The association of H1 with salivary gland polytene chromosomes is regulated by a dynamic, ATP-dependent process. Reducing cellular ATP levels triggers the dissociation of H1 from polytene chromosomes and causes chromosome defects similar to those resulting from the loss of ISWI function. H1 knockdown causes even more severe defects in chromosome structure and a reduction in nucleosome repeat length, presumably due to the failure to incorporate H1 during replication-dependent chromatin assembly. Our findings suggest that ISWI regulates higher-order chromatin structure by modulating the interaction of H1 with interphase chromosomes.

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Figures

F<sc>igure</sc> 1.— — **Figure 1.—**
Loss of histone H1 alters chromosome structure. (A) Reduced histone H1 expression is observed in the salivary glands of *P[w⁺, UAS-His1-dsRNA-8-4]*/+; *da-GAL4/+* larvae, compared to control *da-GAL4/+* larvae, as assayed by protein blotting. Protein sizes can be determined by referring to molecular weight markers alongside the gel. (B–D and F–H) Polytene chromosomes stained with DAPI. Control *da-GAL4/+* chromosomes (B) exhibit normal morphology while *His1* RNAi leads to chromosome decondensation (C–D and F). (D) A magnification of the boxed regions of B, C, and G. *P[w⁺, UAS-His1-dsRNA-8-4]*/+; *da-GAL4/+* chromosomes and the male *Iswi¹/Iswi²* X chromosome are decondensed relative to the control chromosome, but the banding pattern is maintained. (E) Quantification of DNA in *P[w⁺, UAS-His1-dsRNA-8-4]*/+; *da-GAL4/+* and control *da-GAL4/+* chromosomes . (F) Individual chromosome arms are no longer distinguishable in some nuclei. (G) The male X chromosome (arrowhead) is decondensed in *Iswi¹/Iswi²* larvae. (H) Expression of ISWI^K159R leads to disorganized chromatin (arrowhead) and decondensation (arrow) of all chromosomes. (I) Quantification of DNA in *P[w⁺, eyGAL4], P[w⁺, UAS-ISWI^K159R-HA-6His]11-4/H2AvD-GFP* and control *H2AvD-GFP/TM3* chromosomes. Bars, 20 μm.

F<sc>igure</sc> 2.— — **Figure 2.—**
Histone H1 is not required for the expression of ISWI or its binding to chromatin. (A) Levels of ISWI protein are not affected in the salivary glands of *P[w⁺, UAS-His1-dsRNA-8-4]*/+; *da-GAL4/+* larvae, compared to control *da-GAL4/+* larvae, as assayed by protein blotting. A comparable blot was probed with antibodies against histone H3 as a control. Protein sizes can be determined by referring to molecular weight markers alongside the gel. (B and C) Polytene chromosomes of *da-GAL4/+* (B) and *P[w⁺, UAS-His1-dsRNA-8-4]*/+; *da-GAL4/+* (C) larvae were stained with an antibody against ISWI. Polytene chromosomes were prepared and processed in parallel, and images were captured using identical exposure times.

F<sc>igure</sc> 3.— — **Figure 3.—**
Histone H1 is rapidly exchanged in salivary gland nuclei and increases NRL. (A) Quantitative analysis of FRAP experiments. The recovery curve for aphidicolin-treated nuclei are shown in dark shading, the recovery curve for control DMSO-treated nuclei in light shading. Salivary glands were incubated in aphidicolin or DMSO for 4 hr prior to FRAP analysis. (B) Partial micrococcal nuclease digestion of chromatin isolated from salivary glands of *da-GAL4/+* and *P[w⁺, UAS-His1-dsRNA-8-4]*/+; *da-GAL4/+* larvae. Loss of histone H1 leads to a reduction in NRL compared to control. DNA fragment sizes can be determined by referring to the 100-bp ladder alongside the gel. (C) Examples of phenotypes resulting from azide treatment of nuclei expressing H1-CFP or H2AvD-GFP; dashed line identifies the nuclear boundary. Histone H1 dissociates from chromosomes and appears in the nucleoplasm. Azide treatment had no effect on H2AvD association with chromosomes. Bars, 20 μm.

F<sc>igure</sc> 4.— — **Figure 4.—**
Loss of histone H1 increases chromosome volume. (A–D and H–J) Live analysis of nuclei expressing H2AvD-GFP reveals decondensation of *P[w⁺, UAS-His1-dsRNA-8-4]/+; da-GAL4, H2AvD-GFP/+* chromosomes (B) compared to *da-GAL4, H2AvD-GFP/+* chromosomes (A). (D) *P[w⁺, eyGAL4], P[w⁺, UAS-ISWI^K159R-HA-6His]11-4/H2AvD-GFP* chromosomes show disorganized chromatin structure but no increase in chromosome volume compared to control *H2AvD-GFP/TM3* chromosomes (C). Bars, 20 μm. (E) Volume quantification of *P[w⁺, UAS-His1-dsRNA-8-4]/+; da-GAL4, H2AvD-GFP/+* and control *da-GAL4, H2AvD-GFP/+* chromosomes. (F) Volume quantification of *P[w⁺, eyGAL4], P[w⁺, UAS-ISWI^K159R-HA-6His]11-4/H2AvD-GFP* and control *H2AvD-GFP/TM3* chromosomes. (G) Quantification of the change in chromatin compaction relative to control, established by calculating the ratio of volume to DNA for each nucleus. The ratio of control samples was normalized to 1. (H–J) A magnification of arms from *H2AvD-GFP/TM3* chromosomes (H), *P[w⁺, UAS-His1-dsRNA-8-4]/+; da-GAL4, H2AvD-GFP/+* chromosomes (I) and *P[w⁺, eyGAL4], P[w⁺, UAS-ISWI^K159R-HA-6His]11-4/H2AvD-GFP* chromosomes (J). Bars, 5 μm.

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Drosophila ISWI regulates the association of histone H1 with interphase chromosomes in vivo - PubMed