ISWI regulates higher-order chromatin structure and histone H1 assembly in vivo

Abstract

Imitation SWI (ISWI) and other ATP-dependent chromatin-remodeling factors play key roles in transcription and other processes by altering the structure and positioning of nucleosomes. Recent studies have also implicated ISWI in the regulation of higher-order chromatin structure, but its role in this process remains poorly understood. To clarify the role of ISWI in vivo, we examined defects in chromosome structure and gene expression resulting from the loss of Iswi function in Drosophila. Consistent with a broad role in transcriptional regulation, the expression of a large number of genes is altered in Iswi mutant larvae. The expression of a dominant-negative form of ISWI leads to dramatic alterations in higher-order chromatin structure, including the apparent decondensation of both mitotic and polytene chromosomes. The loss of ISWI function does not cause obvious defects in nucleosome assembly, but results in a significant reduction in the level of histone H1 associated with chromatin in vivo. These findings suggest that ISWI plays a global role in chromatin compaction in vivo by promoting the association of the linker histone H1 with chromatin.

Figure 1. ISWI Plays a Global Role in Chromosome Compaction In Vivo

Polytene chromosomes were prepared from larval salivary glands and stained with DAPI. Wild-type female (A) and male (B) polytene chromosomes exhibit normal morphology. The loss of zygotic Iswi function in Iswi¹/Iswi² larvae causes the decondensation of the X chromosome in male (D, arrowhead) but not female (C, arrow) larvae. Expression of wild-type ISWI does not dramatically alter chromosome morphology (E). By contrast, the expression of a dominant-negative ISWI protein (ISWI^K159R) in salivary gland nuclei causes the dramatic decondensation of all chromosomes (F).

Figure 2. ISWI Regulates Chromosome Structure in Diploid Cells

(A–F) Metaphase chromosome spreads isolated from neuroblasts of third-instar larvae grown at 18 °C. (A) Wild-type male metaphase chromosomes. (B) Metaphase chromosomes isolated from male Iswi¹/Iswi² mutant larvae show normal condensation of the X chromosome (arrow). By contrast, mitotic chromosomes (C–E) of larval neuroblasts producing dominant-negative ISWI (ISWI^K159R) appear much less condensed than normal. In these chromosomes, euchromatin is highly decondensed, whereas heterochromatic regions, such as centromeres (open arrowhead in [D]) and the fourth chromosomes (arrowheads in [C] and [E]), appear to condense normally. The production of dominant-negative BRM (BRM^K804R) does not alter the morphology of mitotic chromosomes (F). (G and H) Nuclear cycle 12 embryos grown at 25 °C were stained for α-tubulin (green) and DAPI (red). Metaphase chromosomes in control +; da-GAL4/T(2;3)B3 CyO, TM6B, Tb embryos (G) appear normally condensed, while Iswi²/+; UAS-Iswi^K159R/da-GAL4 embryos (H) show abnormal chromosome morphology and spindle formation.

Figure 3. ISWI Plays a Relatively Global Role in Transcriptional Activation and Repression In Vivo

(A and B) ISWI is associated with hundreds of euchromatic sites in a pattern that is largely complementary to that of Pol II. (A) ISWI (green) and Pol II (red) were detected using antibodies against ISWI and the second largest subunit of Pol II (RPII140). The distributions of ISWI and RPII140 are largely nonoverlapping, as observed in the split image. (B) ISWI (green) and elongating Pol II (red) were detected using antibodies against ISWI and the antibodies against Pol II hyperphosphorylated on Ser2 of the CTD (Pol IIo^ser2). Very little overlap is observed between ISWI and elongating Pol II, as observed in the split image. (C and D) Analysis of physical interactions between ISWI and its potential target genes in polytene chromosomes by immuno-FISH. Wild-type polytene chromosomes were stained with antibodies against ISWI (green) and the elongating form of Pol II (Pol IIo^ser2) (blue). FISH analysis identifies the locus of genes CG18350/Sxl (C) and CG1214/ru (D) in red. CG18350, which is expressed at reduced levels in Iswi mutants, localizes with Pol IIo^ser2 but does not co-localize with ISWI, as indicated by the arrows. CG1214, which is expressed at elevated levels in Iswi mutants, co-localizes with ISWI but not with Pol IIo^ser2, as indicated by the arrows.

Figure 4. Genes That Exhibit Altered Expression in ISWI Mutants Are Broadly Distributed throughout the Genome

The positions of euchromatic genes that exhibit a 2-fold or greater change in expression in the salivary glands of Iswi mutant males are shown. The x-axis corresponds to the relative cytological positions of genes on the X chromosome (X) and the left or right arms of the second (2L and 2R) and third (3L and 3R) chromosomes. Red and green bars mark genes that are repressed and activated by ISWI, respectively. The fold change in gene expression (log₂) in Iswi mutants relative to wild-type is shown on the y-axis. The chromosomes are drawn to scale based on the amount of euchromatin found on each arm.

Figure 5. H4K16 Acetylation Is Not Affected by Loss of ISWI

Salivary gland polytene chromosomes from male third-instar larvae were stained with DAPI and an antibody that specifically recognizes acetylated H4K16 (H4AcK16). The male X chromosome is marked by arrowheads. The level of H4K16 acetylation on the X chromosome of wild-type (A) and Iswi¹/Iswi² (B) larvae is similar. Chromosome decondensation resulting from the expression of ISWI^K159R in salivary gland nuclei is not due to the spread of H4K16 acetylation to the autosomes (C). All three images were captured using identical exposure times.

Figure 6. Loss of ISWI Results in Defects in Chromatin Assembly

(A) The distribution of histones was analyzed on polytene chromosomes. Wild-type (WT) chromosomes immunostained with a pan-histone antibody (MAB052), which has been described as staining all histones, exhibit uniform histone staining of all arms. Iswi¹/Iswi² male autosomes were uniformly stained, while staining of the X chromosome was dramatically reduced (arrowheads). Iswi¹/Iswi² female chromosomes show uniform staining of all chromosomes, including the X chromosome, which is condensed normally (arrows). Chromosomes from cells that have produced a dominant-negative form of ISWI (ISWI^K159R) have dramatically reduced staining. (B) Chromatin isolated from salivary glands of third-instar larvae was partially digested with microccocal nuclease. The open triangles indicate mono-, di-, tri-, and tetra-nucleosomal repeats. The approximate size of the DNA fragments can be visualized by referring to the 100-bp ladder marked alongside the gel. Comparison of digestion patterns of chromatin isolated from wild-type larvae and from larvae expressing ISWI^K159R reveals no obvious changes in nucleosome spacing or sensitivity to digestion. (C) Specificity of the reportedly pan-histone MAB052 antibody was analyzed by protein blotting: 20 ng of histone H1 (H1) purified from calf thymus, 1 μg recombinant Drosophila histone octamers (OCT), and embryo (E) and salivary gland (SG) extracts were resolved on a 12% SDS-polyacrylamide gel. The antibody, which is not really pan-histone, has a strong preference for histone H1 and only weakly recognizes recombinant Drosophila histone H3. In embryo and salivary gland extracts, the antibody exhibits a strong preference for histone H1, and also recognizes an uncharacterized band in salivary gland extracts (asterisk).

Figure 7. Loss of ISWI Results in Loss of Histone H1

(A) A polyclonal antibody (αdH1) specifically recognizes histone H1 in both embryo (E) and salivary gland (SG) extracts by protein blotting. (B) Immunostaining of male Iswi¹/Iswi² polytene chromosomes with antibodies against histone H1 reveals that the levels of histone H1 are dramatically reduced on the decondensed X chromosome (arrowheads). The inserts show an enlarged image of the male X chromosome. (C and D) Wild-type chromosomes exhibit uniform staining with antibodies against histone H1 (C), whereas both the X chromosome and autosomes of larvae expressing ISWI^K159R have dramatically reduced staining (D). The images were captured with comparable exposure times. (E) Histone H1 levels are compared to histone H3 levels in salivary gland chromatin extracts by protein blotting using antibodies that specifically recognize Drosophila histone H1 [54] and histone H3 (Abcam, catalog number ab1791). Chromatin from larvae expressing ISWI^K159R was intentionally overloaded to ensure visualization of the histone H1 band. Consistent with the results presented in (D), the expression of ISWI^K159R leads to a significant (∼5-fold) reduction compared to wild-type (WT) in the ratio of histone H1 to histone H3 associated with chromatin.