The Drosophila NURF remodelling and the ATAC histone acetylase complexes functionally interact and are required for global chromosome organization

Abstract

Drosophila Gcn5 is the catalytic subunit of the SAGA and ATAC histone acetylase complexes. Here, we show that mutations in Gcn5 and the ATAC component Ada2a induce a decondensation of the male X chromosome, similar to that induced by mutations in the Iswi and Nurf301 subunits of the NURF nucleosome remodelling complex. Genetic studies as well as transcript profiling analysis indicate that ATAC and NURF regulate overlapping sets of target genes during development. In addition, we find that Ada2a chromosome binding and histone H4-Lys12 acetylation are compromised in Iswi and Nurf301 mutants. Our results strongly suggest that NURF is required for ATAC to access the chromatin and to regulate global chromosome organization.

Figure 1

ATAC and Iswi interact functionally during Drosophila development. Decreased Gcn5 or Ada2a expression enhances the Iswi dominant-negative phenotype induced by expression of Iswi^K159R in the eye. (A) Enhancement of Iswi^K159R dominant-negative phenotype by Gcn5 and Ada2a mutant alleles. eye-Gal4, UAS-Iswi^K159R/Tm3, Ser females were crossed with RNAi trigger UAS-IR[Gcn5] transgenic males or Tm3, Sb balanced Gcn5^E333st, Gcn5^f02830, Ada2a^Δ189 or Ada2b^Δ842 heterozygous males. Progenies of the indicated genotype were scored for eye defects as class 1 (>50% reduction in size) or class 2 (<50% reduction in size). The eye-Gal4, UAS-Iswi^K159R chromosome is indicated in the table as Iswi^K159R. Results are expressed as percentages relative to the total count of siblings of the indicated genotype. Control Iswi^K159R/Tm3, Sb siblings were counted from the progeny of the eye-Gal4, UAS-Iswi^K159R/Tm3, Ser × Gcn5^E333st/Tm3, Sb cross. Equivalent counts were obtained for Iswi^K159R/Tm3, Sb siblings from the other crosses. Statistical significance was first assessed by a χ² homogeneity test with 5 degrees of freedom for error, which indicated that class-1 and class-2 frequencies vary with the tested genotypes (P<0.0001). Pairwise χ² tests were then performed for each mutant genotype as indicated, taking the Iswi^K159R/Tm3, Sb as reference. χ² and P-values indicate that class-1 and class-2 frequencies differ significantly from the control in all mutant combinations except in Ada2b^Δ842 (P=0.0137). (B) A representative class-1 eye-Gal4, UAS-Iswi^K159R/Gcn5^E333st adult fly is shown together with a representative class-2 eye-Gal4, UAS-Iswi^K159R/Tm3, Sb adult fly.

Figure 2

Gcn5, Ada2a and Nurf301 regulate a common set of genes at the end of the larval phase. Venn diagrams describing the overlap of genes downregulated by at least a factor of three in Gcn5, Ada2a and Nurf301 mutants at the end of the third larval instar.

Figure 3

Gcn5 mutations induce male-specific X-chromosome decondensation. Polytene chromosomes from salivary glands were prepared from males and females homozygous for the (A,B) Gcn5^f02830 or (C,D) Gcn5^E333st mutations and stained with 4,6-diamidino-2-phenylindole (DAPI) or an H4-AcK16 antibody, as indicated. (E) DAPI staining of polytene chromosomes from mle¹/mle¹; Gcn5^E333st/Gcn5^f02830 mutant male larvae. (F) DAPI staining of polytene chromosomes from Act-msl2/+; Gcn5^E333st/Gcn5^f02830 mutant female larvae. White arrows indicate X chromosomes.

Figure 4

Mutation of the ATAC subunit Ada2a induces male-specific X-chromosome bloating. Polytene chromosomes from homozygous (A,B) Ada2a^Δ189 or (C,D) Ada2b^Δ842 third instar mutant larvae were stained with 4,6-diamidino-2-phenylindole. White arrows indicate X chromosomes.

Figure 5

Binding of Ada2a to chromosomes and H4-AcK12 acetylation are impaired by mutations in components of the Iswi-containing NURF remodelling complex. Polytene chromosomes from wild-type (wt), homozygous Iswi² and heteroallelic Nurf301¹/Nurf301³ mutant males were co-stained with 4,6-diamidino-2-phenylindole (blue) and Ada2a, Ada2b, H4-AcK12 or H3-AcK9/14 (red) antibodies, as indicated.