Modulation of ISWI function by site-specific histone acetylation

Abstract

Mutations in Drosophila ISWI, a member of the SWI2/SNF2 family of chromatin remodeling ATPases, alter the global architecture of the male X chromosome. The transcription of genes on this chromosome is increased 2-fold relative to females due to dosage compensation, a process involving the acetylation of histone H4 at lysine 16 (H4K16). Here we show that blocking H4K16 acetylation suppresses the X chromosome defects resulting from loss of ISWI function in males. In contrast, the forced acetylation of H4K16 in ISWI mutant females causes X chromosome defects indistinguishable from those seen in ISWI mutant males. Increased expression of MOF, the histone acetyltransferase that acetylates H4K16, strongly enhances phenotypes resulting from the partial loss of ISWI function. Peptide competition assays revealed that H4K16 acetylation reduces the ability of ISWI to interact productively with its substrate. These findings suggest that H4K16 acetylation directly counteracts chromatin compaction mediated by the ISWI ATPase.

Fig. 1. The dosage compensation complex is necessary for the X chromosome defects observed in ISWI² males. (A) Salivary gland polytene chromosomes from homozygous ISWI² male larvae. Note the bloated appearance of the X chromosome. (B) Salivary gland polytene chromosomes from homozygous mle¹ ISWI² male larvae. Loss of mle function completely suppresses X chromosome defects associated with loss of ISWI function. No staining of the male X chromosome was detected in these larvae using antibodies against acetylated H4K16 (data not shown). (C) Salivary gland polytene chromosomes from OreR male larvae. In all panels, the X chromosome is marked by an arrow.

Fig. 2. The activity of the dosage compensation complex is sufficient for the X chromosome defects resulting from loss of ISWI function. (A) Salivary gland chromosomes from female ISWI²/ISWI²; [H83M2]/+ larvae. Note the bloated appearance of the X chromosome (marked by an arrow) resulting from the expression of the MSL-2 protein in ISWI² females. An extreme example of this phenotype is shown in (B).The X chromosome was recognized in these individuals by staining with an antibody against acetylated H4K16 (data not shown). (C) Salivary gland polytene chromosomes from homozygous ISWI² female larvae. Note that all chromosomes appear normal.

Fig. 3. Elevated MOF expression enhances phenotypes resulting from partial loss of ISWI function in the developing eye. (A) hsp.mof/TM3, Sb males were crossed to ey-Gal4, UAS-ISWI^K159R/TM3, Sb females at 29°C and female progeny were scored for eye defects as follows: 1, wild-type eye; 2, roughness comprising <50% of normal eye area; 3, roughness comprising >50% of normal eye area; 4, eye rough and <50% reduced in size; 5, eye rough and >50% reduced in size; 6, eye absent. Note that the hsp.mof transgene dramatically enhances eye defects associated with ISWI^K159R expression. No enhancement was observed in progeny raised at 18°C. (B) Table of data used to generate graph shown in (A). % Control refers to the number of progeny recovered relative to hsp.mof/TM3, Sb siblings. Note the decreased viability resulting from the expression of both MOF and ISWI^K159R. (C) Representative eyes of hsp.mof/TM3, Sb (class 1), ISWI^K159R/TM3, Sb (class 2) and hsp.mof/ISWI^K159R (class 5).

Fig. 4. The ATPase activity of ISWI is modulated by site-specific acetylation of the H4 N-terminal tail. The ATPase of ISWI is stimulated strongly by the presence of nucleosomes. Synthetic peptides corresponding to the H3 and H4 N-terminus were added to ATPase reactions to determine their potential to compete with the nucleosomal substrate. The competition properties of peptides acetylated at H4K16 (filled circles), H4K12 (open diamonds), H4K8 (open triangles) are best compared to wild-type H4 (open circles) and H3 (open squares) N-termini in reactions containing 200 pmol of peptide. The graph reflects the average of two independent experiments with similar competition profiles. The result has been qualitatively reproduced under a variety of conditions, albeit with different absolute values.