Genetic identification of a network of factors that functionally interact with the nucleosome remodeling ATPase ISWI

Abstract

Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.

Figure 1. An eye assay to identify modifiers of ISWI^K159R.

(A) We assigned individual eyes a score from 1 to 6 based on the severity of the defects ,. Similarly to the ISWI² allele , null mutations for acf1 and E(bx) strongly enhanced eye defects caused by expression of the ISWI^K159R transgene. Our scoring system allowed the visualization of ISWI^K159R eye defects as phenotypic class distributions, helping the identification of modifiers of ISWI^K159R phenotypes that are statistically significant. (B) Outline of the F₁ eye-based screen for dominant modifiers of ISWI^K159R by EP insertions on the second, third and (C) X chromosomes.

Figure 2. Genetic interaction of ISWI with Sin3A and Rpd3.

Loss of Sin3A or Rpd3 function enhances defects caused by the expression of ISWI^K159R in the eye. (A) Four EP insertions mapping on the first intron shared by the multiple transcripts of the Sin3A gene were recovered in our screen. Interactions of EP2387, EP2580, EP866, EP2032 and the null Sin3A⁰⁸²⁶⁹ allele with ISWI^K159R are shown. (B) The null Rpd3⁰⁴⁵⁵⁶ allele, but not EP(3)3672 inserted ∼10Kbp away from the Rpd3 in the Src64B gene enhances ISWI^K159R. The top panels show a graphical histogram representation of the eye scoring data reported in Table 1. The lower panels show genomic mapping data for the EP’s (directional arrows) and Sin3A, Rpd3 alleles (arrowheads) tested.

Figure 3. ISWI co-localize with Sin3A and Rpd3 at many sites on polytene chromosomes.

(A, C, E and G) Distribution of ISWI and DAPI or (B and D) ISWI and Sin3A or (F and H) ISWI and Rpd3 proteins on salivary gland polytene chromosomes from wild-type third instar larvae. (C, D, G and H) Magnifications of boxed areas shown as “split” chromosome images. Displaying multiple staining patterns in a ‘‘split’’ format helps avoiding visual artifacts when high levels of one protein can mask low levels of another ,. ISWI, while present at varying abundances it co-localize with Sin3A and Rpd3 proteins at many sites on polytene chromosomes.

Figure 4. ISWI physically interacts with Sin3A and Rpd3 in embryo and larval stages.

(A) Immunoprecipitation with anti-HA antibodies using embryo protein extracts derived from a line expressing HA-tagged ISWI (HA-ISWI) and from control extracts (ISWI). ISWI is specifically immunoprecipitated from the HA-ISWI extract together with the Rpd3 and Sin3A proteins. (B) Immunoprecipitation with the anti-ISWI antibody, but not with control IgG, immunoprecipitates Sin3A and Rpd3 on embryo protein extracts. Western blot analysis was performed on 10% of the total input extract [I], supernatant [S], wash [W], 5% of the total pellet [P1], and 30% of the total pellet [P] and [P2] using antibodies against ISWI, Sin3A and Rpd3. The arrows indicate two Sin3A isoforms detected by Western. (C) Larval nuclear extracts derived from larvae expressing TAP-tagged ISWI were bound to IgG Agarose beads. The ISWI-CBP fusion, consisting of the ISWI protein fused in frame with the calmodulin binding peptide, was eluted from the resin by cleavage with the TEV protease. The ISWI-CBP fusion protein eluted together with Sin3A and Rpd3 in nuclear extracts purified from larvae expressing TAP-tagged ISWI. The TEV cleaved eluate was rebound to a calmodulin coupled resin and the bound proteins were eluted by stripping the resin with SDS-loading buffer. Densitometric analysis indicate that ∼15% of total Sin3A and Rpd3 present in larval nuclear extracts associate with ISWI. Western blot analysis was performed on 0.05% of the total input extract [I], 3% of the TEV cleaved eluate [E1] and 3% of the SDS stripped eluate [E2], using antibodies against ISWI, Sin3A and Rpd3. Arrows indicate TAP-tagged ISWI [ISWI-TAP], ISWI fused in frame with the calmodulin binding peptide [ISWI-CBP] and endogenous untagged ISWI [ISWI].

Figure 5. ISWI is associated with an HDAC activity co-eluting with Sin3A and Rpd3.

(A) ISWI together with Sin3A and Rpd3 specifically eluted by TEV cleavage from larval nuclear extracts derived from the TAP-tagged ISWI line [ISWI-TAP], but not in control untagged extracts [ISWI]. Proteins eluting by TEV cleavage, containing ISWI-CBP together with Sin3A and Rpd3 were assayed for (B) nucleosome-stimulated ATPase and (C & D) HDAC activity on acetylated histone H4 and H3 substrates. The fraction containing ISWI-CBP [lane 1] but not the control fraction [lane 2] has nucleosome stimulated ATPase. A specific histone H4 and H3 HDAC activity was also found associated with the fraction containing ISWI-CBP [lane 2] but not the control fraction [lane 3]. For the ATPase assay, about 1.5% of Input [I] and TEV cleaved eluates were tested for ATPase activity in the presence of 100 ng of reconstituted recombinant chromatin. The HDAC assays were conducted on 15000 cpm of acetylated histones, with 8% of Input [I] and TEV cleaved eluates.

Figure 6. Loss of ISWI reduces the binding of Sin3A and Rpd3 to chromatin.

(A and G) DAPI and immunostaining of (B) Sin3A or (H) Rpd3 on wild-type salivary gland male polytene chromosomes. (D and J) DAPI staining and distribution of (E) Sin3A or (K) Rpd3 on ISWI mutant salivary gland male polytene chromosomes. Merge of a double immunostainig for ISWI and Sin3A (C and F) or ISWI and Rpd3 (I and L) on wild-type and ISWI mutant salivary gland male polytene chromosomes, respectively. ISWI mutant chromosomes show a significant reduction in the levels of chromatin bound Sin3A and Rpd3 proteins when compared to wild-type chromosomes. The arrows mark the male X chromosome.

Figure 7. Changes in chromatin bound ISWI affects the levels of acetylated histone H3 and H4.

(A) Immunofluorescence detection of ISWI [red] and acetylated histone H4 [green; K5Ac, K8Ac, K12Ac, K16Ac] on wild-type salivary gland polytene chromosomes [upper panel]. The distributions of ISWI and acetylated histone H4 are dissimilar on both autosomes and X chromosome, with few sites of overlap (arrows), as visible in the split-image magnifications. Distribution of ISWI [green] and acetylated histone H3 [red; K9Ac, K14Ac] on wild-type salivary gland polytene chromosomes [lower panel]. ISWI share a good number of sites on both autosomes and X chromosome with the acetylated histone H3 (arrows), as visible in the split-image magnifications. (B) Distributions and levels of acetylated histone H3 [upper pannel], acetylated histone H4 [middle panel] and Sin3A [lower panel] on DAPI stained polytene chromosomes from w1118 strain[wt], ISWI¹/ISWI² mutant [ISWI] and from salivary glands expressing wild-type ISWI [uas-ISWI]. The specificity of αH3Ac and αH4Ac antibodies has been tested by Western on unmodified and acetylated histone substrates (data not shown). Arrows indicate the puffed X chromosome on ISWI¹/ISWI² mutant male chromosomes.