Genome-wide characterization of chromatin binding and nucleosome spacing activity of the nucleosome remodelling ATPase ISWI

Abstract

The evolutionarily conserved ATP-dependent nucleosome remodelling factor ISWI can space nucleosomes affecting a variety of nuclear processes. In Drosophila, loss of ISWI leads to global transcriptional defects and to dramatic alterations in higher-order chromatin structure, especially on the male X chromosome. In order to understand if chromatin condensation and gene expression defects, observed in ISWI mutants, are directly correlated with ISWI nucleosome spacing activity, we conducted a genome-wide survey of ISWI binding and nucleosome positioning in wild-type and ISWI mutant chromatin. Our analysis revealed that ISWI binds both genic and intergenic regions. Remarkably, we found that ISWI binds genes near their promoters causing specific alterations in nucleosome positioning at the level of the Transcription Start Site, providing an important insights in understanding ISWI role in higher eukaryote transcriptional regulation. Interestingly, differences in nucleosome spacing, between wild-type and ISWI mutant chromatin, tend to accumulate on the X chromosome for all ISWI-bound genes analysed. Our study shows how in higher eukaryotes the activity of the evolutionarily conserved nucleosome remodelling factor ISWI regulates gene expression and chromosome organization genome-wide.

Figure 1

ISWI chromatin binding is enriched after the transcription start site (TSS). (A) The density of ISWI-enriched genomic sequences is plot relative to the distance from the TSS of nearby genes. ISWI on average tends to bind with a peak (red arrow) at about ∼300 bp after the TSS. (B) Representative example of a gene bound by ISWI at the level of the promoter–exon region. As expected, ISWI also binds genes whose expression is (C) increased or (D) decreased in ISWI mutant larval tissues relative to wild-type levels (for a complete list, see Supplementary Table S1B and C) (Corona et al, 2007). (E) ISWI binds near the TSS of genes involved in nurse cell apoptosis. ISWI enrichment is proportional to the normalized raw log₂(ChIP^ISWI/input) red signals reported to the right y axis. The thick black bars highlight ISWI peaks. The peak score values of ISWI peaks are reported to the left y axis. Exons are represented by filled-in boxes, introns by a continuous line, while the 5′- and 3′-UTRs by thick lines. The transcription direction for each gene can be deduced by the repeated small blue arrows present in the intron segments. Genes bound by ISWI are shown in red.

Figure 2

Identification of ISWI-binding elements (IBE). (A) DNA consensus motif corresponding to IBE identified with MDscan where correlated with the ‘peak score’ function using the Spearman's correlation index. The Spearman's rank assesses how well the relationship between two variables can be described using a monotonic function. The positive Spearman's correlation index values we obtained indicate that the number of occurrences for every analysed IBE within each ISWI-bound DNA sequence increases as a function of the ‘peak score’, strongly indicating that the identified motifs are likely associated with ISWI binding. (B–F) The top five motifs identified with multiple EM for motif elicitation (MEME) along with their putative D. melanogaster DNA-binding factors are shown. These logos are a graphical representation of DNA multiple sequence alignment. Each logo consists of a series of stacks of the four DNA deoxynucleotide (A, T, G, C), one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position.

Figure 3

Multi-layer model (MLM) nucleosome classification. (A) ‘Well-positioned’ nucleosomes (in green) are shown as peaks of a bell-shaped curve, ‘delocalized’ nucleosomes (in blue) represent single nucleosomes or arrays of nucleosomes with high mobility, while ‘fused’ nucleosomes (in red) reflect a single nucleosome that occupies two distinct close positions. On the left of the arrows are shown examples of nucleosome configuration that may contribute to the log₂(Nuc-DNA/GenDNA) ratio signal shape. The MLM detected all three classes of nucleosomes with similar relative abundances between wild type (B) and ISWI mutant (C) chromatin. The MLM identifies with high reliability well-positioned and delocalized nucleosomes around the well-characterized nucleosome promoter architecture of the (D) hsp26 and (E) hsp27 genes (Thomas and Elgin, 1988; Quivy and Becker, 1996). Delocalized nucleosomes are depicted as blue lines, while well-positioned nucleosomes as green lines.

Figure 4

Differences in nucleosome positioning around the TSS and 3′-end between wild-type and ISWI male chromatin. The average log₂(NucDNA/GenDNA) raw ratio of all the signals coming from wild-type (green) and ISWI mutant (red) male salivary gland chromatin of all genes present in the nucleosome-tiled array is plot relative to the (A) TSS distance or the (B) 3′-end distance. Nucleosome positions were calculated with the MLM for (C) wild-type and (E) ISWI mutant chromatin near the TSS, and for (D) wild-type and (F) ISWI mutant chromatin around the 3′-end. The MLM analysis shows well-positioned nucleosomes as shadings of yellow, linker regions in red, while delocalized nucleosomes in orange. The green and red ovals represent an interpretation of the MLM nucleosome positioning analysis in wild-type and ISWI mutant chromatin, respectively. Filled ovals indicate well-positioned nucleosomes, while shaded ovals show delocalized nucleosomes.

Figure 5

Loss of ISWI causes nucleosome spacing defects after the TSS on ISWI-bound genes. The average log₂(NucDNA/GenDNA) raw ratio of the signals coming from wild-type (green) and ISWI mutant (red) male salivary gland chromatin of all ISWI-bound genes present in the nucleosome positioning array is plot relative to the (A) TSS distance or the (B) 3′-end distance. The nucleosome-free region (NFR) mapping the TSS is indicated by the black arrow. Nucleosome positions were calculated with the MLM for (C) wild-type and (E) ISWI mutant chromatin relative to the TSS, and for (D) wild-type and (F) ISWI mutant chromatin around the 3′-end. The MLM analysis shows well-positioned nucleosomes as shadings of yellow, linker regions in red, while delocalized nucleosomes in orange. The green and red ovals represent an interpretation of the MLM nucleosome positioning analysis in wild-type and ISWI mutant chromatin, respectively. Filled ovals indicate well-positioned nucleosomes, while shaded ovals show delocalized nucleosomes. The double-arrowed red bar indicates the region after the TSS where we observe higher nucleosome mobility in the absence of ISWI.

Figure 6

Analysis of nucleosome positioning at specific gene loci bound by ISWI. The normalized log₂(NucDNA/GenDNA) ratio (blue signal) was used to calculate the MLM nucleosome positions in wild-type (green segments) and ISWI mutant (red segments) chromatin in ISWI-bound genes (A) at the level of the promoter–exon region, and on genes (B) negatively regulated or (C) positively regulated by ISWI (for a complete list, see Supplementary Table S1B and C) (Corona et al, 2007). Exons are represented by filled-in boxes, introns by a continuous line, while the 5′ and 3′-UTRs by thick lines. The transcription direction for each gene can be deduced by the repeated small blue arrows present in the intron segments. The grey bars highlight ISWI-bound region. The thick black bars represent regions of chromatin with nucleosome positioning differences between wild-type and ISWI mutant chromatin. The black arrows point at the nucleosome-free regions (NFR) present at the level of the TSS. (D) Changes in transcription levels, between wild-type and ISWI mutants, are measured by semi-quantitative RT–PCR on some representative examples of ISWI-bound (+) and not-bound (−) genes, where nucleosomes position differences (+) or no-differences (−) are detected. The data were normalized for the house-keeping gene Act5C that is not bound by ISWI and where no nucleosome differences were observed. While genes bound by ISWI with no nucleosome position changes (i.e. CG1179) do not show changes in transcription, genes bound by ISWI (i.e. CG8732 & CG1765; the same analysed in panels (B, C)) showing nucleosome position changes also show a change in the level of their transcripts when comparing wild type with ISWI mutants.

Figure 7

ISWI binds dosage compensated genes to make their TSS nucleosome free. The density of nucleosome positioning changes occurring between wild-type and ISWI mutant chromatin was calculated using the (A) normalized raw log₂(NucDNA/GenDNA) ratio signal or (B) the MLM, and was plot against the distance from the transcription start site (TSS) of ISWI-bound genes. The red arrow indicates where the nucleosome difference density peaks relative to the TSS. (C) Nucleosome position differences between wild-type and ISWI mutant chromatin for all ISWI-bound genes were calculated for each chromosome arm and plot against their expected values, based on the relative representation of the different chromosome sequences in the nucleosome-tiled array. (D) The number of ISWI-bound genes for each chromosome arm was calculated and plot against their expected values, based on the known number of genes mapping each chromosome. The P-values calculated comparing the expected with the observed values are shown on top of each histogram pair. Nucleosome positions were calculated with the MLM for (E) wild-type and (G) ISWI mutant chromatin relative to the TSS of ISWI- and MSL-bound genes, and for (F) wild-type and (H) ISWI mutant chromatin relative to the TSS of genes not bound neither by ISWI nor MSL. The MLM analysis shows well-positioned nucleosomes as shadings of yellow, linker regions in red, while delocalized nucleosomes in orange. The green and red ovals represent an interpretation of the MLM nucleosome positioning analysis in wild-type and ISWI mutant chromatin, respectively. Filled ovals indicate well-positioned nucleosomes, while shaded ovals show delocalized nucleosomes. The black arrow indicates the nucleosome-free region at the TSS of wild-type ISWI- and MSL-bound genes. The red double-arrowed bar indicates the region at the TSS where we observe nucleosome occupancy in the absence of ISWI in ISWI- and MSL-bound genes.