(a) An initial group of sequence-dependent targets recruit the complex and lead to upregulation of transcription via local acetylation, which is sufficient for marginal survival. A second class of genes is targeted by spreading mediated by the MSL3 chromodomain, partly via the recognition of H3K36Me mark at 3’ ends of active genes. (b) In the chromodomain mutants, targeting by spreading is defective. The complex is not stabilized at 3’ ends of targets and diffuses away.

(a) The number of genes within bound clusters on the X chromosome was compared among experiments in a Venn diagram. 98% (418/432) of ΔCD bound genes and 96% (327/340) of SYD62A bound genes are also WT sites; 92% (313/340) of SYD62A bound genes belong to the ΔCD set. (b) Distributions of WT log-ratios for genes from different categories. Among all X-linked genes (black), those identified as bound in WT (magenta) have high log-ratios. Importantly, most of the genes bound in ΔCD (green) or SYD62A (blue) mutants have the highest signals in WT. The color scheme corresponds to that in Fig. 2a; for this plot, we defined the signal for a gene as the maximum log-ratio over the gene. (c) Scatter-plot of ChIP-chip signals between WT and ΔCD at probe level. If the probe is equally bound in both WT and ΔCD experiments, the representative point should fall near the diagonal. The presence of two separate clusters of red probes on the X suggests that there are two types of bound sites in WT: chromodomain-independent (cluster 1) and chromodomain-dependent sites (cluster 2).

Chromosomes from roX1 roX2 deficient 3^rd instar larvae with the GMroX2-26D8 transgene on chromosome 2L were stained for either the TAP epitope (red) alone or for both anti-MSL2 (red) and anti-MSL3 (green) whose overlap is shown by yellow. Anti-MSL3 staining shows both TAP-tagged and untagged MSL3 proteins. The transgene insertion is indicated by a star and the chromosomes are aligned by the location of the transgene for comparison. (a) When a transgene harboring roX2 is put on an autosome in the absence of both roX genes on the X, the MSL complex spreads extensively around the transgene insertion. (b) In the presence of the WT transgene (red), 100% of nuclei show extensive spreading. Three representative chromosomes are shown. (c) ΔCD (red) shows mosaic spreading. Three representative nuclei are shown. One shows spreading, albeit limited, whereas the other two show binding to the transgene but no spreading. Endogenous complexes (yellow) are also affected. (d) W59G (red) also shows mosaic spreading, but a majority of the nuclei show limited spreading. (e) SYD62A generally fails to spread and interferes with endogenous spreading.

A range of concentrations of recombinant MSL3 proteins was tested for binding to nucleosomes in gel shift assays. (a) Binding to recombinant nucleosomes methylated at H3K36. SYD62A and W59G point mutants can still bind unmodified nucleosomes albeit to a lesser extent than the WT (compare lanes 4, 7 and 10). WT has higher affinity for modified nucleosomes (compare lanes 4 and 14). The point mutants show no difference in binding to modified vs. unmodified nucleosomes (compare lanes 7 and 17 for SD62A; 10 and 20 for W59G). (b) WT MSL3 can bind native nucleosomes efficiently in gel shift assays. W59G has decreased binding and ΔCD has no detectable binding. (c) Chromatin entry sites are preferentially found in H3K36me3-enriched regions. Sequences matching the consensus for MSL recognition elements (MREs) were ordered by their enrichment for H3K36me3 and grouped into 10 bins (x-axis). The likelihood of an MRE being in a functional chromatin entry site (CES) (y-axis) increases as the level of H3K36me3 enrichment increases. H3K36me3 enrichment was computed by averaging the signal of the 3 probes nearest to each MRE.

(a) Schematic of the structure of the MSL3TAP transgenic protein, with mutations indicated below. To force expression of only the short isoform, the start codon for MSL3 was changed to a serine (M1S). (b) Complementation analysis. Rescue frequencies were calculated by comparing the number of msl3¹ homozygous mutant males expressing one copy of the transgene to their brothers with the TM3, Sb, msl3⁺ balancer. Number of progeny scored for each genotype is noted on the right. Chromodomain mutant males also exhibited a 2-day developmental delay with respect to their msl3⁺ brothers. (c) Protein expression was assayed on Western blots, using anti-PAP antibody against the TAP epitope. All but the LYT30A construct were stably expressed. (d) Chromodomain mutants assemble into MSL complexes. S2 cells expressing msl3tap constructs were used to immunoprecipitate MSL3TAP. Immunoprecipitations (IP) were then analyzed by Western blot for MSL1 and MOF. Even lanes: Input sample; odd lanes: IP sample. The lower band in the MSL1 IP may be a degradation product. (e) Chromodomain mutants are indistinguishable from WT in their binding patterns on the male X chromosome at the resolution of polytene staining (red: MSL3TAP). Stainings were done in the absence of endogenous msl3. From top to bottom: WT; SYD62A; ΔCD; W59G.

(a) 200kb of sample binding data from WT and mutants. The chromodomain mutant proteins bind in specific peaks, but fail to cover all WT sites. The H3K36 methylation profile from male tissue culture cells (S2) over the same region is shown for comparison. The H3K36me3 profile is very similar to the profile for WT MSL binding, extending beyond the chromatin entry sites. Boxes below the profiles represent annotated genes; red: transcribed, black: non-transcribed. (b) GA-rich motif identified by MEME from the SYD62A binding peaks. (c) The distribution of distances to the nearest chromatin entry site (CES) for the probes bound by WT (magenta), the ΔCD mutant (green) and the SYD62A mutant (navy), or the average of 10 random circular permutations of WT (light blue). The number of probes bound by the mutants (y axis) falls more sharply than WT as one moves farther from CES (X axis). (d) Alternative representation of the data in (c). The y-axis represents the % of bound probes (i.e. density) rather than absolute count. The binding of chromodomain mutants is clustered at or near the chromatin entry sites as shown by increased density of binding. (e) Representation of ChIP-chip data of MSL3TAP binding on polytene chromosomes shows that patterns are not distinguishable at 30kb resolution. Each black bar represents a cluster bound by MSL3TAP after merging neighboring clusters with gaps smaller than 30kb.