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. 2013 Feb 21;3(2):309-18.
doi: 10.1016/j.celrep.2013.01.008. Epub 2013 Feb 8.

Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes

Hyangyee Oh  1 , Matthew Slattery, Lijia Ma, Alex Crofts, Kevin P White, Richard S Mann, Kenneth D Irvine
Affiliations

Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes

Hyangyee Oh et al. Cell Rep. .

Abstract

The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkie's association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie's transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie.

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Figures

Fig. 1
Fig. 1. Localization of Yki and GAF on chromosomes in wing discs
A) Comparison of the percentage DNA near promoters (within 3 kb upstream of a transcription start site), intron, exon, and intergenic regions within the whole genome, and within the Yki-bound fraction. B) Comparison of the distribution of mRNA levels (blue) and proximal promoter (within 100 bp of transcription start) H3K4me3 modification (yellow) amongst between Yki target genes as and non-Yki targets. Units of expression for RNA-seq are Fragments per Kilobase of transcript per Million mapped reads (FPKM). H3K4me3 wing ChIP-seq data are from (Pérez-Lluch et al., 2011). C) Overlap between Yki and GAF binding sites, numbers indicate the numbers of peaks. Numbers in the overlap differ because one peak for one protein can overlap two peaks of the other. D) Plot of ChIP peaks at three loci regulated by Yki. Transcription units of targets are in purple, transcription units of neighboring genes are in black. Regions called as Yki peaks are identified by gray bars. Yki-responsive enhancers that have been identified at these loci (Oh and Irvine, 2011; Wu et al., 2008; Zhang et al., 2008) are indicated in orange at bottom, a previously identified Hth and Yki-binding region (Peng et al., 2009) is indicated in light blue. E) Motifs and significance scores for DNA-binding proteins at Yki-bound chromatin. De novo motif analysis identified variants on the GAF binding motif (GAGA) as the highest scoring. SMAD2 (Mad) and E2F binding motifs are enriched within all Yki binding regions, whereas TEAD1 (Sd) motifs are most enriched within Yki binding regions without a GAF binding site. F) Analysis of DNA within Yki-bound wing peaks using a degenerate Sd-binding motif consistent with the published literature (Halder and Carroll, 2001): [AT][AG][AG]AAT[GT][CT]. 2285 regions do not contain a Sd motif, and the remainder do. 360 regions contain clustered Sd motifs (2 motifs separated by 20 or fewer base pairs) and 3246 regions contain one or more isolated motifs. G) GSEA analysis of the correlation between relative expression changes and Yki binding. Genes are ordered according to their relative fold change between wild-type and wtsP2. Log2-transformed fold change values for expressed genes are plotted in the bottom graph (see Methods). The GSEA running enrichment score is represented in the top graph (green line). Genes that increase in wtsP2 are significantly enriched for Yki target genes (p <0.005; FDR q-value <0.005); genes that decrease in wtsP2 are not enriched for Yki targets. See also Fig. S1.
Fig. 2
Fig. 2. Yki associates with GAF and Mor
A) Silver-stained gel displaying proteins precipitated from S2 cell nuclear extracts by anti-Yki antibodies or preimmune-serum, as indicated. Red, green and blue asterisks indicate positions of Yki, GAF and Mor bands, as determined by immunoblotting or mass-spectrometry. B,C) Western blots on S2 cell nuclear extract (input), or proteins immunoprecipitated from S2 cell nuclear extract by anti-Yki antibodies or preimmune-serum, and detected by anti-Yki (top panels), anti-GAF (B, bottom panel), or anti-Mor (C, bottom panel). Nuclear Yki was barely detectible in the input lanes, but enriched by immunopurification, and is normally detected as a doublet. Three bands of GAF were detected by anti-GAF antibody. D,E) Western blots showing co-immunoprecipitation of Yki3SA:Flag or Yki-WW3SA:Flag with GAF:V5 (D) or Mor:V5 (E) from S2 cell nuclear extracts, GFP:Flag is a negative control. Upper panels (Input) show blots on nuclear lysates, lower panels (V5-IP) show blots (anti-Flag) on material precipitated by anti-V5 beads. F) Western blot showing results of GST pull-down assays. GST:GAF:V5, GST:Mor:V5, or GST were immobilized on glutathione-agarose beads and incubated with bacterially-expressed GFP:V5, Yki:V5, Yki-WW:V5, or Yki-N:V5. Protein complexes were washed, resolved by SDS-PAGE, and bound proteins - detected by anti-V5 or anti-GST antibodies, as indicated.
Fig. 3
Fig. 3. Influence of GAF, BRM, and Mediator on Yki target genes
A–J) Projections through 3–5 confocal sections of wing discs; panels marked by prime symbols show separated channels. Yellow asterisks identify regions with normal gene expression, white asterisks identify regions with expression of RNAi lines and altered target gene expression. A–B) en-Gal4 UAS-GFP ex-lacZ UAS-Dcr2, with (A) UAS-RNAi-Trl[vdrc106433] (B) UAS-RNAi-Dalao[TRiP.JF02116] showing expression of ex-lacZ (magenta), and with posterior cells marked by GFP (green). C–E) en-Gal4 bs-GFP; UAS-Dcr2 and with (C) UAS-RNAi-Trl[vdrc106433], (D) UAS-RNAi-Mor[vdrc110712], (E) UAS-RNAi-MED23[vdrc105247], showing expression of Diap1 (red) and bs-GFP (green), and with posterior cells marked by Dcr2 (blue). F) en-Gal4 UAS-GFP; UAS-RNAi-Trl[vdrc106433] UAS-Dcr2 brC12-lacZ, showing expression of brC12-lacZ (red) and Wg (blue), and with posterior cells marked by GFP (green). G) Wild type control showing expression of ex-lacZ (magenta). (H) Wild type control showing expression of Diap1(red). (I) Wild type control showing expression of bs-GFP (green). J) Wild type control showing expression of Wg (blue). K–M) Histograms showing results of luciferase assays (depicted as average firefly/renilla ratio from triplicate experiments, error bars indicate standard deviation) K) using br2.5-luciferase, L) ex-luciferase, M) DRE-luciferase, reporters in S2 cells transfected to express Yki, TkvQ235D (T), Mad (M) or Medea (Me) as indicated. dsRNAs for RNAi against the specific genes were also added as indicated. See also Figs S2,S3.
Fig. 4
Fig. 4. GAF, BRM, and Mediator are required for Yki activity
Projections through 3–5 confocal sections of wing discs; panels marked by prime symbols show separated channels. Yellow asterisks identify regions with normal gene expression, white asterisks identify regions with expression of RNAi lines and altered target gene expression. A–C) en-Gal4 bs-GFP; UAS-Yki:V5S250A UAS-Dcr2 and with (A) UAS-lacZ (control) (B) UAS-RNAi-Trl[vdrc106433], (C) UAS-RNAi-Mor[vdrc110712], showing expression of Diap1 (red) and bs-GFP (green), and with posterior cells marked by Yki (blue). D–G) en-Gal4 bs-GFP; UAS-RNAi- wts[vdrc9928] UAS-Dcr2 and with (D) UAS-lacZ (E) UAS-RNAi-Trl[vdrc106433], (F) UAS-RNAi-Mor[vdrc110712] or (G) UAS-RNAi-MED23[vdrc105247], showing expression of Diap1 (red) and bs-GFP (green), and with posterior cells marked by Dcr2 (blue). See also Fig. S4.
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