Fig 2: Hdac6 KD does not induce Tip60-p400 acetylation or ubiquitination.Tip60-p400 complex purified from control or Hdac6 KD Tip60-H3F ESCs or untagged ESCs Western blotted with antibodies recognizing acetylated lysine (left) or ubiquitin (right). Note that the background band in the ubiquitin western blot is also found in the untagged control, and is therefore non-specific.DOI: http://dx.doi.org/10.7554/eLife.01557.025

Fig 3: Overlapping effects of Hdac6 KD and Tip60 KD on gene expression in ESCs.(A) Scatter plot of gene expression (Log2 (fold change) relative to control KD ESCs) upon Tip60 KD relative to Hdac6 KD. Genes misregulated upon Tip60 KD (adjusted p<0.1) are shown in red. (B) Western blot showing the levels of p400, Dmap1, Hdac6 and Tip60 (FLAG) upon Tip60 or Hdac6 KD. β-actin is shown as a loading control. (C) Unsupervised hierarchical clustering of genes misregulated (adjusted p<0.1) upon Tip60 KD. Up-regulated genes are indicated in yellow and downregulated genes are indicated in blue. The first cluster (#1) includes 200 genes that were upregulated upon Tip60 KD but not Hdac6 KD, the second cluster (#2) includes 867 genes upregulated in both Tip60 KD and Hdac6 KD ESCs, the third cluster (#3) includes 277 genes that were downregulated only in Tip60 KD cells and the forth cluster (#4) includes 424 genes that were downregulated in both Tip60 KD and Hdac6 KD ESCs. (D) Hdac6-dependent target genes are biased toward genes repressed by Tip60. Genes repressed or activated by Tip60 were split based on their Hdac6-dependence, and each group was plotted as the Log2 (ratio) of genes observed in each group relative to the expected number of genes if Hdac6-dependence was randomly distributed. Asterisk indicates statistically significant enrichment (p<10–20). (E) Validation of microarray datasets. The expression levels of genes from each cluster were measured by RT-qPCR in the indicated KDs and expressed as Log2 (fold change) values relative to control (GFP) KD ESCs after normalization. Data shown are mean ± SD of three technical replicates from one representative experiment of two biological replicates performed.DOI: http://dx.doi.org/10.7554/eLife.01557.011

Fig 4: Hdac6 KD does not cause general disruption of Tip60-p400 complex.(Left) Silver stain of Tip60-p400 complex purified from control or Hdac6 KD Tip60-H3F ESCs or untagged ESCs, as indicated. (Right) Tip60-p400 complex purified from control or Hdac6 KD ESCs Western blotted for indicated proteins.DOI: http://dx.doi.org/10.7554/eLife.01557.021

Fig 5: Reduced levels of Tip60 binding to differentiation genes upon Hdac6 KD.(Left) Example genomic loci showing Tip60 binding as measured by ChIP-seq upon GFP KD or Hdac6 KD. The first two panels show that Tip60 binding to the promoter regions of highly expressed genes (Rps9, Rpl27a) is relatively unaffected by Hdac6 KD. The second two panels show that Tip60 binding to the promoter regions of differentiation genes (Nodal, Nefl) is strongly reduced downstream of the TSS upon Hdac6 KD. (Right) Anti-FLAG ChIP-qPCR data are shown for the indicated ESCs at the promoter-proximal regions of the target genes shown. Note that Speer2 is a negative control locus to which Tip60-p400 does not bind. Data shown are mean ± SD of three technical replicates from one representative experiment of two biological replicates performed.DOI: http://dx.doi.org/10.7554/eLife.01557.027