MOESM3 of The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

Additional File 3: Figure S3. Isolated nuclei were extracted with 0.1% TX-100 and increasing NaCl concentrations, and soluble (S) and insoluble (P) nuclear fractions analysed by western blotting using anti-Ty antibodies to visualize c-Myb and c-Myb D152V. Densitometric analysis of salt extractions are shown in upper row for c-Myb (middle row) and c-Myb D152V (bottom row)

MOESM2 of The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

Additional File 2: Figure S2. The same experiment as shown in Fig. 3, but here RNA was analysed for expression of lysozyme (LYZ, Gallus gallus (Chicken)) measured by qRT-PCR. The values of RNA expression were normalized to the relative amount of the reference gene hprt

MOESM1 of The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

Additional File 1: Figure S1. Peptide arrays containing 384 histone tail modification combinations incubated with GST-c-Myb-R3 (left) or GST-c-Myb-R3-D152V (right) and detected with anti-GST primary antibody

HP1c interacts with two Zn-finger containing proteins Woc and ROW.

<p>(A) Comassie staining of a flag affinity purification from nuclear extracts of SL2 cells (lane 1) or SL2 cells stably transfected with an expression vector for HP1c (lane 2). Major proteins are indicated in bold letters, proteins that were co-purified to a various degree in different preps are indicated in italics. Signals derived from the M2-antibody or proteins that are also present in the controlare indicated with an asterisk. (B) GST pull down assays using GST (lane 2), GST-HP1a (lane 3) or GST-HP1c (lane 4) as a bait and in vitro translated Su(var)3–9 (top panel) ACF1 (second panel) or ROW (bottom panel) as prey (2.5% of the input material is shown in lane 1). To ensure equal loading the SDS-PAA gel was stained with coomassie blue (bottom panel). (C) Specificity of the HP1c antibody. Western Blot on purified recombinant HP1 isoforms using the monoclonal HP1c antibody used in this study (top panel) and an anti-GST antibody (bottom panel). (D) Whole extract of wt or HP1c−/− mutant flies were subjected to SDS-PAGE and blotted using an anti HP1c (top panel) or an anti tubulin antibody. (E) Immunoprecipitation assays using nuclear extracts of early Drosophila embryos (0–12 h). Co-precipitated proteins were detected by Western Blotting. A mock immunoprecipitation using a non specific antibody was performed as a control (lane 2).</p

HP1c colocalizes with Woc on polytene chromosomes.

<p>(A) Salivary gland polytene chromosomes from wild type larvae stained with α-HP1c and α-woc (upper panel). Enlargement and generation of split images allows a detailed analysis of HP1c and woc localization (lower panel). (B) Salivary gland polytene chromosomes from woc-mutant larvae stained with α-HP1c and α-HP2 as a control. (C) Salivary gland polytene chromosomes from HP1c-mutant larvae stained with α-HP1c and α-woc. In the merged images, woc is depicted in green, HP1c in red. DNA was stained with DAPI (blue).</p

Woc and HP1c coordinate HP1c expression on a transcriptional level.

<p>(A) Western blot using whole cell extracts of either wild type or woc-mutant Drosophila 3^rd instar larvae. (B) RT-PCR analysis using total RNA isolated from 3^rd instar larvae. HP1c expression analysis was performed with wild type larvae and larvae from two fly strains carrying different heteroallelic combinations of Woc mutant alleles. Primers for the ribosomal protein RLP12 spanning an intron were used as a control. (C) Drosophila SL2 cells transfected with (right panel) or without (left panel) an expression construct for a FLAG-tagged version of HP1c driven by an actin promoter were subjected to woc RNAi. Protein levels were determined by Western Blotting with the indicated antibodies. The asterisks indicates the endogenous HP1c (D) Whole cell extracts from <i>Drosphila</i> SL2 cells transfected with either HP1aFLAG or HP1cFLAG were prepared. Endogenous HP1c levels were determined by immunoblotting with an HP1c specific antibody (lower panel). Expression of the FLAG-tagged HP1 isoforms was determined as a control. (E) RT-PCR analysis of total RNA using a primer pair specific for endogenous HP1c. RNA was isolated from Drosophila SL2 cells that were either non-transfected or transfected with the indicated expression plasmids. (F) Model of HP1c action to modulate its own transcription by counteracting Woc mediated transcriptional activation.</p

H2A.Z maps to Hox loci in wild-type and PRC1 mutant ESCs.

<p>Log2 of ChIP:input for: H2A.Z, H3K27me3, EZH2 and Ring1B from WT (+/+) (top) and <i>Ring1B^−/−</i> (bottom) ESCs using a custom tiling microarray. Data is shown for the four paralogous murine hox loci (Hoxa, Hoxb, Hoxc and Hoxd) and their flanking genomic regions. The data represent a mean of 2 biological replicates. RefSeq gene annotations and CGIs are from the July 2007 (mm9) Build 37 assembly of the mouse genome (genome.ucsc.edu).</p

Identification of Ring1B associated peptides in mouse ESCs.

<p>A) IPs of Ring1B from nuclear extracts of WT and <i>Ring1B^−/−</i> cells rescued with Ring1BI53A, resolved by 4–20% SDS-PAGE and stained with colloidal blue. Proteins that had a significant number of detected peptides by mass spectrometry analysis are listed. Peptides also detected in the control IgG lane were subtracted. Asterisks indicate the heavy and light Ig chains. B) IP of Ring1B from nuclear extracts of WT and <i>Ring1B^−/−</i> cells rescued with full length Ring1B or Ring1BI53A resolved by 12% SDS-PAGE and immunoblotted with α-Dmap1. C) Immunoblot of p400 IP from WT and <i>Ring1B^−/−</i> ESCs using p400 and Ring1B antibodies. D) IP of endogenous p400 from nuclear extracts of WT ESCs resolved by 4–20% SDS PAGE. Polypeptides stained by colloidal blue were identified by mass spectrometry. Listed are proteins with significant number of peptide hits. E) Size exclusion chromatography of nuclear extract from WT ESCs. Every other fraction (50%) were resolved by 4–15% SDS-PAGE and immunoblotted with anti-p400, anti-Dmap1, anti-Mll2, anti-Menin, anti-Ring1B, anti-Rybp, anti-Ezh2 and anti-Suz12. The upper panel represent fraction number, Input (In), black triangles indicate the retention of molecular weight standards in mega Dalton (MDa) or kilo Dalton (kDa).</p

H2A.Z enrichment is not dependent on PRC2.

<p>A) Immunoblots for H3K27me3 and H3 in <i>Eed^−/−</i> and matched WT ESCs B) Immunoblots for H2A.Z and H2A in <i>Eed^−/−</i> and matched WT ESCs. C) ChIP for control IgG or H2A.Z at the promoters of <i>Hoxb1, Hoxb13, Gata4, Pou5f1</i> and <i>Nanog,</i> assayed by qRT-PCR, in WT (grey) or <i>Eed^−/−</i> (black) ESCs. Enrichment is shown as mean % input bound ± s.e.m. over three biological replicates. IgG is shown in white bars. D) Immunoblots for H3K27me3 and H3 in <i>Suz12^−/−</i> and WT (<i>Eed^+/+)</i> ESCs E) Immunoblots for H2A.Z and H2A in <i>Suz12^−/−</i> and WT (<i>Eed^+/+)</i> ESCs. F) ChIP for control IgG or H2A.Z at the promoters of <i>Hoxb1, Hoxb13, Hoxd10, Gata4, Cdx2, Pou5f1</i> and <i>Nanog,</i> assayed by qRT-PCR, in WT (grey) or <i>Suz12^−/−</i> (black) ESCs. Enrichment is shown as mean % input bound ± s.e.m. over three biological replicates. IgG is shown in white bars.</p

H2A.Z localises to CpG islands and overlap with EZH2 and Ring1B.

<p>A) Venn diagram showing overlap of genes displaying H2A.Z with EZH2 and Ring1B enrichment at TSS ± 500 bp within arrayed regions (total 240 unique TSS) in WT ESCs. B) Box plots showing Log2 H2A.Z ChIP/Input of over TSSs (±250 bp), CpG islands (CGI), exons, introns and transcription end sites (TES ± 250 bp) across all probes or Hox loci in WT ESCs. C) As in (E) but for EZH2, H3K27me3 and Ring1B.</p