Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells

The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.


Bra -/-
Each antibody incubation was done at 4°C for 30 minutes in blocking buffer (made in Cytoperm buffer) and was followed by 3 washes in Cytoperm buffer. Lastly, cells were re-suspended in PBS supplemented with 1% BSA. The appropriate isotype controls, primary antibody omission controls and cells-only controls, as well as single positive samples for each channel were included as controls.

Immunofluorescence
Cells were fixed for 15 minutes at room temperature in 4% paraformaldehyde (PFA) (Sigma) and then washed three times with PBS. Cells were blocked and permeabilised with PBS containing 10% donkey serum (Serotec) and 0.1% Triton-X (Sigma) for 30 minutes at room temperature. Subsequently, cells were incubated overnight at 4°C with primary antibodies diluted 1/100 in PBS with 1% donkey serum [goat anti-BRACHYURY (R&D Systems, AF2085) and rabbit anti-EOMES (abcam, ab23345)]. Cells were then washed three times with PBS and incubated with the appropriate secondary antibody (Alexa Fluor, Invitrogen) diluted 1/300 in PBS with 1% donkey for 1-2 hours at room temperature. The cells were washed three times with PBS. Nuclear stain was provided by adding DAPI in the first wash at a 1/10000 dilution.

Endogenous Protein Co-Immunoprecipitation
All buffers contained 20 mM Hepes, 0.2 mM PMSF, 0.5 mM EDTA, Complete protease inhibitor mix (Roche), 0.2 mM DTT and varying concentrations of Glycerol (G), KCl (K) or Sucrose (S), where K20 stands for 20 mM KCl, N50 for 50 mM NaCl, S250 for 250 mM Sucrose, etc. Cells were harvested in Cell Dissociation Buffer (Gibco) and lysed in ice-cold K20G5 using a Dounce homogenizer with 20 strokes. Nuclei were washed in isotonic K20S250, re-suspended in two volumes of K10G20 and extracted by addition of 400mM NaCl, douncing and subsequent rotation for 1h at 4°C. Chromatin and debris were separated from the nuclear extract (supernatant) by ultracentrifugation at 12000g and then dialyzed against K100N50 to establish near physiological salt conditions. Co-immunoprecipitation was carried out with antibodies indicated in the figure legends [R&D systems: SMAD1 (AF2039), SMAD2/3 (AF3797), control IgG (AB-108-C)] followed by ProteinG beads (Roche) and final washes in K100G10+0.02%Tween20. Samples were subsequently eluted in LDS sample buffer (Invitrogen) and run on a SDS polyacrylamide gel in preparation for analysis by immunoblotting. Volumes loaded: 5%-10% (Input), 50% (Immunoprecipitate).

Total RNA extraction
Cells were washed briefly with PBS before adding cell lysis buffer (RLT buffer, QIAGEN). Total RNA was then extracted using the RNeasy Mini kit (QIAGEN) following the manufacturer's instructions. During RNA purification, spin columns were incubated with RNase-free DNase (QIAGEN, 79254) to remove residual DNA. After washing, the purified RNA was eluted in 30 µl of DEPC-treated water (Ambion). Samples were assessed for RNA concentration and quality using a NanoDrop spectrometer. Purified RNA was stored at -80ºC for further analyses.

Sample preparation for Illumina BeadArrays
Total RNA was extracted from cultured cells using the RNeasy Mini Kit (QIAGEN) as described above. RNA was then amplified, biotin labelled, and hybridized to Human WG-6 expression BeadArrays (Illumina) using a BeadScanner/BeadStation (Illumina) according to the manufacturer's standard protocols (Illumina) at the Microarray Resources Centre, Department of Pathology, University of Cambridge. Three biological replicates were hybridized for each experimental condition.

qRT-PCR
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) mixtures were prepared using Fast SYBR Green Master Mix (Applied Biosystems, 4385614), according to the manufacturer's instructions. PCR reactions were performed in a 7500 Fast Real-Time PCR System (Applied Biosystems) following the manufacturer's instructions. Three µl of diluted cDNA (1/200) from each sample were analysed in duplicate wells for specific gene expression. Expression values were then normalized by the expression of the "house-keeping" gene Porphobilinogen Deaminase (PBGD) in each sample, which was analysed in the same PCR plate. Relative gene expression was assessed using the formula 2 -ΔCt , where the Ct value corresponds to the cycle (within the exponential phase of the PCR reaction) at which the fluorescence crosses the optimal threshold established by Applied Biosystems software before DNA concentration begins to plateau. The ΔCt is the difference between the Ct of the gene of interest and the Ct of PBGD. Error bars on all qPCR graphs represent standard deviation from three independent biological replicates. Student's t-tests (two-tailed assuming non-equal variance) were performed. Primer sequences can be found in Table S6.

Preparation of Illumina ChIP-seq libraries
Preparation of ChIP-seq libraries was carried out using the ChIP-seq DNA sample kit (Illumina, IP-102-1001) according to manufacturer's instructions. Minor modifications included: 1) using 5 ng of DNA as starting material [DNA concentration was measured using a Qubit fluorometer (Invitrogen) according to the manufacturer's instructions], 2) diluting the Illumina adaptor mix 40x before use, as recommended by Schmidt et al. (Schmidt et al., 2009), 3) using the DNA Clean & Concentrator-5 kit (Zima Research) throughout the protocol, 4) performing the final size selection of DNA fragments (200bp-250bp) using the E-gel SizeSelect (2% agarose) system (Invitrogen).

Sequencing of Illumina ChIP-seq libraries
Processing of the samples was carried out at the MRC National Institute for Medical Research in London. The quality assessment of the libraries was carried out using the Agilent 2100 Bioanalyser to verify 1) the absence of primer dimers and 2) that the selected DNA fragments had a single-peak distribution with the desired size (around 200 bp). The libraries were finally sequenced in a Genome Analyzer (Illumina), according to manufacturer's instructions.

ChIP-qPCR
One µl of eluted DNA from each ChIP sample was analysed in duplicate and normalized to the Input. Target enrichment was assessed using the formula (1+Primer Efficiency) -ΔCt , as previously described (Aparicio et al., 2004;Mukhopadhyay et al., 2008). The ΔCt is the difference between the Ct of the ChIP sample and the Ct of the Input sample. Primer sequences can be found in Table S6.

Illumina BeadArray analysis -Data processing
Data provided in raw bead-level format was imported into the R statistical programming environment (http://www.r-project.org/). The background was corrected with the RMA algorithm (Irizarry et al., 2003) and summarized via the BeadArray package of the Bioconductor (http://www.bioconductor.org) suite of open-source bioinformatics software. Once imported and processed as described, profiles were log 2 transformed and quantile normalised using the Bioconductor:limma package.

Illumina BeadArray analysis -Differential expression
For each microarray probe-set, significant difference in expression between groups of sample profiles, including those from Bernardo et al. (2011) (FLyA-or FLyB-treated hESCs, 36 hours), was assessed using the moderated t-statistic of (Wettenhall and Smyth, 2004), as implemented in Bioconductor:limma. To correct for multiple hypothesis testing on such a scale, significance p-values obtained for all probe-sets were corrected using the false discovery rate (FDR) method of Storey and Tibshirani (2003) and differential expression deemed significant at a false discovery rate (FDR) of 5% (q < 0.05). Shared EntrezGene IDs were used to quantify intersection between differentially regulated genes and BRACHYURY-bound regions (see ChIP-seq methods) and to create corresponding Venn diagrams.

Illumina BeadArray analysis -Data visualization
Heatmaps of comparative gene expression were created via application of the heatmap.2 method from Bioconductor:gplots to gene expression profiles as described above. For each probe-set, log 2 expression intensities were scaled to have zero mean and unit standard deviation across all samples present. In the case wherein a gene was represented by more than one microarray probe-set, a single probe-set was selected for display according to highest mean expression across all samples regardless of sample group (highest average signal).   Table S1: ChIP-seq data analysis of BRA-bound regions (peak location and gene annotation) Click here to Download Table S1  Table S6