Hill lab Developmental Signalling Laboratory
: Regulation of transcription by activated Smad complexes
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The main function of the TGF-β superfamily/Smad pathways is to induce new programmes of gene expression, but apart from previous work in my lab showing that Smads activate transcription by remodelling the chromatin template, little is understood about how exactly they achieve this.
Our work in this area is focused on Nodal signalling, which is not only critical for embryonic development, and for maintaining pluripotency of human ES cells, but is exploited by cancer cells to promote tumour progression and metastasis.
We are using the embryonic carcinoma cell line, P19, which respond to Nodal and Activin both acutely and chronically as a model system. As well as being a model for cancer, these cells express a combination of pluripotency factors and mesendoderm markers in response to Activin/Nodal.
To characterise the transcriptional responses to Activin we performed RNA-seq on P19 in the non-signalling state, in cells treated with Activin for short (1 h) or extended (8 h) times, and in the untreated state (autocrine signalling). This has allowed us to define four main classes of response genes (transiently induced; induced sustained; delayed induced; repressed), which is enabling us to define different classes of enhancers that are co-regulated.
In the same conditions we have also performed ChIP-seq analysis for Smad2, for total histone H3, for two different histone modifications characteristic of active chromatin (H3K27ac and H3K9ac), and for two different forms of RNA Polymerase II (initiating state phosphorylated at Ser 5, and elongating state phosphorylated at Ser 2) (Figure 2).
Integration of the RNA-seq data with all of these ChIP-seq datasets has revealed the mechanism whereby activated Smad2-containing complexes find their targets in chromatin and activate transcription (Coda et al., 2017 Elife 6:e22474.
Footprinting analysis and motif enrichment is now being used to define what other cofactors bind with Smad2 at enhancers of Activin-responsive genes, distinguishing between those that bind with Smad2 1 h after Activin stimulation and those that are synthesised in response to Activin and bind with Smad2 at later time points. This is yielding a number of interesting candidates which are being experimentally verified.
Figure 1: ChIP-seq for Smad2 and RNA-polymerase over time in response to Activin. IGV browser display of the Lefty1/Lefty2 genomic locus, showing tracks and MACS-called peaks for Smad2 and tracks for RNA Polymerase II (Ser5P). The two major enhancers containing Smad2 binding sites (SBSs) are indicated.
