A Poised Chromatin Platform for TGF-β Access to Master Regulators

Abstract

Specific chromatin marks keep master regulators of differentiation silent, yet poised for activation by extracellular signals. We report that nodal TGF-β signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. TRIM33-Smad2/3 binds the histone marks H3K9me3 and K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. Binding is through the PHD-Bromo cassette of TRIM33. In the crystal structure of this cassette bound to histone H3 peptides, PHD recognizes K9me3 and Bromo an adjacent K18ac. Binding of TRIM33-Smad2/3 to H3K9me3 displaces the chromatin compacting factor HP1γ and makes nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal cues use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.

Transcriptional Control by the SMADs

“…It is likely that this sequence of events is repeated numerous times during the process of transcriptional activation by the SMADs, with the likelihood that the histone acetylation is also reversed by HDACs. In another study, a different model of TIF1g action was proposed, which does not involve its E3 ubiquitin ligase activity at all (Xi et al 2011). In that study in mouse ESCs, TIF1g was shown to interact with phosphorylated SMAD2/3 in the absence of SMAD4 and to be recruited to upstream regions of the mesendoderm genes, Gsc and Mixl1, by virtue of an interaction between the PHD finger-bromodomain with H3 tails that are unmethylated at Lys4, acetylated at Lys18, and trimethylated at Lys9.…”

The bromodomain interaction module

“…Subfamily V of the human BRDs contains the transcriptional repressor tripartite motif-containing 66 (TRIM66) [71], the tripartite motif-containing 33 (TRIM33) [72], the transcriptional regulator transcriptional intermediary factor 1 (TIF1a) [73], the transcriptional regulators nuclear auto-antigen Sp-100 (SP100) [74], nuclear autoantigen Sp-110 (SP110) [75] and SP140 nuclear body protein (SP140) [76] as well as the SP140-like protein (LOC93349), the transcriptional repressor bromodomain adjacent to zinc finger domain 2A (BAZ2A) [77] and the bromodomain adjacent to zinc finger domain 2B (BAZ2B) [78]. The main characteristic of this sub-class of bromodomains is the existence of a PHD/BRD tandem module which seems to be a necessary structural motif for peptide recognition as well as protein stability [73,79]. The BRD of TIF1a binds acetylated histone H3 peptides with weak micromolar affinity which is enhanced when longer peptides and the tandem PHD/BRD module are used, suggesting a cooperativity between modules even though peptides may not be methylated in order to promote binding to the PHD finger [73].…”

“…The arrangement of the two modules suggests a non-cooperative mode of peptide binding between them. (D) Binding of a tri-methylated and acetylated peptide (H3K9 me3 K14 ac ) to the tandem PHD/BRD modules of TRIM33 [79]. The two modules cooperatively bind the peptide demonstrating an avidity effect.…”

“…The deep and relatively hydrophobic cavity where K ac epitopes dock offers a drugable pocket for the development of small molecule inhibitors of the BRD interaction with acetylated proteins [94]. Additionally, the diversity of the loop regions surrounding the central cavity results in large variations in surface [79] electrostatic properties suggesting that specific targeting of BRDs is possible [33]. Early BRD inhibitors with low micromollar affinities have been shown to disrupt the interaction of PCAF to the HIV-1 TAT with selectivity over the BRDs of CREBBP and TIF1b [95] or the interaction between CREBBP and p53 [96].…”

Activin/Nodal signalling before implantation: setting the stage for embryo patterning

“…Gsc, which encodes the transcription factor and mesendoderm regulator Goosecoid, is a poised gene whose expression is induced once differentiation is underway [91]. Work in the Massagué laboratory showed that the stimulation of ESCs with Activin leads to the formation of companion Smad4 -Smad2/3 and TRIM33-Smad2/3 complexes, which are both required to activate Gsc expression.…”