A network of transcription factors governs the dynamics of NODAL/Activin transcriptional responses.

SMAD2, an effector of the NODAL/Activin signalling pathway, regulates developmental processes by sensing distinct chromatin states and interacting with different transcriptional partners. However, the network of factors that controls SMAD2 chromatin binding and shapes its transcriptional programme over time is poorly characterised. Here, we combine ATAC-seq with computational footprinting to identify temporal changes in chromatin accessibility and transcription factor activity upon NODAL/Activin signalling. We show that SMAD2 binding induces chromatin opening genome wide. We discover footprints for FOXI3, FOXO3 and ZIC3 at the SMAD2-bound enhancers of the early response genes, Pmepa1 and Wnt3, respectively, and demonstrate their functionality. Finally, we determine a mechanism by which NODAL/Activin signalling induces delayed gene expression, by uncovering a self-enabling transcriptional cascade whereby activated SMADs, together with ZIC3, induce the expression of Wnt3. The resultant activated WNT pathway then acts together with the NODAL/Activin pathway to regulate expression of delayed target genes in prolonged NODAL/Activin signalling conditions. This article has an associated First Person interview with the first author of the paper.

A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress.

Repetitive sequences derived from transposons make up a large fraction of eukaryotic genomes and must be silenced to protect genome integrity. Repetitive elements are often found in heterochromatin; however, the roles and interactions of heterochromatin proteins in repeat regulation are poorly understood. Here we show that a diverse set of C. elegans heterochromatin proteins act together with the piRNA and nuclear RNAi pathways to silence repetitive elements and prevent genotoxic stress in the germ line. Mutants in genes encoding HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 also show functionally redundant sterility, increased germline apoptosis, DNA repair defects, and interactions with small RNA pathways. Remarkably, fertility of heterochromatin mutants could be partially restored by inhibiting cep-1/p53, endogenous meiotic double strand breaks, or the expression of MIRAGE1 DNA transposons. Functional redundancy among factors and pathways underlies the importance of safeguarding the genome through multiple means.

Distinct modes of SMAD2 chromatin binding and remodeling shape the transcriptional response to NODAL/Activin signaling.

NODAL/Activin signaling orchestrates key processes during embryonic development via SMAD2. How SMAD2 activates programs of gene expression that are modulated over time however, is not known. Here we delineate the sequence of events that occur from SMAD2 binding to transcriptional activation, and the mechanisms underlying them. NODAL/Activin signaling induces dramatic chromatin landscape changes, and a dynamic transcriptional network regulated by SMAD2, acting via multiple mechanisms. Crucially we have discovered two modes of SMAD2 binding. SMAD2 can bind pre-acetylated nucleosome-depleted sites. However, it also binds to unacetylated, closed chromatin, independently of pioneer factors, where it induces nucleosome displacement and histone acetylation. For a subset of genes, this requires SMARCA4. We find that long term modulation of the transcriptional responses requires continued NODAL/Activin signaling. Thus SMAD2 binding does not linearly equate with transcriptional kinetics, and our data suggest that SMAD2 recruits multiple co-factors during sustained signaling to shape the downstream transcriptional program.

TGF-ß signaling to chromatin: how Smads regulate transcription during self-renewal and differentiation.

Ligands of the TGF-ß superfamily (including the TGF-ßs, Nodal and BMPs) play instructive roles during embryonic development. This is achieved by regulation of genes important for both maintaining pluripotency and germ layer specification and differentiation. Here we review how the TGF-ß superfamily ligands signal to the chromatin to regulate transcription during development. The effectors of the pathway, the Smad transcription factors, are regulated in a combinatorial and spatiotemporal manner. This occurs via post-translational modifications affecting stability, localization and activity, as well as through interactions with other transcription factors and chromatin modifying enzymes, which occur on DNA. Expression profiling and Chromatin Immunoprecipitation have defined Smad target genes and binding sites on a genome-wide scale, which vary between cell types and differentiation stages. This has led to the insight that Smad-mediated transcriptional responses are influenced by the presence of master transcription factors, such as OCT4, SOX2 and NANOG in embryonic stem cells, interaction with other signal-induced factors, as well as by the general chromatin remodeling machinery. Interplay with transcriptional repressors and the polycomb group proteins also regulates the balance between expression of self-renewal and mesendoderm-specific genes in embryonic stem cells and during early development.

Recruitment of TIF1γ to chromatin via its PHD finger-bromodomain activates its ubiquitin ligase and transcriptional repressor activities.

The interplay between sequence-specific DNA-binding transcription factors, histone-modifying enzymes, and chromatin-remodeling enzymes underpins transcriptional regulation. Although it is known how single domains of chromatin "readers" bind specific histone modifications, how combinations of histone marks are recognized and decoded is poorly understood. Moreover, the role of histone binding in regulating the enzymatic activity of chromatin readers is not known. Here we focus on the TGF-ß superfamily transcriptional repressor TIF1γ/TRIM33/Ectodermin and demonstrate that its PHD finger-bromodomain constitutes a multivalent histone-binding module that specifically binds histone H3 tails unmethylated at K4 and R2 and acetylated at two key lysines. TIF1γ's ability to ubiquitinate its substrate Smad4 requires its PHD finger-bromodomain, as does its transcriptional repressor activity. Most importantly, TIF1γ's E3 ubiquitin ligase activity is induced by histone binding. We propose a model of TIF1γ activity in which it dictates the residence time of activated Smad complexes at promoters of TGF-ß superfamily target genes.