BackgroundTranscription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse.ResultsHere we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET.ConclusionsThese data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0897-0) contains supplementary material, which is available to authorized users.
Th17 cells are critically involved in host defense, inflammation, and autoimmunity1–5. TGF-β is instrumental in Th17 differentiation by cooperating with IL-66,7. Yet, the mechanism of how TGF-β enables Th17 differentiation remains elusive. Here we reveal that TGF-β licenses Th17 differentiation by releasing Ski-Smad4-complex suppressed RORγt expression. We found serendipitously that, unlike wild-type T cells, Smad4-deficient T cells differentiated into Th17 cells in the absence of TGF-β signaling in a RORγt-dependent manner. Ectopic Smad4 expression suppressed the RORγt expression and Th17 differentiation of Smad4-deficient T cells. Unexpectedly however, TGF-β neutralized Smad4 mediated suppression without affecting Smad4 binding to Rorc locus. Proteomic analysis revealed that Smad4 interacted with Ski, a transcriptional repressor degraded upon TGF-β stimulation. Ski controlled the histone acetylation/de-acetylation of Rorc locus and Th17 differentiation via Smad4 because ectopic Ski expression inhibited H3K9Ac of Rorc locus, Rorc expression and Th17 differentiation in a Smad4-dependent manner. Therefore, TGF-β-induced disruption of Ski releases Ski-Smad4 complex imposed suppression of RORγt to license Th17 differentiation. This study reveals a critical mechanism by which TGF-β controls Th17 differentiation and uncovers Ski-Smad4 axis as a potential therapeutic target for treating Th17 related diseases.
GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss-of function despite a dearth of information regarding their effect on disease course or their mechanistic impact on the breast cancer transcriptional network. Here, we address molecular and clinical features associated with GATA3 mutations. A novel classification scheme defines distinct clinical features for patients bearing breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2). An engineered ZnFn2 mutant cell line by CRISPR–Cas9 reveals that mutation of one allele of the GATA3 second zinc finger (ZnFn2) leads to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding correlates with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that ZnFn2 mutations impact breast cancer through gain and loss-of function.
GATA3 is a highly conserved, essential transcription factor expressed in a number of tissues, including the mammary gland. GATA3 expression is required for normal development of the mammary gland where it is estimated to be the most abundant transcription factor in luminal epithelial cells. In breast cancer, GATA3 expression is highly correlated with the luminal transcriptional program. Recent genomic analysis of human breast cancers has revealed high frequency mutation in GATA3 in luminal tumors, suggesting ‘driver’ function(s). Here we discuss mutation of GATA3 in breast cancer, and the potential mechanism(s) by which mutation may lead to a growth advantage in cancer.
Chromatin remodeling is of crucial importance during brain development. Pathogenic alterations of several chromatin remodeling ATPases have been implicated in neurodevelopmental disorders. We describe an index case with a de novo missense mutation in CHD3, identified during whole genome sequencing of a cohort of children with rare speech disorders. To gain a comprehensive view of features associated with disruption of this gene, we use a genotype-driven approach, collecting and characterizing 35 individuals with de novo CHD3 mutations and overlapping phenotypes. Most mutations cluster within the ATPase/helicase domain of the encoded protein. Modeling their impact on the three-dimensional structure demonstrates disturbance of critical binding and interaction motifs. Experimental assays with six of the identified mutations show that a subset directly affects ATPase activity, and all but one yield alterations in chromatin remodeling. We implicate de novo CHD3 mutations in a syndrome characterized by intellectual disability, macrocephaly, and impaired speech and language.
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