Global Regulator SATB1 Recruits β-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner

et al. (2010) Global Regulator SATB1 Recruits b-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner. PLoS Biol 8(1): e1000296. doi:10.1371/journal.pbio.1000296 Global Regulator SATB1 Recruits b-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner Dimple Notani 0 Kamalvishnu P. Gottimukkala 0 Ranveer S. Jayani 0 Amita S. Limaye 0 Madhujit V. Damle 0 Sameet Mehta 0 Prabhat Kumar Purbey 0 Jomon Joseph 0 Sanjeev Galande 0 Roel Nusse, Stanford University School of Medicine, Howard Hughes Medical Institute, United States of America 0 1 National Centre for Cell Science , Ganeshkhind, Pune , India , 2 Centre for Modelling and Simulation, University of Pune , Ganeshkhind, Pune , India In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of b-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) to activate target genes. Wnt/b -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1), the T lineage-enriched chromatin organizer and global regulator, interacts with b-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon b-catenin signalling. GATA-3 is a T helper type 2 (TH2) specific transcription factor that regulates production of TH2 cytokines and functions as TH2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4+ T cells, suggesting that SATB1 influences TH2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1), an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature TH2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for TH2 differentiation. Knockdown of b-catenin also produced similar results, confirming the role of Wnt/b-catenin signalling in TH2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits b-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating TH2 cells in a Wnt-dependent manner. SATB1 coordinates TH2 lineage commitment by reprogramming gene expression. The SATB1:b-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1 orchestrates TH2 lineage commitment by mediating Wnt/b-catenin signalling. This report identifies a new global transcription factor involved in b-catenin signalling that may play a major role in dictating the functional outcomes of this signalling pathway during development, differentiation, and tumorigenesis. - Funding: This work was supported by grant from the the Wellcome Trust, United Kingdom. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: ACF, ATP utilizing chromatin assembly and remodelling factor; Arm, Armadillo; BIO, 6-bromoindirubin-39-Oxime; CBP, CREB Binding Protein; CD, Cut Domain; ChIP, Chromatin Immunoprecipitation; CREB, cAMP responsive element binding protein; CT, threshold cycle; DAPI, 49,6-Diamidino-2-PhenylIndole; DKK1, Dickkopf 1; DL1, Delta-like Ligand 1; DMSO, Dimethyl Sulfoxide; DN, double negative; DP, double positive; EMSA, electrophoretic mobility shift assay; FCS, Foetal Calf Serum; FlT3, FMS-like Tyrosine Kinase 3; Fz, frizzled; GSK-3 b, Glycogen Synthase 3 beta; GST, Glutathione S Transferase; H3K9, Histone 3 Lysine 9; HAT, Histone Acetyl Transferase; HD, Homeo Domain; HDAC1, Histone Deacetylase 1; HEK, Human Embryonic Kidney; HMG, High Mobility Group; IFNc, Interferon gamma; IL-10, Interleukin 10; IL-12, Interleukin 12; IL-13, Interleukin 13; IL-4, Interleukin 4; IL-7, Interleukin 7; IP, Immunoprecipitation; LEF, Lymphoid Enhancer Binding Factor 1; LiCl, Lithium Chloride; MAR, Matrix Attachment Regions; MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); PBS, Phosphate Buffered Saline; PCAF, p300/CBP-associated factor; PDZ, Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and Zonula occludens-1 protein (zo-1); RFP, red fluorescent protein; RPMI, Roswell Park Memorial Institute; RT-PCR, Reverse Transcription-Polymerase Chain Reaction; SATB1, Special AT rich Binding protein 1; SBS, SATB1 Binding Sequences; SDS, sodium dodecyl sulfate; SP, single positive; STAT4, Signal Transducer and activator of Transcription 4; STAT6, Signal Transducer and activator of Transcription 6; TH, T Helper; TBP, TATA Binding Protein; TCF, T Cell Factor; TCR, T cell Receptor; WB, Western Blotting; Wnt, Wingless type Wnt growth factors regulate a variety of developmental processes by altering specific gene expression patterns [1]. Wnt proteins are secreted molecules that coordinate cell-to-cell interactions in many different cell types by binding to a member of the Frizzled (Fz) family of transmembrane receptors [2]. Binding of Wnt to Fz (Table S1) elicits a complex cascade of molecular events culminating in the inhibition of the negative regulatory kinase GSK-3b [3]. Phosphorylation of b-catenin by GSK-3b targets it for degradation via the bTrCP ubiquitin ligase-proteasome pathway [4]. Dephosphorylated b-catenin accumulates inside the nucleus [5] where it associates with the lymphoid enhancer factor/T cell factor (LEF/TCF) transcription factors to induce target gene transcription [6]. In vertebrates, bcatenin acts as a transcriptional activator, which is required to overcome the transcriptional repression by repressor complexes [7]. The C-terminus of b-catenin is indispensable for the transactivation function, presumably since it harbours binding sites for transcriptional coactivators such as p300/CBP and TBP [7,8]. Thus, recruitment of chromatin remodelling factors on TCFs genomic targets to modulate the gene transcription appears to be the principal function of stabilized b-catenin [8]. In vertebrates the canonical Wnt signalling culminates in b-catenin moving into the nucleus where it activates transcription of target genes. Wnt/b-catenin signalling is essential for the thymic maturation and differentiation of nave T cells. Here we show that SATB1, a T cell lineageenriched chromatin organizer and global regulator, binds to b-catenin and recruits it to SATB1s genomic binding sites so that genes formerly repressed by SATB1 are upregulated by Wnt signalling. Some of the genes known to be regulated by SATB1 (such as genes encoding cytokines and the transcription factor GATA3) are required for differentiation of Th2 cells, an im (...truncated)