Phosphorylation-dependent regulation of SATB1, the higher-order chromatin organizer and global gene regulator.

The chromatin organizer SATB1 regulates distant genes by selectively tethering matrix attachment regions (MARs) to the nuclear matrix. Post-translational modifications (PTMs) are important regulators of functional activities of proteins. Recently, a phosphorylation-dependent molecular switch that provided insights into the molecular mechanism of transcriptional repression and activation by SATB1 was discovered. SATB1 is specifically phosphorylated by protein kinase C (PKC) at serine 185 in vivo, and this modification leads to repression of transcription by SATB1 via increased association with the histone deacetylase 1 (HDAC1) corepressor. In this chapter, we describe methods for overexpression and purification of full length SATB1 protein and for its in vitro phosphorylation. We also describe method for in vivo phosphorylation of SATB1 upon immunoprecipitation using anti-SATB1. Finally, we describe a functional assay to monitor the effect of phosphorylation on transcription activity of SATB1 in vivo using MAR-linked reporter assay, in the presence and absence of PKC inhibitors.

Global regulator SATB1 recruits beta-catenin and regulates T(H)2 differentiation in Wnt-dependent manner.

In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) to activate target genes. Wnt/beta -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 beta-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 beta-catenin signalling. GATA-3 is a T helper type 2 (T(H)2) specific transcription factor that regulates production of T(H)2 cytokines and functions as T(H)2 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 T(H)2 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 T(H)2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H)2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H)2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H)2 cells in a Wnt-dependent manner. SATB1 coordinates T(H)2 lineage commitment by reprogramming gene expression. The SATB1:beta-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 T(H)2 lineage commitment by mediating Wnt/beta-catenin signalling. This report identifies a new global transcription factor involved in beta-catenin signalling that may play a major role in dictating the functional outcomes of this signalling pathway during development, differentiation, and tumorigenesis.

Acetylation-dependent interaction of SATB1 and CtBP1 mediates transcriptional repression by SATB1.

Special AT-rich binding protein 1 (SATB1) acts as a global regulator of gene expression by recruiting various corepressor or coactivator complexes, thereby establishing a unique chromatin structure at its genomic targets in a context-dependent manner. Although SATB1 acts predominantly as a repressor via recruitment of histone deacetylase 1 (HDAC1) complexes, the precise mechanism of global repression is not clear. Here we report that SATB1 and C-terminal binding protein 1 (CtBP1) form a repressor complex in vivo. The interaction occurs via the CtBP1 interaction consensus motif PVPLS within the PDZ-like domain of SATB1. The acetylation of SATB1 upon LiCl and ionomycin treatments disrupts its association with CtBP1, resulting in enhanced target gene expression. Chromatin immunoprecipitation analysis indicated that the occupancy of CtBP1 and HDAC1 is gradually decreased and the occupancy of PCAF is elevated at the SATB1 binding sites within the human interleukin-2 and mouse c-Myc promoters. Moreover, gene expression profiling studies using cells in which expression of SATB1 and CtBP1 was silenced indicated commonly targeted genes that may be coordinately repressed by the SATB1-CtBP1 complex. Collectively, these results provide a mechanistic insight into the role of SATB1-CtBP1 interaction in the repression and derepression of SATB1 target genes during Wnt signaling in T cells.