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

Chromatin organizer SATB1 and Wnt transducer β-catenin form a complex and regulate expression of GATA3 and TH2 cytokines in Wnt-dependent manner and orchestrate TH2 lineage commitment

The third dimension of gene regulation: organization of dynamic chromatin loopscape by SATB1

Compartmentalized distribution of functional components is a hallmark of the eukaryotic nucleus. Technological advances in recent years have provided unprecedented insights into the role of chromatin organization and interactions of various structural-functional components toward gene regulation. SATB1, the global chromatin organizer and transcription factor, has emerged as a key factor integrating higher-order chromatin architecture with gene regulation. Studies in recent years have unraveled the role of SATB1 in organization of chromatin 'loopscape' and its dynamic nature in response to physiological stimuli. SATB1 organizes the MHC class-I locus into distinct chromatin loops by tethering MARs to nuclear matrix at fixed distances. Silencing of SATB1 mimics the effects of IFNγ treatment on chromatin loop architecture of the MHC class-I locus and altered expression of genes within the locus. At genome-wide level, SATB1 seems to play a role in organization of the transcriptionally poised chromatin

Displacement of SATB1-Bound Histone Deacetylase 1 Corepressor by the Human Immunodeficiency Virus Type 1 Transactivator Induces Expression of Interleukin-2 and Its Receptor in T Cells

One hallmark of human immunodeficiency virus type 1 (HIV-1) infection is the dysregulation of cytokine gene expression in T cells. Transfection of T cells with human T-cell leukemia type 1 or 2 transactivator results in the induction of the T-cell-restricted cytokine interleukin-2 (IL-2) and its receptor (IL-2Rα). However, no T-cell-specific factor(s) has been directly linked with the regulation of IL-2 and IL-2Rα transcription by influencing the promoter activity. Thymocytes from SATB1 (special AT-rich sequence binding protein 1) knockout mice have been shown to ectopically express IL-2Rα, suggesting involvement of SATB1 in its negative regulation. Here we show that SATB1, a T-cell-specific global gene regulator, binds to the promoters of human IL-2 and IL-2Rα and recruits histone deacetylase 1 (HDAC1) in vivo. SATB1 also interacts with Tat in HIV-1-infected T cells. The functional interaction between HIV-1 Tat and SATB1 requires its PDZ-like domain, and the binding of the HDAC1 corepressor occurs through the same. Furthermore, Tat competitively displaces HDAC1 that is bound to SATB1, leading to increased acetylation of the promoters in vivo. Transduction with SATB1 interaction-deficient soluble Tat (Tat 40-72) and reporter assays using a transactivation-negative mutant (C22G) of Tat unequivocally demonstrated that the displacement of HDAC1 itself is sufficient for derepression of these promoters in vivo. These results suggest a novel mechanism by which HIV-1 Tat might overcome SATB1-mediated repression in T cells

pC6-2/caspase-6 system to purify glutathione-S-transferase-free recombinant fusion proteins expressed in Escherichia coli

Glutathione-S-transferase (GST) fusion protein expression vectors are often employed for the expression and purification of proteins in Escherichia coli. GST is then removed by site-specific proteolysis using thrombin. However, the presence of internal thrombin cleavage sites in expressed proteins can severely affect the purification of intact proteins. Cysteine-dependent aspartate-specific proteases (caspases) are efficient enzymes with defined substrate specificity. Unlike most of the proteases used for the removal of affinity tags, caspases do not leave any amino acids at the amino-terminus of cleaved proteins. We have engineered the caspase-6 site VEMD in a pGEX vector to give the pC6-2 vector. The caspase-6 can be easily removed after cleavage. Here, we describe the detailed protocol for purifying proteins using our pC6-2/caspase-6 expression and purification system. The cleavage by caspase-6 occurs in <30 min and the entire procedure can be completed in 2 d

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

Functional interaction between PML and SATB1 regulates chromatin-loop architecture and transcription of the MHC class I locus

International audienceThe function of the subnuclear structure the promyelocytic leukaemia (PML) body is unclear largely because of the functional heterogeneity of its constituents. Here, we provide the evidence for a direct link between PML, higher-order chromatin organization and gene regulation. We show that PML physically and functionally interacts with the matrix attachment region (MAR)-binding protein, special AT-rich sequence binding protein 1 (SATB1) to organize the major histocompatibility complex (MHC) class I locus into distinct higher-order chromatin-loop structures. Interferon gamma (IFNgamma) treatment and silencing of either SATB1 or PML dynamically alter chromatin architecture, thus affecting the expression profile of a subset of MHC class I genes. Our studies identify PML and SATB1 as a regulatory complex that governs transcription by orchestrating dynamic chromatin-loop architecture

Phosphorylation of SATB1, a global gene regulator, acts as a molecular switch regulating its transcriptional activity In vivo

SATB1 regulates gene expression by acting as a "docking site" for several chromatin remodeling enzymes and also by recruiting corepressors (HDACs) or coactivators (HATs) directly to promoters. However, how these contrasting effectors act at the level of SATB1 is not clear. We show here that phosphorylation by PKC acts as a switch to determine whether SATB1 interacts with HDAC1 or PCAF. Phosphorylation and dephosphorylation of SATB1 exerted opposing effects on MAR-linked reporter activity in vivo. SATB1 interacted with both CBP/p300 and PCAF HATs; however, these interactions resulted in the acetylation of the PDZ-like domain of SATB1 by PCAF but not by CBP/p300 and resulted in loss of its DNA binding activity. Using the T cell activation model, we provide mechanistic insights into how IL-2 transcription is reciprocally governed by the phosphorylation status of SATB1 and propose that a similar mechanism may dictate the ability of SATB1 to function as a global regulator