Down-regulation of the global regulator SATB1 by statins in COLO205 colon cancer cells

Special AT-rich sequence binding protein 1 (SATB1) regulates the expression of more than 1,000 genes in tumor cells. SATB1 expression has been implicated in metastasis, and its silencing results in reduced cancer progression and the reversion of metastatic cells to normal appearance. Therefore, any compound causing down-regulation of SATB1 expression or activity may be exploited for its therapeutic potential in terms of cancer regression. Earlier studies showed that the 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase inhibitors (statin drugs), which are widely used to treat hypercholesterolemia, possess other pleotropic activities. These are now increasingly gaining attention for their cancer prevention abilities. However, the downstream interplay of the molecular mechanisms of such anti-cancer activities is unclear. Here, we show that SATB1 is down-regulated by statins in a time- and dose-dependent manner in COLO205 cells. This effect was statin-specific as the down-regulation of SATB1 was brought about by hydrophobic statins, such as simvastatin and fluvastatin, but not by hydrophilic pravastatin. Notably, treatment with mevalonate, an intermediate in the cholesterol and isoprenoid biosynthetic pathways, led to the inhibition of SATB1 down-regulation and cytotoxicity mediated by statins. Treatment with the proteasome inhibitors lactacystine and MG-132 inhibited the statin-mediated down-regulation of SATB1, suggesting that regulation occurs at the post-translational level. Thus, our results demonstrate a novel molecular mechanism for the anti-cancer activity of statin drugs in colon cancer cells, without invoking significant cytotoxicity

N-terminal PDZ-like domain of chromatin organizer SATB1 contributes towards its function as transcription regulator

The special AT-rich DNA-binding protein 1 (SATB1) is a matrix attachment region (MAR)-binding protein that acts as a global repressor via recruitment of CtBP1:HDAC1-containing co-repressors to its binding targets. The N-terminal PSD95/Dlg-A/ZO-1 (PDZ)-like domain of SATB1 mediates interactions with several chromatin proteins. In the present study, we set out to address whether the PDZ-domain-mediated interactions of SATB1 are critical for its in vivo function as a global repressor. We reasoned that since the N-terminal PDZ-like domain (amino acid residues 1-204) lacks DNA binding activity, it would fail to recruit the interacting partners of SATB1 to its genomic binding sites and hence would not repress the SATB1-regulated genes. Indeed, in vivo MAR-linked luciferase reporter assay revealed that overexpression of the PDZ-like domain resulted in de-repression, indicating that the PDZ-like domain exerts a dominant negative effect on genes regulated by SATB1. Next, we developed a stable dominant negative model in human embryonic kidney (HEK) 293T cells that conditionally expressed the N-terminal 1-204 region harbouring the PDZ-like domain of SATB1. To monitor the effect of sequestration of the interaction partners on the global gene regulation by SATB1, transcripts from the induced and uninduced clones were subjected to gene expression profiling. Clustering of expression data revealed that 600 out of 19000 genes analysed were significantly upregulated upon overexpression of the PDZ-like domain. Induced genes were found to be involved in important signalling cascades and cellular functions. These studies clearly demonstrated the role of PDZ domain of SATB1 in global gene regulation presumably through its interaction with other cellular proteins

SATB1 dictates expression of multiple genes including IL-5 involved in human T helper cell differentiation

Special AT-rich binding protein 1 (SATB1) is a global chromatin organizer and a transcription factor regulated by interleukin-4 (IL-4) during the early T helper 2 (Th2) cell differentiation. Here we show that SATB1 controls multiple IL-4 target genes involved in human Th cell polarization or function. Among the genes regulated by SATB1 is that encoding the cytokine IL-5, which is predominantly produced by Th2 cells and plays a key role in the development of eosinophilia in asthma. We demonstrate that, during the early Th2 cell differentiation, IL-5 expression is repressed through direct binding of SATB1 to the IL-5 promoter. Furthermore, SATB1 knockdown-induced upregulation of IL-5 is partly counteracted by down-regulating GATA3 expression using RNAi in polarizing Th2 cells. Our results suggest that a competitive mechanism involving SATB1 and GATA3 regulates IL-5 transcription, and provide new mechanistic insights into the stringent regulation of IL-5 expression during human Th2 cell differentiation. (Blood. 2010;116(9):1443-1453

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

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

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