Global chromatin organizer SATB1 acts as a context-dependent regulator of the Wnt/Wg target genes.

Special AT-rich binding protein-1 (SATB1) integrates higher-order chromatin architecture with gene regulation, thereby regulating multiple signaling pathways. In mammalian cells SATB1 directly interacts with ß-catenin and regulates the expression of Wnt targets by binding to their promoters. Whether SATB1 regulates Wnt/wg signaling by recruitment of ß-catenin and/or its interactions with other components remains elusive. Since Wnt/Wg signaling is conserved from invertebrates to humans, we investigated SATB1 functions in regulation of Wnt/Wg signaling by using mammalian cell-lines and Drosophila. Here, we present evidence that in mammalian cells, SATB1 interacts with Dishevelled, an upstream component of the Wnt/Wg pathway. Conversely, ectopic expression of full-length human SATB1 but not that of its N- or C-terminal domains in the eye imaginal discs and salivary glands of third instar Drosophila larvae increased the expression of Wnt/Wg pathway antagonists and suppressed phenotypes associated with activated Wnt/Wg pathway. These data argue that ectopically-provided SATB1 presumably modulates Wnt/Wg signaling by acting as negative regulator in Drosophila. Interestingly, comparison of SATB1 with PDZ- and homeo-domain containing Drosophila protein Defective Proventriculus suggests that both proteins exhibit limited functional similarity in the regulation of Wnt/Wg signaling in Drosophila. Collectively, these findings indicate that regulation of Wnt/Wg pathway by SATB1 is context-dependent.

Structure function relations in PDZ-domain-containing proteins: Implications for protein networks in cellular signalling.

Protein scaffolds as essential backbones for organization of supramolecular signalling complexes are a recurrent theme in several model systems. Scaffold proteins preferentially employ linear peptide binding motifs for recruiting their interaction partners. PDZ domains are one of the more commonly encountered peptide binding domains in several proteins including those involved in scaffolding functions. This domain is known for its promiscuity both in terms of ligand selection, mode of interaction with its ligands as well as its association with other protein interaction domains. PDZ domains are subject to several means of regulations by virtue of their functional diversity. Additionally, the PDZ domains are refractive to the effect of mutations and maintain their three-dimensional architecture under extreme mutational load. The biochemical and biophysical basis for this selectivity as well as promiscuity has been investigated and reviewed extensively. The present review focuses on the plasticity inherent in PDZ domains and its implications for modular organization as well as evolution of cellular signalling pathways in higher eukaryotes.

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.

Studying histone modifications and their genomic functions by employing chromatin immunoprecipitation and immunoblotting.

Histones are one of the most abundant and highly conserved proteins in eukaryotes. Apart from serving as structural entities for orderly compaction of genomes, they play an instrumental role in the regulation of many important biological processes involving DNA such as transcription, DNA repair, and the cell cycle. Histone modifications have been implicated in maintaining the transcriptionally poised state of important genesin embryonic stem cells. Histone modifications are believed to be responsible for compartmentalization of chromatin into active and inactive domains. Hence, the tools and techniques required for studying these proteins are of utmost importance to biologists. This chapter provides a brief review of the posttranslational modifications of the N-terminal tails of histones and their biological roles, followed by step-by-step protocols for the most common techniques employed to study them. Here, we describe chromatin immunoprecipitation (ChIP) for studying the genomic functions of the most widely studied histone modifications, namely, histone H3 lysine 9 acetylation and histone H3 lysine 9 trimethylation that are typically associated with transcriptional activation and repression, respectively. Special emphasis has been given on the method of preparation of sonicated chromatin prior to immunoprecipitation since this single step affects the success of ChIP greatly and is often poorly described in published protocols. Protocol for histone isolation by acid-extraction and detection by Coomassie staining has also been described. We also describe the protocol for immunoblot analysis of histones using antibodies against key histone modifications. This chapter will serve as a useful resource in the study of histones and their posttranslational modifications.