Research in Galande laboratory is focused on studying how the dynamic changes in higher order chromatin assembly govern gene expression in a spatial and temporal manner. We are also interested in understanding dynamic changes in chromatin architecture under various immunological signaling processes such as activation and differentiation of T cells and pathogenesis of diseases involving T cells. This is currently observed by studying the T cell prevalent special AT-rich binding protein 1 (SATB1) as a paradigm. Our laboratory identified involvement of a T-lineage-enriched host factor SATB1 during the early events in HIV-1 life cycle (Kumar et al., 2005). A phosphorylation-dependent molecular switch in SATB1 was identified providing mechanistic insights into the ability of SATB1 to act as a global regulator of gene expression (Kumar et al., 2006). We also demonstrated that functional interaction between SATB1 and PML oncoprotein regulates the chromatin loop architecture and transcription of the MHC class I locus (Kumar et al., 2007). Recently, we showed that SATB1 interacts with two components of Wnt signaling, namely CtBP1 and b-catenin, and regulates Wnt targets (Purbey et al., 2009; Notani et al., 2010). These reports establish SATB1 as a new global transcription factor involved in b-catenin signaling that may play a major role in dictating the functional outcomes of this signaling pathway during development, differentiation, and tumorigenesis. To summarize, we are specifically interested in unraveling the third dimension of gene regulation! Our research interests and outcome of recent projects can be summarized as follows: The third dimension of gene regulation: It's all in the looping! Non-random and compartmentalized distribution of functional components is a hallmark of the nucleus in multicellular organisms. In these organisms, DNA is organized with help of basic proteins into an orderly packaged compact structure called chromatin. Out of the thousands of protein-coding 'genes'or DNA segments that each cell contains, only a fraction is used at any given time, and those genes that are seldomly used are packaged much tightly as compared to the ones that are 'expressed' or used during the lifetime of a cell. Moreover, the various hierarchical states of the packaging are interconvertible depending upon the physiological need of the cell and also contribute in variety of ways to achieve stringent regulation of gene activity. Dynamic nature of chromatin loops is one such mechanism wherein the structural transitions lead to functional consequences. Technological advances in recent years have provided unprecedented insights into the role of chromatin organization and interactions of various structural-functional components towards gene regulation. The global chromatin organizer SATB1 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. At genome-wide level, SATB1 seems to play a role in organization of the 'transcriptionally poised' chromatin, the part of chromatin that contains genes that are required for execution of specific cellular processes (Fig. 1). We are currently pursuing the implications of these findings towards development and differentiation of the cells of the immune system.
Figure 1. Global chromatin organization by SATB1. (a) Mouse thymocyte stained with DAPI to reveal heterochromatin blobs or the chromatin territories (CTs). (b) Thymocyte stained with anti-SATB1 to reveal 'cage-like' network structure circumscribing the CTs. (c) Schematic representation of an enlarged sector from the thymocyte depicting occupancy of SATB1 at various locations within the decondensed and active chromatin loops that fill the space within the highly condensed CTs. (Reproduced from Galande et al., 2007). SATB1 is the only chromatin associated protein which harbors a PDZ-like signaling domain. PDZ domains are protein interaction modules that are implicated in most of the cell signaling. PDZ together with homeodomain-containing protein SATB1, provide a framework, which supports assembly of regulatory protein complexes onto a discrete set of genomic targets. Currently, we are testing a hypothesis, that, SATB1 is a chromatin level functional end point for the PDZ-mediated signal transduction, Which further results in coordinated gene regulation through PDZ-mediated interactions of SATB1 (Fig. 2. depicts our working hypothesis).
Figure 2. Schematic representation of the signal transduction pathway involving PDZ domain-containing proteins. SATB1 is depicted in green color within the nucleus. PDZ domains are indicated in magenta. Interestingly, we found that in HIV-1 infected cells SATB1 is targeted by Tat, the viral transactivator, such that genes under the control of SATB1 including IL-2 and its receptor are upregulated. The functional interaction between HIV-1 Tat and SATB1 necessitates its PDZ-like domain and the HDAC1 co-repressor also binds through the same. Furthermore, Tat competitively displaces HDAC1 which is bound to SATB1, leading to increased acetylation of histones at the promoters in vivo. These results clearly indicate a novel mechanism by which HIV-1 Tat might overcome SATB1-mediated repression in T-cells (Kumar et al., 2005). To gain insight into DNA recognition and transcription activity by SATB1 we have recently determined its optimal DNA-binding sequence by random oligonucleotide selection. The consensus SATB1-binding sequence (CSBS) comprises a palindromic sequence in which two identical AT-rich half-sites are arranged as inverted repeats flanking a central cytosine or guanine. Binding studies using homeodomain (HD)-lacking SATB1 and binding target with increased spacer between the two half-sites led us to propose a model for SATB1-DNA complex in which the HDs bind in an antiparallel fashion to the palindromic consensus element via minor groove, bridged by the PDZ-like dimerization domain (Fig. 3). CSBS-driven in vivo reporter analysis indicated that SATB1 acts as a repressor upon binding to the CSBS and most of its derivatives and the extent of repression is proportional to SATB1's binding affinity to these sequences. These studies provide mechanistic insights into the mode of DNA binding and its effect on the regulation of transcription by SATB1 (Purbey et al., 2008).
Figure 3. Schematic diagram depicting the mode of binding of SATB1 with its consensus DNA binding sequence. For details, see Purbey et al., 2008.
Recent research highlights Global Regulator SATB1 Recruits b-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner In vertebrates the canonical Wnt signaling culminates in b-catenin moving into the nucleus where it activates transcription of target genes. Wnt/b-catenin signaling is essential for the thymic maturation and differentiation of naive T cells. We show that SATB1 binds to b-catenin and recruits it to SATB1's genomic binding sites so that genes formerly repressed by SATB1 are upregulated by Wnt signaling. 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 important subset of helper T cells. Specifically we show that siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4+ T cells. Inhibiting Wnt signaling led to downregulation of GATA-3 and of signature TH2 cytokines such as IL-4, IL-10, and IL-13. Knockdown of b-catenin also produced similar results, thus together these data confirm the role of Wnt/b-catenin signaling in TH2 differentiation. Our data demonstrate that SATB1 orchestrates TH2 lineage commitment by modulating Wnt/b-catenin signaling (Notani et al., PLoS Biology, 2010, For full article follow the link- (http://biology.plosjournals.org/perlserv/?request=get-document&doi>=10.1371/journal.pbio.1000296).
Figure 4. SATB1 and b-catenin colocalize in the thymocyte nuclei. Indirect immunofluorescence staining thymocytes using antibodies to SATB1 (red) and b-catenin (green) was performed. DNA counterstaining was performed using DAPI (blue). For details, see Notani et al., 2010.
Technology development: An improved protein purification system We have developed a novel cassette for expression and purification of proteins (Purbey et al., 2006). This system overcomes several disadvantages of the typically employed protocols for expression and purification of tagged proteins, and hence is of tremendous research and commercial potential. We have obtained a US patent for the same. Purbey PK, Jayakumar CP, Patole MS, and Galande S. сn improved purification systemҮ Patent # US 7,604,980 B2, 2009.
1. Sameet Mehta (Postdoctoral Fellow) 2. Ranveer Jayani (Senior Research Fellow) 3. Praveena R.L. (Senior Research Fellow) 4. Kamal Vishnu Prasad Gottimukkala (Senior Research Fellow) 5. Sunita Singh(Senior Research Fellow) 6. Rafeeq Ahmed Mir (Junior Research Fellow) 7. Shashikant Gawai (Project Assistant 8. Trupti Bhankhede (Project Assistant) 9. Amruthrao Deshmukh (Senior Research Fellow) 10. Rahul Kumar Jangid (Junior Research Fellow) 11. Nitin Sonawane (Technician) 12. Tanuja Bankar (Technician) 13. Sarmistha Kalanke (Project Assistant) 14. Santosh Botre (Lab assistant) Project Trainees 1. Rasika Lohokare 2. Mithila Burute
1. Pavan Kumar P (Rockefeller, USA) 2. Prabhat Kumar Purbey (UCLA, USA) 3. Amita Limaye (VCCRI, Sydney, Australia) 4. Dimple Notani (UCSD, USA) 5. Shiny Titus (Postdoctoral Research Associate) 6. Suman Prasad ( Postdoctoral Research Associate) 7. Madhujit Damle ( Postdoctoral Research Associate)
The work in our laboratory is supported by grants from the Department of Biotechnology, New Delhi, India, Department of Science and Technology, and The Wellcome Trust, UK. Sanjeev Galande is an International Senior Research Fellow of the Wellcome Trust, UK.
This picture has almost everyone (past and current members) in them!
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