Sanjeev Galande

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

The 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 γ (IFNγ) 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.

Poly(ADP-ribose) Polymerase and Ku Autoantigen Form a Complex and Synergistically Bind to Matrix Attachment Sequences

Genomic sequences with a cluster of ATC sequence stretches where one strand consists exclusively of well mixed As, Ts, and Cs confer high base unpairing propensity under negative superhelical strain. Such base unpairing regions (BURs) are typically found in scaffold or matrix attachment regions (SARs/MARs) that are thought to contribute to the formation of the loop domain structure of chromatin. Several proteins, including cell type-specific proteins, have been identified that bind specifically to double-stranded BURs either in vitro or in vivo. By using BUR-affinity chromatography to isolate BUR-binding proteins from breast cancer SK-BR-3 cells, we almost exclusively obtained a complex of poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK). Both PARP and DNA-PK are activated by DNA strand breaks and are implicated in DNA repair, recombination, DNA replication, and transcription. In contrast to the previous notion that PARP and Ku autoantigen, the DNA-binding subunit of DNA-PK, mainly bind to free ends of DNA, here we show that both proteins individually bind BURs with high affinity and specificity in an end-independent manner using closed circular BUR-containing DNA substrates. We further demonstrate that PARP and Ku autoantigen form a molecular complexin vivo and in vitro in the absence of DNA, and as a functional consequence, their affinity to the BURs are synergistically enhanced. ADP-ribosylation of the nuclear extract abrogated the BUR binding activity of this complex. These results provide a mechanistic link toward understanding the functional overlap of PARP and DNA-PK and suggest a novel role for these proteins in the regulation of chromatin structure and function.

p300-mediated acetylation of histone H3 lysine 56 functions in DNA damage response in mammals

The packaging of newly replicated and repaired DNA into chromatin is crucial for the maintenance of genomic integrity. Acetylation of histone H3 core domain lysine 56 (H3K56ac) has been shown to play a crucial role in compaction of DNA into chromatin following replication and repair in Saccharomyces cerevisiae. However, the occurrence and function of such acetylation has not been reported in mammals. Here we show that H3K56 is acetylated and that this modification is regulated in a cell cycle-dependent manner in mammalian cells. We also demonstrate that the histone acetyltransferase p300 acetylates H3K56 in vitro and in vivo, whereas hSIRT2 and hSIRT3 deacetylate H3K56ac in vivo. Further we show that following DNA damage H3K56 acetylation levels increased, and acetylated H3K56, which is localized at the sites of DNA repair. It also colocalized with other proteins involved in DNA damage signaling pathways such as phospho-ATM, CHK2, and p53. Interestingly, analysis of occurrence of H3K56 acetylation using ChIP-on-chip revealed its genome-wide spread, affecting genes involved in several pathways that are implicated in tumorigenesis such as cell cycle, DNA damage response, DNA repair, and apoptosis.

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.

Early Stress Evokes Age-Dependent Biphasic Changes in Hippocampal Neurogenesis, Bdnf Expression, and Cognition

BACKGROUND Adult-onset stressors exert opposing effects on hippocampal neurogenesis and cognition, with enhancement observed following mild stress and dysfunction following severe chronic stress. While early life stress evokes persistent changes in anxiety, it is unknown whether early stress differentially regulates hippocampal neurogenesis, trophic factor expression, and cognition across the life span. METHODS Hippocampal-dependent cognitive behavior, neurogenesis, and epigenetic regulation of brain-derived neurotrophic factor (Bdnf) expression was examined at distinct time points across the life span in rats subjected to the early stress of maternal separation (ES) and control groups. We also examined the influence of chronic antidepressant treatment on the neurogenic, neurotrophic, and cognitive changes in middle-aged ES animals. RESULTS Animals subjected to early stress of maternal separation examined during postnatal life and young adulthood exhibited enhanced hippocampal neurogenesis, decreased repressive histone methylation at the Bdnf IV promoter along with enhanced BDNF levels, and improved performance on the stress-associated Morris water maze. Strikingly, opposing changes in hippocampal neurogenesis and epigenetic regulation of Bdnf IV expression, concomitant with impairments on hippocampal-dependent cognitive tasks, were observed in middle-aged ES animals. Chronic antidepressant treatment with amitriptyline attenuated the maladaptive neurogenic, epigenetic, transcriptional, and cognitive effects in middle-aged ES animals. CONCLUSIONS Our study provides novel insights into the short- and long-term consequences of ES, demonstrating both biphasic and unique, age-dependent changes at the molecular, epigenetic, neurogenic, and behavioral levels. These results indicate that early stress may transiently endow animals with a potential adaptive advantage in stressful environments but across a life span is associated with long-term deleterious effects.

SATB1 Cleavage by Caspase 6 Disrupts PDZ Domain-Mediated Dimerization, Causing Detachment from Chromatin Early in T-Cell Apoptosis

ABSTRACT SATB1 is expressed primarily in thymocytes and orchestrates temporal and spatial expression of a large number of genes in the T-cell lineage. SATB1 binds to the bases of chromatin loop domains in vivo, recognizing a special DNA context with strong base-unpairing propensity. The majority of thymocytes are eliminated by apoptosis due to selection processes in the thymus. We investigated the fate of SATB1 during thymocyte and T-cell apoptosis. Here we show that SATB1 is specifically cleaved by a caspase 6-like protease at amino acid position 254 to produce a 65-kDa major fragment containing both a base-unpairing region (BUR)-binding domain and a homeodomain. We found that this cleavage separates the DNA-binding domains from amino acids 90 to 204, a region which we show to be a dimerization domain. The resulting SATB1 monomer loses its BUR-binding activity, despite containing both its DNA-binding domains, and rapidly dissociates from chromatin in vivo. We found this dimerization region to have sequence similarity to PDZ domains, which have been previously shown to be involved in signaling by conferring protein-protein interactions. SATB1 cleavage during Jurkat T-cell apoptosis induced by an anti-Fas antibody occurs concomitantly with the high-molecular-weight fragmentation of chromatin of ∼50-kb fragments. Our results suggest that mechanisms of nuclear degradation early in apoptotic T cells involve efficient removal of SATB1 by disrupting its dimerization and cleavage of genomic DNA into loop domains to ensure rapid and efficient disassembly of higher-order chromatin structure.

HIstome—a relational knowledgebase of human histone proteins and histone modifying enzymes

Histones are abundant nuclear proteins that are essential for the packaging of eukaryotic DNA into chromosomes. Different histone variants, in combination with their modification ‘code’, control regulation of gene expression in diverse cellular processes. Several enzymes that catalyze the addition and removal of multiple histone modifications have been discovered in the past decade, enabling investigations of their role(s) in normal cellular processes and diverse pathological conditions. This sudden influx of data, however, has resulted in need of an updated knowledgebase that compiles, organizes and presents curated scientific information to the user in an easily accessible format. Here, we present HIstome, a browsable, manually curated, relational database that provides information about human histone proteins, their sites of modifications, variants and modifying enzymes. HIstome is a knowledgebase of 55 human histone proteins, 106 distinct sites of their post-translational modifications (PTMs) and 152 histone-modifying enzymes. Entries have been grouped into 5 types of histones, 8 types of post-translational modifications and 14 types of enzymes that catalyze addition and removal of these modifications. The resource will be useful for epigeneticists, pharmacologists and clinicians. HIstome: The Histone Infobase is available online at http://www.iiserpune.ac.in/∼coee/histome/ and http://www.actrec.gov.in/histome/.

Direct interaction with and activation of p53 by SMAR1 retards cell-cycle progression at G2/M phase and delays tumor growth in mice.

The tumor‐suppressor p53 is a multifunctional protein mainly responsible for maintaining genomic integrity. p53 induces its tumor‐suppressor activity by either causing cell‐cycle arrest (G1/S or G2/M) or inducing cells to undergo apoptosis. This function of wild‐type p53 as “guardian of the genome” is presumably achieved by forming molecular complexes with different DNA targets as well as by interacting with a number of cellular proteins, e.g., Mdm2, Gadd45, p21, 14‐3‐3σ, Bax and Apaf‐1. Upon activation, p53 activates p21, which in turn controls the cell cycle by regulating G1 or G2 checkpoints. Here, we report SMAR1 as one such p53‐interacting protein that is involved in delaying tumor progression in vivo as well as in regulating the cell cycle. SMAR1 is a newly identified MARBP involved in chromatin‐mediated gene regulation. The SMAR1 gene encodes at least 2 alternatively spliced variants: SMAR1L (the full‐length form) and SMAR1S (the shorter form). We report that expression of SMAR1S, but not of SMAR1L, mRNA was decreased in most of the human cell lines examined, suggesting selective silencing of SMAR1S. Overexpression of SMAR1S in mouse melanoma cells (B16F1) and their subsequent injection in C57BL/6 mice delays tumor growth. Exogenous SMAR1S causes significant retardation of B16F1 cells in the G2/M phase of the cell cycle compared to SMAR1L. SMAR1S activates p53‐mediated reporter gene expression in mouse melanoma cells, breast cancer cells (MCF‐7) and p53 null cells (K562), followed by activation of its downstream effector, p21. We further demonstrate that SMAR1 physically interacts and colocalizes with p53. These data together suggest that SMAR1 is the only known MARBP that delays tumor progression via direct activation and interaction with tumor‐suppressor p53.

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

ABSTRACT 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.

Functional and regulatory characteristics of eukaryotic type II DNA topoisomerase.

DNA topoisomerases are ubiquitous nuclear enzymes that govern the topo-logical interconversions of DNA by transiently breaking/rejoining the phosphodiester backbone of one (type I) or both (type II) strands of the double helix. Consistent with these functions, topoisomerases play key roles in many aspects of DNA metabolism. Type II DNA topoisomerase (topo II) is vital for various nuclear processes, including DNA replication, chromosome segregation, and maintenance of chromosome structure. Topo II expression is regulated at multiple stages, including transcriptional, posttranscriptional, and posttranslational levels, by a multitude of signaling factors. Topo II is also the cellular target for a variety of clinically relevant anti-tumor drugs. Despite significant progress in our understanding of the role of topo II in diverse nuclear processes, several important aspects of topo II function, expression, and regulation are poorly understood. We have focused this review specifically on eukaryotic DNA topoisomerase II, with an emphasis on functional and regulatory characteristics.