ABSTRACT
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.
Subject(s)
Blotting, Western/methods , Chromatin Immunoprecipitation/methods , Genome/physiology , Histones/metabolism , Histones/physiology , Protein Processing, Post-Translational , Animals , Histone Code/physiology , Histones/analysis , Histones/chemistry , Humans , Models, Biological , Protein Processing, Post-Translational/physiologyABSTRACT
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.
Subject(s)
DNA Damage/physiology , Histones/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Checkpoint Kinase 2 , DNA Repair/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HeLa Cells , Histones/genetics , Humans , Jurkat Cells , Mice , NIH 3T3 Cells , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae , Signal Transduction/physiology , Sirtuin 2/genetics , Sirtuin 2/metabolism , Sirtuin 3/genetics , Sirtuin 3/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , p300-CBP Transcription Factors/geneticsABSTRACT
In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of beta-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) to activate target genes. Wnt/beta -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1), the T lineage-enriched chromatin organizer and global regulator, interacts with beta-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon beta-catenin signalling. GATA-3 is a T helper type 2 (T(H)2) specific transcription factor that regulates production of T(H)2 cytokines and functions as T(H)2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4(+) T cells, suggesting that SATB1 influences T(H)2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1), an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature T(H)2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for T(H)2 differentiation. Knockdown of beta-catenin also produced similar results, confirming the role of Wnt/beta-catenin signalling in T(H)2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits beta-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating T(H)2 cells in a Wnt-dependent manner. SATB1 coordinates T(H)2 lineage commitment by reprogramming gene expression. The SATB1:beta-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1 orchestrates T(H)2 lineage commitment by mediating Wnt/beta-catenin signalling. This report identifies a new global transcription factor involved in beta-catenin signalling that may play a major role in dictating the functional outcomes of this signalling pathway during development, differentiation, and tumorigenesis.
Subject(s)
Matrix Attachment Region Binding Proteins/physiology , Th2 Cells/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , Binding Sites , CD4-Positive T-Lymphocytes/metabolism , Cell Differentiation , Cell Lineage , Chromatin Immunoprecipitation , E1A-Associated p300 Protein/metabolism , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Humans , Promoter Regions, Genetic , Protein Structure, Tertiary , Signal Transduction , Th2 Cells/cytology , beta Catenin/chemistryABSTRACT
Retroviral integration has recently been shown to be nonrandom, favoring transcriptionally active regions of chromatin. However, the mechanism for integration site selection by retroviruses is not clear. We show here the occurrence of Alu-like motifs in the sequences flanking the reported viral integration sites that are significantly different from those obtained from the randomly picked sequences from the human genome, suggesting that unique primary sequence features exist in the genomic regions targeted by human immunodeficiency virus type 1 (HIV-1). Additionally, these sequences were preferentially bound by SATB1, the T lineage-restricted chromatin organizer, in vitro and in vivo. Alu repeats make up nearly 10% of the human genome and have been implicated in the regulation of transcription. To specifically isolate sequences flanking the viral integration sites and also harboring both Alu-like repeats and SATB1-binding sites, we combined chromatin immunoprecipitation with sequential PCRs. The cloned sequences flanking HIV-1 integration sites were specifically immunoprecipitated and amplified from the pool of anti-SATB1-immunoprecipitated genomic DNA fragments isolated from HIV-1 NL4.3-infected Jurkat T-cell chromatin. Moreover, many of these sequences were preferentially partitioned in the DNA associated tightly with the nuclear matrix and not in the chromatin loops. Strikingly, many of these regions were disfavored for integration when SATB1 was silenced, providing unequivocal evidence for its role in HIV-1 integration site selection. We propose that definitive sequence features such as the Alu-like motifs and SATB1-binding sites provide a unique chromatin context in vivo which is preferentially targeted by the HIV-1 integration machinery.
Subject(s)
Alu Elements/genetics , Chromatin/genetics , Chromatin/metabolism , HIV-1/genetics , Matrix Attachment Region Binding Proteins/metabolism , Virus Integration/genetics , Amino Acid Motifs/genetics , Binding Sites/genetics , Cell Line , HIV-1/pathogenicity , Humans , Jurkat Cells , Protein Binding/geneticsABSTRACT
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.