ABSTRACT
Histone deacetylases (HDACs) play crucial roles during mammalian development and for cellular homeostasis. In addition, these enzymes are promising targets for small molecule inhibitors in the treatment of cancer and neurological diseases. Conditional HDAC knock-out mice are excellent tools for defining the functions of individual HDACs in vivo and for identifying the molecular targets of HDAC inhibitors in disease. Here, we describe the generation of tissue-specific HDAC knock-out mice and delineate a strategy for the generation of conditional HDAC knock-in mice.
Subject(s)
Blastocyst/enzymology , Chromatin/metabolism , Epigenesis, Genetic , Genetic Vectors/metabolism , Histone Deacetylase 1/genetics , Mouse Embryonic Stem Cells/enzymology , Animals , Blastocyst/cytology , Blotting, Southern , CRISPR-Cas Systems , Chromatin/chemistry , Chromosomes, Artificial, Bacterial/chemistry , Chromosomes, Artificial, Bacterial/metabolism , Crosses, Genetic , Female , Gene Knock-In Techniques , Genetic Vectors/chemistry , Histone Deacetylase 1/deficiency , Homologous Recombination , Integrases/genetics , Integrases/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mouse Embryonic Stem Cells/cytology , Organ Specificity , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolismABSTRACT
Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology.
Subject(s)
Aurora Kinases/metabolism , Embryonic Development , Histone Deacetylase 1/metabolism , Mitosis , Zebrafish/embryology , Acetylation , Animals , Genes, Regulator , Histones/metabolism , PhosphorylationABSTRACT
CD8 coreceptor expression is dynamically regulated during thymocyte development and is tightly controlled by the activity of at least 5 different cis-regulatory elements. Despite the detailed characterization of the Cd8 loci, the regulation of the complex expression pattern of CD8 cannot be fully explained by the activity of the known Cd8 enhancers. In this study, we revisited the Cd8ab gene complex with bioinformatics and transgenic reporter gene expression approaches to search for additional Cd8 cis-regulatory elements. This led to the identification of an ECR (ECR-4), which in transgenic reporter gene expression assays, directed expression preferentially in CD44(hi)CD62L(+) CD8(+) T cells, including innate-like CD8(+) T cells. ECR-4, designated as Cd8 enhancer E8VI, was bound by Runx/CBFß complexes and Bcl11b, indicating that E8VI is part of the cis-regulatory network that recruits transcription factors to the Cd8ab gene complex in CD8(+) T cells. Transgenic reporter expression was maintained in LCMV-specific CD8(+) T cells upon infection, although short-term, in vitro activation led to a down-regulation of E8VI activity. Finally, E8VI directed transgene expression also in CD8αα(+) DCs but not in CD8αα-expressing IELs. Taken together, we have identified a novel Cd8 enhancer that directs expression in CD44(hi)CD62L(+) CD8(+) T cells, including innate-like and antigen-specific effector/memory CD8(+) T cells and in CD8αα(+) DCs, and thus, our data provide further insight into the cis-regulatory networks that control CD8 expression.
Subject(s)
CD8 Antigens/genetics , CD8-Positive T-Lymphocytes/metabolism , Conserved Sequence , Dendritic Cells/metabolism , Gene Expression Regulation , Regulatory Sequences, Nucleic Acid/genetics , Animals , Base Sequence , CD8 Antigens/biosynthesis , Chromosome Mapping , Core Binding Factor Alpha 3 Subunit/metabolism , Dogs , Enhancer Elements, Genetic , Genes, Reporter , Humans , Hyaluronan Receptors/analysis , Immunologic Memory , L-Selectin/analysis , Mice , Mice, Transgenic , Molecular Sequence Data , Protein Interaction Mapping , Rats , Repressor Proteins/metabolism , Sequence Alignment , Sequence Homology, Nucleic Acid , Species Specificity , T-Lymphocyte Subsets/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
Molecular mechanisms that maintain lineage integrity of helper T cells are largely unknown. Here we show histone deacetylases 1 and 2 (HDAC1 and HDAC2) as crucial regulators of this process. Loss of HDAC1 and HDAC2 during late T cell development led to the appearance of major histocompatibility complex (MHC) class II-selected CD4(+) helper T cells that expressed CD8-lineage genes such as Cd8a and Cd8b1. HDAC1 and HDAC2-deficient T helper type 0 (TH0) and TH1 cells further upregulated CD8-lineage genes and acquired a CD8(+) effector T cell program in a manner dependent on Runx-CBFß complexes, whereas TH2 cells repressed features of the CD8(+) lineage independently of HDAC1 and HDAC2. These results demonstrate that HDAC1 and HDAC2 maintain integrity of the CD4 lineage by repressing Runx-CBFß complexes that otherwise induce a CD8(+) effector T cell-like program in CD4(+) T cells.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/metabolism , Th1 Cells/immunology , Animals , Cell Differentiation/genetics , Cell Lineage/genetics , Cells, Cultured , Core Binding Factor alpha Subunits/metabolism , Core Binding Factor beta Subunit/metabolism , Cytokines/metabolism , Cytotoxicity, Immunologic/genetics , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Histone Deacetylase 1/genetics , Histone Deacetylase 2/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein BindingABSTRACT
The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage-dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue. Strikingly, deletion of a single Hdac2 allele in HDAC1 knockout mice results in severe epidermal defects, including alopecia, hyperkeratosis, hyperproliferation and spontaneous tumour formation. These mice display impaired Sin3A co-repressor complex function, increased levels of c-Myc protein, p53 expression and apoptosis in hair follicles (HFs) and misregulation of HF bulge stem cells. Surprisingly, ablation of HDAC1 but not HDAC2 in a skin tumour model leads to accelerated tumour development. Our data reveal a crucial function of HDAC1/HDAC2 in the control of lineage specificity and a novel role of HDAC1 as a tumour suppressor in the epidermis.
