ABSTRACT
Previous transgenic-reporter and targeted-deletion studies indicate that the subset-specific expression of CD8αß heterodimers is controlled by multiple enhancer activities, since no silencer elements had been found within the locus. We have identified such a silencer as L2a, a previously characterized â¼ 220 bp nuclear matrix associating region (MAR) located â¼ 4.5 kb upstream of CD8α. L2a transgenes driven by the E8(I) enhancer showed no reporter expression in thymic subsets or T cells in splenic, inguinal and mesenteric lymph node peripheral T cells. Deletion of L2a resulted in significant reporter de-repression, even in the CD4(+)CD8(+) double positive (DP) thymocyte population. L2a contains binding sites for two MAR-interacting proteins, SATB1 and CDP. We found that that binding of these factors was markedly influenced by the content and spacing of L2a sub-motifs (L and S) and that SATB1 binds preferentially to the L motif both in vitro and in vivo. A small fraction of the transgenic CD8 single positive (SP) thymocytes and peripheral CD8(+) T cells bypassed L2a-silencing to give rise to variegated expression of the transgenic reporter. Crossing the L2a-containing transgene onto a SATB1 knockdown background enhanced variegated expression, suggesting that SATB1 is critical in overcoming L2a-silenced transcription.
Subject(s)
CD8 Antigens/biosynthesis , Gene Expression Regulation/immunology , Homeodomain Proteins/metabolism , Matrix Attachment Region Binding Proteins/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Silencer Elements, Transcriptional/genetics , Animals , Base Sequence , CD8 Antigens/genetics , Cell Separation , Electrophoretic Mobility Shift Assay , Flow Cytometry , Gene Expression , Homeodomain Proteins/genetics , Matrix Attachment Region Binding Proteins/genetics , Mice , Mice, Transgenic , Molecular Sequence Data , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Repressor Proteins/geneticsABSTRACT
BACKGROUND: Disrupting the balance of histone lysine methylation alters the expression of genes involved in tumorigenesis including proto-oncogenes and cell cycle regulators. Methylation of lysine residues is commonly catalyzed by a family of proteins that contain the SET domain. Here, we report the identification and characterization of the SET domain-containing protein, Smyd2. RESULTS: Smyd2 mRNA is most highly expressed in heart and brain tissue, as demonstrated by northern analysis and in situ hybridization. Over-expressed Smyd2 localizes to the cytoplasm and the nucleus in 293T cells. Although accumulating evidence suggests that methylation of histone 3, lysine 36 (H3K36) is associated with actively transcribed genes, we show that the SET domain of Smyd2 mediates H3K36 dimethylation and that Smyd2 represses transcription from an SV40-luciferase reporter. Smyd2 associates specifically with the Sin3A histone deacetylase complex, which was recently linked to H3K36 methylation within the coding regions of active genes in yeast. Finally, we report that exogenous expression of Smyd2 suppresses cell proliferation. CONCLUSION: We propose that Sin3A-mediated deacetylation within the coding regions of active genes is directly linked to the histone methyltransferase activity of Smyd2. Moreover, Smyd2 appears to restrain cell proliferation, likely through direct modulation of chromatin structure.
Subject(s)
Gene Expression Regulation , Histone-Lysine N-Methyltransferase/physiology , Histones/chemistry , Methyltransferases/chemistry , Amino Acid Sequence , Animals , Cell Line , Chromatin/chemistry , Histone-Lysine N-Methyltransferase/chemistry , Humans , Lysine/chemistry , Methyltransferases/biosynthesis , Mice , Molecular Sequence Data , NIH 3T3 Cells , Protein Structure, Tertiary , Repressor Proteins/chemistry , Sequence Homology, Amino Acid , Sin3 Histone Deacetylase and Corepressor ComplexABSTRACT
The vertebrate heart is assembled during embryogenesis in a modular manner from different populations of precursor cells. The right ventricular chamber and outflow tract are derived primarily from a population of progenitors known as the anterior heart field. These regions of the heart are severely hypoplastic in mutant mice lacking the myocyte enhancer factor 2C (MEF2C) and BOP transcription factors, suggesting that these cardiogenic regulatory factors may act in a common pathway for development of the anterior heart field and its derivatives. We show that Bop expression in the developing heart depends on the direct binding of MEF2C to a MEF2-response element in the Bop promoter that is necessary and sufficient to recapitulate endogenous Bop expression in the anterior heart field and its cardiac derivatives during mouse development. The Bop promoter also directs transcription in the skeletal muscle lineage, but only cardiac expression is dependent on MEF2. These findings identify Bop as an essential downstream effector gene of MEF2C in the developing heart, and reveal a transcriptional cascade involved in development of the anterior heart field and its derivatives.
Subject(s)
Gene Expression Regulation, Developmental , Heart Ventricles/embryology , Mice/embryology , Muscle Proteins/metabolism , Myogenic Regulatory Factors/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Blotting, Southern , DNA, Complementary/genetics , DNA-Binding Proteins , Electrophoretic Mobility Shift Assay , In Situ Hybridization , MEF2 Transcription Factors , Mice, Mutant Strains , Molecular Sequence Data , Muscle Proteins/genetics , Muscle, Skeletal/metabolism , Oligonucleotides , Promoter Regions, Genetic/genetics , Transcription Factors/genetics , Transgenes/geneticsABSTRACT
Studies have suggested that binding of the SATB1 protein to L2a, a matrix association region located 4.5 kb 5' to the mouse CD8alpha gene, positively affects CD8 expression in T cells. Therefore, experiments were performed to determine the effect on T cell development of reduced expression of SATB1. Because homozygous SATB1-null mice do not survive to adulthood due to non-thymus autonomous defects, mice were produced that were homozygous for a T cell-specific SATB1-antisense transgene and heterozygous for a SATB1-null allele. Thymic SATB1 protein was reduced significantly in these mice, and the major cellular phenotype observed was a significant reduction in the percentage of CD8SP T cells in thymus, spleen, and lymph nodes. Mice were smaller than wild type but generally healthy, and besides a general reduction in cellularity and a slight increase in surface CD3 expression on CD8SP thymocytes, the composition of the thymus was similar to wild type. The reduction in thymic SATB1 does not lead to the variegated expression of CD8-negative single positive thymocytes seen upon deletion of several regulatory elements and suggested by others to reflect failure to activate the CD8 locus. Thus, the present results point to an essential role for SATB1 late in the development and maturation of CD8SP T cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Matrix Attachment Region Binding Proteins/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Animals , Base Sequence , CD3 Complex/metabolism , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Cell Proliferation , Humans , Matrix Attachment Region Binding Proteins/deficiency , Matrix Attachment Region Binding Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , RNA, Antisense/genetics , T-Lymphocyte Subsets/cytology , Thymus Gland/cytology , Thymus Gland/metabolismABSTRACT
Several pathological conditions involve alterations in expression of neuronal nicotinic acetylcholine receptors (nAChRs). Although some studies have addressed processes involved with muscle nAChR expression, knowledge of the regulation of neuronal nAChRs is particularly sparse. The following studies were designed to investigate cellular mechanisms involved with expression of neuronal alpha3beta4* nAChRs. Catecholamine secretion assays and receptor binding studies coupled with receptor alkylation were used to study the nAChR regulation and turnover. Alkylation of adrenal nAChRs results in a rapid and complete loss of receptor-mediated neurosecretion and surface [(3)H]epibatidine binding sites. After alkylation, both neurosecretory function and nAChR binding slowly (24-48 h) return to prealkylation levels. When cells are treated with the protein synthesis inhibitor puromycin, after alkylation, receptor-mediated neurosecretion does not recover. Long-term treatment (24-48-h) with puromycin, in the absence of alkylation, results in a slow, time-dependent shift to the right, followed by a downward shift, in the nicotine concentration-response curve, documenting a disappearance of surface nAChRs. Puromycin treatment alone also results in a loss to both surface and intracellular [(3)H]epibatidine binding sites. nAChR beta4 subunit levels are significantly decreased after treatment with puromycin. These data support a constitutive turnover of adrenal alpha3beta4* nAChRs, requiring continual de novo synthesis of new receptor protein.
Subject(s)
Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Receptors, Nicotinic/biosynthesis , Animals , Cattle , Cells, Cultured , Gene Expression Regulation/physiology , Membrane Proteins/genetics , Protein Binding/physiology , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolismSubject(s)
Bacterial Proteins , DNA-Binding Proteins/metabolism , Fibroblasts/metabolism , Flow Cytometry/methods , Genes, Reporter , Luciferases , Spectrometry, Fluorescence/methods , Transcription Factors/metabolism , Transfection/methods , Animals , Cell Line , Fibroblasts/cytology , Mice , Quality Control , Spectrometry, Fluorescence/standardsABSTRACT
The m-Bop protein encoded by the mouse Bop gene is strongly expressed in heart and skeletal muscle, and recent studies with Bop knockout mice have demonstrated that m-Bop is essential for cardiogenesis in vivo and can act as a HDAC-dependent repressor in vitro. In the present studies, m-Bop was observed to interact with skNAC, a reported transcriptional activator specific to heart and skeletal muscle. The amino-terminal S region of the split S-ET domain of m-Bop as well as the MYND domain were required for interaction with skNAC in both the two-hybrid system and in coimmunoprecipitation experiments from cultured mammalian cells. As shown previously for interaction of the MYND domain-containing transcriptional corepressor, BS69, with several viral and cellular oncoproteins, a PXLXP motif in skNAC was required for interaction with m-Bop. Similar kinetics of induction and localization of m-Bop and skNAC during the induction of myogenesis in cultured C2C12 cells suggests a possible associated role for these proteins during this process.
Subject(s)
Heart/growth & development , Muscle Proteins , Trans-Activators/physiology , Transcription Factors/physiology , Animals , Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Line , Co-Repressor Proteins , DNA-Binding Proteins , Humans , Kinetics , Mice , Mice, Knockout , Molecular Chaperones , Muscle, Skeletal/growth & development , Protein Binding , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
Many transcription factors regulate specific temporal-spatial events during cardiac differentiation; however, the mechanisms that regulate such events are largely unknown. Using a modified subtractive hybridization method to identify specific genes that influence early cardiac development, we found that Bop is expressed specifically in cardiac and skeletal muscle precursors before differentiation of these lineages. Bop encodes a protein containing MYND and SET domains, which have been shown to regulate transcription by mediating distinct chromatin modifications. We show that m-Bop is a histone deacetylase-dependent transcriptional repressor. Targeted deletion of Bop in mice disrupted maturation of ventricular cardiomyocytes and interfered with formation of the right ventricle. Normal expression of Hand2, a transcription factor essential for right ventricular development, in cardiomyocyte precursors is dependent upon m-Bop. These results indicate that m-Bop is essential for cardiomyocyte differentiation and cardiac morphogenesis.