ABSTRACT
Adaptive immune responses to inhaled allergens are induced following CCR7-dependent migration of precursor of dendritic cell (pre-DC)-derived conventional DCs (cDCs) from the lung to regional lymph nodes. However, monocyte-derived (moDCs) in the lung express very low levels of Ccr7 and consequently do not migrate efficiently to LN. To investigate the molecular mechanisms that underlie this dichotomy, we studied epigenetic modifications at the Ccr7 locus of murine cDCs and moDCs. When expanded from bone marrow precursors, moDCs were enriched at the Ccr7 locus for trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with transcriptional repression. Similarly, moDCs prepared from the lung also displayed increased levels of H3K27me3 at the Ccr7 promoter compared with migratory cDCs from that organ. Analysis of DC progenitors revealed that epigenetic modification of Ccr7 does not occur early during DC lineage commitment because monocytes and pre-DCs both had low levels of Ccr7-associated H3K27me3. Rather, Ccr7 is gradually silenced during the differentiation of monocytes to moDCs. Thus, epigenetic modifications of the Ccr7 locus control the migration and therefore the function of DCs in vivo. These findings suggest that manipulating epigenetic mechanisms might be a novel approach to control DC migration and thereby improve DC-based vaccines and treat inflammatory diseases of the lung.
Subject(s)
Dendritic Cells/immunology , Epigenesis, Genetic , Histones/genetics , Lung/immunology , Monocytes/immunology , Receptors, CCR7/genetics , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Cell Differentiation , Cell Lineage/immunology , Cell Movement , Cell Proliferation , Dendritic Cells/cytology , Histones/immunology , Lung/cytology , Lymph Nodes/cytology , Lymph Nodes/immunology , Methylation , Mice , Mice, Transgenic , Monocytes/cytology , Primary Cell Culture , Promoter Regions, Genetic , Receptors, CCR7/immunology , Signal Transduction , Transcription, GeneticABSTRACT
Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR ß-chain gene triggers feedback inhibition of V(ß)-to-DJ(ß) recombination in double-positive (DP) thymocytes, which correlates with reduced V(ß) chromatin accessibility and a locus conformational change that separates V(ß) from DJ(ß) gene segments. We previously generated a Tcrb allele that maintained V(ß) accessibility but was still subject to feedback inhibition in DP thymocytes. We have now further analyzed the contributions of chromatin accessibility and locus conformation to feedback inhibition using two novel TCR alleles. We show that reduced V(ß) accessibility and increased distance between V(ß) and DJ(ß) gene segments both enforce feedback inhibition in DP thymocytes.
Subject(s)
Gene Frequency/immunology , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor/genetics , Membrane Glycoproteins/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , Recombination, Genetic/immunology , Animals , Chromatin/genetics , Chromatin/immunology , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic/genetics , Enhancer Elements, Genetic/immunology , Feedback, Physiological , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor/genetics , Mice , Mice, 129 Strain , Mice, Knockout , Mice, Transgenic , Nucleic Acid ConformationABSTRACT
Accessibility of chromosomal recombination signal sequences to the RAG protein complex is known to be essential for V(D)J recombination at Ag receptor loci in vivo. Previous studies have addressed the roles of cis-acting regulatory elements and germline transcription in the covalent modification of nucleosomes at Ag receptor loci. However, a detailed picture of nucleosome organization at accessible and inaccessible recombination signal sequences has been lacking. In this study, we have analyzed the nucleosome organization of accessible and inaccessible Tcrb and Tcra alleles in primary murine thymocytes in vivo. We identified highly positioned arrays of nucleosomes at Dbeta, Jbeta, and Jalpha segments and obtained evidence indicating that positioning is established at least in part by the regional DNA sequence. However, we found no consistent positioning of nucleosomes with respect to recombination signal sequences, which could be nucleosomal or internucleosomal even in their inaccessible configurations. Enhancer- and promoter-dependent accessibility was characterized by diminished abundance of certain nucleosomes and repositioning of others. Moreover, some changes in nucleosome positioning and abundance at Jalpha61 were shown to be a direct consequence of germline transcription. We suggest that enhancer- and promoter-dependent transcription generates optimal recombinase substrates in which some nucleosomes are missing and others are covalently modified.
Subject(s)
Genes, T-Cell Receptor/genetics , Nucleosomes/genetics , Receptors, Antigen, T-Cell/genetics , Regulatory Sequences, Nucleic Acid/immunology , Transcription, Genetic/immunology , Animals , Base Sequence , Enhancer Elements, Genetic/immunology , Mice , Mice, Transgenic , Molecular Sequence Data , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The DNA methyltransferase, Dnmt3a, is dynamically regulated throughout mammalian B cell development and upon activation by antigenic stimulation. Dnmt3a inactivation in hematopoietic stem cells has been shown to drive B cell-related malignancies, including chronic lymphocytic leukemia, and associates with specific DNA methylation patterns in transformed cells. However, while it is clear that inactivation of Dnmt3a in hematopoietic stem cells has profound functional effects, the consequences of Dnmt3a inactivation in cells of the B lineage are unclear. To assess whether loss of Dnmt3a at the earliest stages of B cell development lead to DNA methylation defects that might impair function, we selectively inactivated Dnmt3a early in mouse B cell development and then utilized whole genome bisulfite sequencing to generate base-resolution profiles of Dnmt3a+/+ and Dnmt3a-/- naïve splenic B cells. Overall, we find that global methylation patterns are largely consistent between Dnmt3a+/+ and Dnmt3a-/- naïve B cells, indicating a minimal functional effect of DNMT3A in mature B cells. However, loss of Dnmt3a induced 449 focal DNA methylation changes, dominated by loss-of-methylation events. Regions found to be hypomethylated in Dnmt3a-/- naïve splenic B cells were enriched in gene bodies of transcripts expressed in B cells, a fraction of which are implicated in B cell-related disease. Overall, the results from this study suggest that factors other than Dnmt3a are the major drivers for methylome maintenance in B cell development.
Subject(s)
B-Lymphocytes/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , Epigenesis, Genetic , Gene Expression Regulation , Animals , Bone Marrow Cells/metabolism , CpG Islands , DNA Methyltransferase 3A , Disease Susceptibility , Gene Knockout Techniques , Genome , Genomics/methods , Mice , Mice, Knockout , Phenotype , Spleen/cytology , Whole Genome SequencingABSTRACT
Cells of the adaptive immune response undergo dynamic epigenetic changes as they develop and respond to immune challenge. Plasticity is a necessary prerequisite for the chromosomal dynamics of lineage specification, development, and the immune effector function of the mature cell types. The alterations in DNA methylation and histone modification that characterize activation may be integral to the generation of immunologic memory, thereby providing an advantage on secondary exposure to pathogens. While the immune system benefits from the dynamic nature of the epigenome, such benefit comes at a cost - increased likelihood of disease-causing mutation.