Epigenomic Research Of Dendritic Cell Differentiation And Maturation

Dendritic cells (DCs) are the most important professional antigen-presenting cells. Bone marrow progenitor cell-derived immature DCs (imDCs), which are characterized by high endocytic activity and low T-cell activation potential, are present in small quantities in locations that are in contact with the external environment to capture antigens and process them into peptides. Monocytes are a kind of blood precursors of imDCs. When activated by pathogens through pattern recognition receptors (PRRs) such as Toll-like receptor 4 (TLR4) signaling, imDC will become maturation characterized by highly expressing kinds of cytokines and co-stimulatory factors and high ability to present antigens to and activate T cells.Sets of epigenetic information such as DNA methylation, histone modification and chromatin remodeling, which constitute additional layers of transcription regulation, have been discovered to play key roles in setting up and maintaining unique cell identity during eukaryotes development and differentiation. DNA methylation, whereby a methyl group is added to the 5′position of the cytosine pyrimidine ring which imparts stable and heritable signals upon the DNA code, is commonly associated with chromatin remodeling and gene silencing in a tissue- or cell-type-specific manner. Histones have a variety of covalent modifications in N-terminal tails, including acetylation, methylation, phosphorylation, sumoylation, ubiquitination and ADP ribosylation, and could interact with DNA and recruit transcriptional regulator to control the higher order chromatin structure and gene expression. To comprehensively reveal complex regulatory network of epigenetic regulators across an incredibly varied background of developmental stages, tissue types, and disease states, genome-wide studies of epigenetic profiles of variety of cells and tissues have been planned and extensively performed, especially in human beings.Immunological research develops and expands rapidly in the decades. New insights have been achieved in the better understanding of cellular and molecular mechanisms for immune recognition, immune response and immune regulation. More and more new tools, especially omics techniques, have been applied to study the development and functions of the immune system. Epigenetic regulation has been proposed as one of critical mechanisms in immune cell differentiation and function, allowing an specific gene expression pattern for lineage commitment and appropriate immune responses against infection. From the point of view of epigenome, systematically analyzing genome-wide chromatin modifications provides immunologists with powerful tools for data-based immunological research. Recently, more and more DNA methylomes and histone modifications in T cells and B cells have been widely revealed through microarray and deep sequencing in combination with traditional epigenetic techniques such as bisulfite PCR and Chromatin IP (ChIP). However, epigenetic regulation of dendritic cell differentiation and function has not been extensively investiagted, although there are reports about epigenetic regulation of cytokine and MHCII expression in DC activation during infection.To address the question for the epigenetic regulation of dendritic cell differentiation and function, we utilized the epigenomic tools to systematically analyze chromatin regulation during DC differentiation and maturation. We studied dynamic profiles of DNA methylation, histone modification, transcriptome and nuclear-enriched small RNome for DCs, and provided data-based clues to further study the important roles of chromatin regulators in differentiation and maturation of DCs.Part I. Epigenetic profiles of human monocytes and monocyte-derived immature DCs (imDCs) and mature DCs (mDCs)Monocytes, monocyte-derived imDCs, LPS-induced mDCs from one healthy donor were prepared for whole genomic MethylC-Seq. We also performed genome-wide distribution of two key histone modifications, H3K4me3 and H3K27me3, which had been suggested to play regulatory roles in differentiation and activation of immune cells, using ChIP-sequencing (ChIP-seq). Digital gene expression profiling (DGEP) and small RNA sequencing were also conducted for transcription level analysis. Thus, we collected high quality and comprehensive data sets on genomic, epigenetic and RNA levels of the three kinds of cells we studied.We firstly detected the methylated cytosines (mCs) in the whole genome context of the three kinds of cells as previously reported, and found above 99.5% were in the CG context in these cells, among which, 80% mCGs were 80–100% methylated. Through profiling analysis for each of chromosomes, we found that there were slightly more highly methylated mCs in monocytes than those in other two kinds of cells (imDCs and mDCs). To observe the relationship of DNA methylation and histone modifications in the same cell, we further analyzed DNA methylation levels in H3K4me3 and H3K27me3 islands. DNA methylation was slightly higher in H3K27me3 islands. And when H3K4me3 and H3K27me3 co-localized, DNA methylation levels decreased, even lower than those in H3K4me3 enriched domains especially in monocytes and imDCs. We also performed comprehensive analysis of the DNA methylomes for distinct gene-associated and intergenic genomic features separately for the three kinds of cells.Then, according to our DGEP data, the entire transcription units of human protein-coding genes were categoried and subjected to analysis of regional mean DNA methylation levels and histone modification tag densities. High expressed genes have lower mean DNA methylation level and H3K27me3 around TSS. And high expressed miRNAs have lower DNA methylation level in their transcribed regions. To investigate the important roles of epigenetic factors in regulating differentiation and maturation of human DCs, we deeply analyzed our multi-level sequencing data from the point of view of systematic biology. Firstly, we analyzed expression patterns of protein-coding genes and miRNAs. Secondly, through PCA analysis, we compared the contribution of DNA methylation, histone modifications and miRNAs. Thirdly, we analysed the DNA methylation level of DCs related pathway genes, and found, most promoters of these genes underwent DNA demethylation during monocyte differentiation to imDC. Fourthly, we clustered the DNA methylation and histone modification, and found there were same dynamic trends for many locations between DNA methylation and H3K27me3.Although genome-wide profiling and PCA analysis indicated that the level of DNA methylation did not change extensively and frequently, some key genes in cell development and differentiation like transcription factors might be the targets of DNA methylation. When imDCs were matured by LPS, we identified transcription factors EGR1, FLI1 and HSF4 with continuous de novo methylated CpG sites in the upstream regions of their TSS which might be implicated in DNA methylation mediated transcription silencing. We then choose EGR1 for functional study of DCs maturation. To investigate the regulatory role of EGR1 in DCs differentiation and maturation, we analyzed the genome-wide EGR1 binding sites in immature DC via ChIP-seq. KEGG pathway enrichment analysis of possible EGR1 targets revealed that these targets enriched pathways were associated with cell-cell interaction. And further gain-and-loss study of EGR1 function through lentivirus infection showed that EGR1 could inhibit CD86, CD48 and CD58 expression in DCs matured by LPS. Therefore, when DCs matured by LPS signaling, EGR1 was silenced by DNA methylation leading to the high expression of membrane co-stimulatory molecules, which subsequently favor the maturation of DCs and activation of T cell responses.Part II. New kind of nucleus-localized, Dicer-independent small RNAs, nlsR-3 inhibits iNOS expression during LPS response via chromatin regulators.Small RNAs have been found to play more and more important roles in regulating gene expression at transcription level in recent years. However, there is no definite kind of small RNAs especially conducting chromatin regulation for gene expression in mammals identified to date. In this aprt of study, we identified a novel class of nuclear localized small RNAs (nlsRNAs) which derived from nuclear RNA precursors through nuclear and cytoplasmic RNA isolation and sequencing of mouse bone marrow-derived mDCs. These small RNAs were partially dicer-dependant and did not associate with AGO family proteins, which were quite different from microRNAs targeting messanger RNAs in cytoplasm. We further found that one of these small RNAs inhibited inducible nitric oxide synthase (iNOS) expression at chromatin level in a sequence dependent manner during LPS response. This nlsRNA could induce targeting of Mi-2βwhich was a repressive chromatin remodeler to the iNOS promoter and mediate trimethylation of H3K27 which was a repressive histone modification there.Taken together, we have comprehensively studied the profiles of DNA methylation, histone modification, transcriptome and nuclear small RNome during DCs differentiation and maturation through epigenomic techniques. Furthermore, we revealed the role of epigenetic factors in regulating DCs maturation through targeting transcription factors and iNOS. Establishment of this database of DCs has provided us with a platform to mining key epigenetic regulator for DCs identity and function and will indicate new clues to design drug targets in a view of chromatin regulation.