Broad domains of histone marks in the highly compact Paramecium macronuclear genome.

The unicellular ciliate Paramecium contains a large vegetative macronucleus with several unusual characteristics, including an extremely high coding density and high polyploidy. As macronculear chromatin is devoid of heterochromatin, our study characterizes the functional epigenomic organization necessary for gene regulation and proper Pol II activity. Histone marks (H3K4me3, H3K9ac, H3K27me3) reveal no narrow peaks but broad domains along gene bodies, whereas intergenic regions are devoid of nucleosomes. Our data implicate H3K4me3 levels inside ORFs to be the main factor associated with gene expression, and H3K27me3 appears in association with H3K4me3 in plastic genes. Silent and lowly expressed genes show low nucleosome occupancy, suggesting that gene inactivation does not involve increased nucleosome occupancy and chromatin condensation. Because of a high occupancy of Pol II along highly expressed ORFs, transcriptional elongation appears to be quite different from that of other species. This is supported by missing heptameric repeats in the C-terminal domain of Pol II and a divergent elongation system. Our data imply that unoccupied DNA is the default state, whereas gene activation requires nucleosome recruitment together with broad domains of H3K4me3. In summary, gene activation and silencing in Paramecium run counter to the current understanding of chromatin biology.

Resident memory CD4+ T lymphocytes mobilize from bone marrow to contribute to a systemic secondary immune reaction.

Resident memory T lymphocytes (TRM ) of epithelial tissues and the Bm protect their host tissue. To what extent these cells are mobilized and contribute to systemic immune reactions is less clear. Here, we show that in secondary immune reactions to the measles-mumps-rubella (MMR) vaccine, CD4+ TRM are mobilized into the blood within 16 to 48 h after immunization in humans. This mobilization of TRM is cognate: TRM recognizing other antigens are not mobilized, unless they cross-react with the vaccine. We also demonstrate through methylome analyses that TRM are mobilized from the Bm. These mobilized cells make significant contribution to the systemic immune reaction, as evidenced by their T-cell receptor Vß clonotypes represented among the newly generated circulating memory T-cells, 14 days after vaccination. Thus, TRM of the Bm confer not only local, but also systemic immune memory.

Human IgA-Expressing Bone Marrow Plasma Cells Characteristically Upregulate Programmed Cell Death Protein-1 Upon B Cell Receptor Stimulation.

The functions of bone marrow plasma cells (BMPC) beyond antibody production are not fully elucidated and distinct subsets of BMPC suggest potential different functions. Phenotypic differences were identified for human BMPC depending on CD19 expression. Since CD19 is a co-stimulatory molecule of the B-cell-receptor (BCR), and IgA+ and IgM+ BMPC express the BCR on their surface, we here studied whether CD19 expression affects cellular responses, such as BCR signaling and the expression of checkpoint molecules. We analyzed 132 BM samples from individuals undergoing routine total hip arthroplasty. We found that both CD19+ and CD19- BMPC expressed BCR signaling molecules. Notably, the BCR-associated kinase spleen tyrosine kinase (SYK) including pSYK was higher expressed in CD19+ BMPC compared to CD19- BMPC. BCR stimulation also resulted in increased kinase phosphorylation downstream of the BCR while expression of CD19 remained stable afterwards. Interestingly, the BCR response was restricted to IgA+ BMPC independently of CD19 expression. With regard to the expression of checkpoint molecules, CD19- BMPC expressed higher levels of co-inhibitory molecule programmed cell death protein-1 (PD-1) than CD19+ BMPC. IgA+ BMPC characteristically upregulated PD-1 upon BCR stimulation in contrast to other PC subsets and inhibition of the kinase SYK abrogated PD-1 upregulation. In contrast, expression of PD-1 ligand, B and T lymphocyte attenuator (BTLA) and CD28 did not change upon BCR activation of IgA+ BMPC. Here, we identify a distinct characteristic of IgA+ BMPC that is independent of the phenotypic heterogeneity of the subsets according to their CD19 expression. The data suggest that IgA+ BMPC underlie different regulatory principles and/or exert distinct regulatory functions.

Unique and assay specific features of NOMe-, ATAC- and DNase I-seq data.

Chromatin accessibility maps are important for the functional interpretation of the genome. Here, we systematically analysed assay specific differences between DNase I-seq, ATAC-seq and NOMe-seq in a side by side experimental and bioinformatic setup. We observe that most prominent nucleosome depleted regions (NDRs, e.g. in promoters) are roboustly called by all three or at least two assays. However, we also find a high proportion of assay specific NDRs that are often 'called' by only one of the assays. We show evidence that these assay specific NDRs are indeed genuine open chromatin sites and contribute important information for accurate gene expression prediction. While technically ATAC-seq and DNase I-seq provide a superb high NDR calling rate for relatively low sequencing costs in comparison to NOMe-seq, NOMe-seq singles out for its genome-wide coverage allowing to not only detect NDRs but also endogenous DNA methylation and as we show here genome wide segmentation into heterochromatic B domains and local phasing of nucleosomes outside of NDRs. In summary, our comparisons strongly suggest to consider assay specific differences for the experimental design and for generalized and comparative functional interpretations.

DNA methylation signatures of monozygotic twins clinically discordant for multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system with a modest concordance rate in monozygotic twins, which strongly argues for involvement of epigenetic factors. We observe highly similar peripheral blood mononuclear cell-based methylomes in 45 MS-discordant monozygotic twins. Nevertheless, we identify seven MS-associated differentially methylated positions (DMPs) of which we validate two, including a region in the TMEM232 promoter and ZBTB16 enhancer. In CD4 + T cells we find an MS-associated differentially methylated region in FIRRE. Additionally, 45 regions show large methylation differences in individual pairs, but they do not clearly associate with MS. Furthermore, we present epigenetic biomarkers for current interferon-beta treatment, and extensive validation shows that the ZBTB16 DMP is a signature for prior glucocorticoid treatment. Taken together, this study represents an important reference for epigenomic MS studies, identifies new candidate epigenetic markers, and highlights treatment effects and genetic background as major confounders.

A comprehensive analysis of 195 DNA methylomes reveals shared and cell-specific features of partially methylated domains.

BACKGROUND: Partially methylated domains are extended regions in the genome exhibiting a reduced average DNA methylation level. They cover gene-poor and transcriptionally inactive regions and tend to be heterochromatic. We present a comprehensive comparative analysis of partially methylated domains in human and mouse cells, to identify structural and functional features associated with them. RESULTS: Partially methylated domains are present in up to 75% of the genome in human and mouse cells irrespective of their tissue or cell origin. Each cell type has a distinct set of partially methylated domains, and genes expressed in such domains show a strong cell type effect. The methylation level varies between cell types with a more pronounced effect in differentiating and replicating cells. The lowest level of methylation is observed in highly proliferating and immortal cancer cell lines. A decrease of DNA methylation within partially methylated domains tends to be linked to an increase in heterochromatic histone marks and a decrease of gene expression. Characteristic combinations of heterochromatic signatures in partially methylated domains are linked to domains of early and middle S-phase and late S-G2 phases of DNA replication. CONCLUSIONS: Partially methylated domains are prominent signatures of long-range epigenomic organization. Integrative analysis identifies them as important general, lineage- and cell type-specific topological features. Changes in partially methylated domains are hallmarks of cell differentiation, with decreased methylation levels and increased heterochromatic marks being linked to enhanced cell proliferation. In combination with broad histone marks, partially methylated domains demarcate distinct domains of late DNA replication.