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Limfòcits b; Metilació de l'ADN; Genoma Linfocitos b; Metilación de ADN; Genoma B-lymphocytes; DNA methylation; Genome Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns. Spanish Ministry of Science, Innovation and Universities [SAF2017-88086-R to E.B.]; cofunded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe. E.B is supported by Instituto de Salud Carlos III (ISCIII), Ref. AC18/00057, associated with i-PAD project (ERARE European Union program); P.L. and C.P. are supported by the German Cancer Aid project CO-CLL [70113869]; B.G. is funded by the Deutsche Forschungsgemeinschaft [GR1617/14-1/iPAD, SFB1160/2_B5, RESIST–EXC 2155–Project ID 390874280, CIBSS–EXC-2189–Project ID 390939984]; BMBF [GAIN 01GM1910A]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities [SAF2017-88086-R].

We are very grateful to Dr. José Luis Sardina for useful feedback. We thank CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (MICINN ; grant number PID2020-117212RB-I00/AEI/10.13038/501100011033). E.M.-C. is funded with RESTORE project (EU H2020 Research and Innovation Programme, number 779316) and Spanish projects PI17/01521 and PI20/01313, integrated in the Plan Nacional de I+D+I and co-supported by the ISCIII-Subdirección General de Evaluación and FEDER. O.M.-P. holds an i-PFIS PhD fellowship (grant number IFI17/00034) from Acción Estratégica en Salud 2013-2016 ISCIII, co-financed by Fondo Social Europeo. F.F. holds a PhD fellowship from the INsTRuCT Consortium, which receives. Innovative Training Network subsidy from the EU H2020 program. We are very grateful to Dr. Jos? Luis Sardina for useful feedback. We thank CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (MICINN; grant number PID2020-117212RB-I00/AEI/10.13038/501100011033). E.M.-C. is funded with RESTORE project (EU H2020 Research and Innovation Programme, number 779316) and Spanish projects PI17/01521 and PI20/01313, integrated in the Plan Nacional de I+D+I and co-supported by the ISCIII-Subdirecci?n General de Evaluaci?n and FEDER. O.M.-P. holds an i-PFIS PhD fellowship (grant number IFI17/00034) from Acci?n Estrat?gica en Salud 2013?2016 ISCIII, co-financed by Fondo Social Europeo. F.F. holds a PhD fellowship from the INsTRuCT Consortium, which receives. Innovative Training Network subsidy from the EU H2020 program. F.C.-M. and E.B. conceived and designed the study; F.C.-M. A.G.F.-B. G.G.-T. O.M.-P. L.C. L.B. F.F. and T.L. performed the differentiation, chromatin immunoprecipitation, co-immunoprecipitation experiments, and immunological assays; F.C.-M. performed the bioinformatic analyses; F.C.-M. A.G.F.-B. G.G.-T. E.M.-C. and E.B. analyzed results; J.R.-U. and E.B. supervised the study; F.C.-M. T.L. and E.B. wrote the manuscript; all authors participated in discussions and interpreting the results. The authors declare no competing interests. The active form of vitamin D, 1,25-dihydroxyvitamin D3, induces a stable tolerogenic phenotype in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites, and promotes epigenetic and transcriptional remodeling. In this study, we report the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis in vitro. Differentiation to tolerogenic DCs associates with activation of the IL-6-JAK-STAT3 pathway. We show that JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other to form a complex with methylcytosine dioxygenase TET2. Most importantly, pharmacological inhibition of JAK2 reverts vitamin D-induced tolerogenic properties of DCs. This interplay among VDR, STAT3, and TET2 opens up possibilities for modulating DC immunogenic properties in clinics.

International audience; Faithful chromosome segregation requires that the sister chromatids be disjoined completely. Defective disjunction can lead to the persistence of histone-free threads of DNA known as ultra-fine bridges (UFBs) that connect the separating sister DNA molecules during anaphase. UFBs arise at specific genomic loci and can only be visualized by detection of associated proteins such as PICH, BLM, topoisomerase IIIα, and RPA. However, it remains unknown how these proteins work together to promote UFB processing. We used a combination of ensemble biochemistry and new single-molecule assays to reconstitute key steps of UFB recognition and processing by these human proteins in vitro. We discovered characteristic patterns of hierarchical recruitment and coordinated biochemical activities that were specific for DNA structures modeling UFBs arising at either centromeres or common fragile sites. Our results describe a mechanistic model for how unresolved DNA replication structures are processed by DNA-structure-specific binding factors in mitosis to prevent pathological chromosome nondisjunction.