A lncRNA identifies Irf8 enhancer element in negative feedback control of dendritic cell differentiation

Dendritic cells (DC) are professional antigen presenting cells that develop from multipotent progenitors (MPP) and DC committed common DC progenitors (CDP) and further differentiate into different subsets: classical DC type 1 and 2 (cDC1 and cDC2, respectively) and plasmacytoid DC (pDC). In this study MPP, CDP, cDC1, cDC2 and pDC were obtained in a two-step in vitro culture system according to Felker et al., J. Immunol. 185, 5326-5335, 2010. Briefly, mouse bone marrow cells were first amplified with a specific cytokine cocktail and then induced to differentiate into DC with Flt3 ligand. MPP, CDP, cDC1, cDC2 and pDC were obtained by FACS sorting as follows: MPP: Gr1- CD117hi CD135low/-; CDP: Gr1- CD117int CD135+ CD115+; cDC1: CD11c+ CD11blow/- XCR1+; cDC2: CD11c+ CD11b+ XCR1- and pDC: CD11c+ CD11b- B220+. FACS sorted cells were then subjected to Omni-ATAC-seq, nuclear-titrated (NuTi) Capture-C targeting Irf8 promoter, and RNA-seq analysis. ATAC-seq data of cDC1 and pDC are published (Li et al., Genome Biol. 20, 45, 2019; GSE118221). ATAC-seq data of MPP, CDP and cDC2, NuTi Capture-C and RNA-seq data of MPP, CDP, cDC1, cDC2 and pDC are published here. CD11c+ CD11b+ B220- cDC and CD11c+ CD11b- B220+ pDC were obtained by FACS sorting and subjected to ChIP-seq analysis of IRF8 and are published here. ATAC-seq analysis of mouse multipotent progenitors (MPP) and DC committed common DC progenitors (CDP), classical dendritic cells type 1 and type 2 (cDC1 and cDC2, respectively) and plasmacytoid dendritic cells (pDC) was performed by Omni-ATAC-seq according to Corces et al., Nat. Methods. 14, 959-962, 2017 with minor modifications as described in Li et al., Genome Biol. 20, 45, 2019 (GSE118221). NuTi Capture-C analysis (MPP, CDP, cDC1, cDC2 and pDC) was performed with a protocol modified from Downes et al., Nat. Commun. 12, 1-15, 2021 and Downes et al., Nat. Protoc. 17, 445-475, 2022. Design of Irf8 promoter viewpoint was with the design tool Oligo (https://oligo.readthedocs.io/en/latest/index.html) and resulted in 2 oligonucleotide probes targeting Irf8 promoter. Oligonucleotide probes were positioned adjacent to the DpnII cut sites on a restriction fragment spanning the Irf8 promoter (chr8:123,259,948-123,260,530). RNA-seq analysis (MPP, CDP, cDC1, cDC2 and pDC) and ChIP-seq analysis (cDC and pDC) was done as described in Allhoff et al., Nucleic Acids Res. 44, e153, 2016 (GSE73143).