Project description:We have comprehensively interrogated wild type B1 and B2 B lineage cells and their progenitors. In mice bearing a conditional deletion of Dnmt3a in the B lineage, we integrated a variety of whole-genome profiling approaches including WGBS, TAB-Seq, RNA-seq, ATAC-Seq and CUT&RUN. The results reveal a stable “foundational methylome” in all B cells that establishes lymphocyte lineage identity. Superimposed on this foundational methylome is a “dynamic methylome” which is differentially modulated in B1 and B2 B cells by the coincident activity/recruitment of DNMT3A and TET enzymes.
Project description:B2 cells were purified from spleen by CD19 Microbeads. B1 cells were purified from peritoneal cavity lavage by CD19 microbeads, followed by a depletion of CD23+ cells. RNA was prepared and differential gene expression analysis performed.
Project description:We have comprehensively interrogated wild type B1 and B2 B lineage cells and their progenitors. In mice bearing a conditional deletion of Dnmt3a in the B lineage, we integrated a variety of whole-genome profiling approaches including WGBS, TAB-Seq, RNA-seq, ATAC-Seq and CUT&RUN. The results reveal a stable “foundational methylome” in all B cells that establishes lymphocyte lineage identity. Superimposed on this foundational methylome is a “dynamic methylome” which is differentially modulated in B1 and B2 B cells by the coincident activity/recruitment of DNMT3A and TET enzymes.
Project description:We have comprehensively interrogated wild type B1 and B2 B lineage cells and their progenitors. In mice bearing a conditional deletion of Dnmt3a in the B lineage, we integrated a variety of whole-genome profiling approaches including WGBS, TAB-Seq, RNA-seq, ATAC-Seq and CUT&RUN. The results reveal a stable “foundational methylome” in all B cells that establishes lymphocyte lineage identity. Superimposed on this foundational methylome is a “dynamic methylome” which is differentially modulated in B1 and B2 B cells by the coincident activity/recruitment of DNMT3A and TET enzymes.
Project description:The experiment aimed at determining the genes that are under the control of the Ikaros transcription factor in mouse splenic B1 and B2 B lymphocyte subsets. To this aim, we used Ikf/f R26-CreERT2+ (Cre+) or Ikf/f R26-CreERT2- (Cre-) mice, which correspond to mice with floxed null alleles for Ikzf1 (Heizmann et al., JEM 210:2823-32, 2013) that were crossed with the R26-CreERT2 mice, which harbor a knock-in of the cDNA encoding the tamoxifen (TAM) inducible CreERT2 recombinase in the Rosa26 gene (Badea et al, J. Neurosci 23:2314-22, 2003). 6-8 week-old mice were injected daily with TAM for 3d (50mg/kg). Mice were sacrificed 10d after the first injection, and splenic B1 and B2 B cell populations sorted by flow cytometry. Splenic B1 and B2 cells from mice with induced deletion of Ikaros
Project description:Three innate (B1-B, NKT, CD8aaT cells) and adaptive (B2-B, CD4T, CD8abT cells) cell-types were sorted by FACS. Three biological replicates for NKT, CD4T, CD8aaT, CD8abT cells and two biological replicates for B1 and B2 cells were generated and the expression profiles were determined using Affymetrix Mu74Av2 chip. Comparisons between the sample groups allow the identification of genes differentially expressed between the innate and adaptive cell-types. Experiment Overall Design: 3 biological replicates for NKT, CD4T, CD8aaT, CD8abT cells and 2 biological replicates for B1, B2 cells were analyzed
Project description:Humoral immunity in mammals relies on the function of two developmentally and functionally distinct B cell subsets - B1 and B2 cells. While B2 cells are responsible for the adaptive response to environmental antigens, B1 cells regulate the production of polyreactive and low affinity antibodies for innate humoral immunity. The molecular mechanism of B cell specification into different subsets is understudied. We identified lysine methyltransferase NSD2 (MMSET/WHSC1) as a critical regulator of B1 cell development. In contrast to its minor impact on B2 cells, deletion of the catalytic domain of NSD2 in primary B cells impairs the generation of B1 lineage. Thus, NSD2, a histone H3 K36 dimethylase, is the first-in-class epigenetic regulator of a B cell lineage in mice.
Project description:We identified a novel long non-coding RNA Lx8-SINE B2, that is a marker of pluripotency. Depletion of Lx8-SINE B2 impacts embryonic stem cell self-renewal. RNA-seq analysis of Lx8-SINE B2 depletion revealed that a number of glycolytic genes with decreased expression. Mechanistically, we found that the Lx8-SINE B2 activates the glycolysis pathway by binding to Eno1. Collectively, our data suggest that Lx8-SINE B2 maintains the self-renewal of mESCs through glycolysis.
