The transcription factors EBF1 and Pax5 have been linked to activation of the B cell lineage program and irreversible loss of alternative lineage potential (commitment), respectively. Here we conditionally deleted Ebf1 in committed pro-B cells after transfer into alymphoid mice. We found that those cells converted into innate lymphoid cells (ILCs) and T cells with variable-diversity-joining (VDJ) rearrangements of loci encoding both B cell and T cell antigen receptors. As intermediates in lineage conversion, Ebf1-deficient CD19(+) cells expressing Pax5 and transcriptional regulators of the ILC and T cell fates were detectable. In particular, genes encoding the transcription factors Id2 and TCF-1 were bound and repressed by EBF1. Thus, both EBF1 and Pax5 are required for B lineage commitment by repressing distinct and common determinants of alternative cell fates.

Lymphopoiesis requires the activation of lineage-specific genes embedded in naive, inaccessible chromatin or in primed, accessible chromatin. The mechanisms responsible for de novo gain of chromatin accessibility, known as "pioneer" function, remain poorly defined. Here, we showed that the EBF1 C-terminal domain (CTD) is required for the regulation of a specific gene set involved in B cell fate decision and differentiation, independently of activation and repression functions. Using genome-wide analysis of DNaseI hypersensitivity and DNA methylation in multipotent Ebf1(-/-) progenitors and derivative EBF1wt- or EBF1ΔC-expressing cells, we found that the CTD promoted chromatin accessibility and DNA demethylation in previously naive chromatin. The CTD allowed EBF1 to bind at inaccessible genomic regions that offer limited co-occupancy by other transcription factors, whereas the CTD was dispensable for EBF1 binding at regions that are occupied by multiple transcription factors. Thus, the CTD enables EBF1 to confer permissive lineage-specific changes in progenitor chromatin landscape.

B-cell fate determination requires the action of transcription factors that operate in a regulatory network to activate B-lineage genes and repress lineage-inappropriate genes. However, the dynamics and hierarchy of events in B-cell programming remain obscure. To uncouple the dynamics of transcription factor expression from functional consequences, we generated induction systems in developmentally arrested Ebf1 −/− pre-pro-B cells to allow precise experimental control of EBF1 expression in the genomic context of progenitor cells. Consistent with the described role of EBF1 as a pioneer transcription factor, we show in a time-resolved analysis that EBF1 occupancy coincides with EBF1 expression and precedes the formation of chromatin accessibility. We observed dynamic patterns of EBF1 target gene expression and sequential up-regulation of transcription factors that expand the regulatory network at the pro-B-cell stage. A continuous EBF1 function was found to be required for Cd79a promoter activity and for the maintenance of an accessible chromatin domain that is permissive for binding of other transcription factors. Notably, transient EBF1 occupancy was detected at lineage-inappropriate genes prior to their silencing in pro-B cells. Thus, persistent and transient functions of EBF1 allow for an ordered sequence of epigenetic and transcriptional events in B-cell programming.

BackgroundTelomeres are nucleoprotein complexes at the end of linear eukaryotic chromosomes which maintain the genome integrity by regulating telomere length, preventing recombination and end to end fusion events. Multiple proteins associate with telomeres and function in concert to carry out these functions. Rap1 interacting factor 1 (Rif1), was identified as a protein involved in telomere length regulation in yeast. Rif1 is conserved upto mammals but its function has diversified from telomere length regulation to maintenance of genome integrity.ResultsWe have carried out detailed bioinformatic analyses and identified Rif1 homologues in 92 organisms from yeast to human. We identified Rif1 homologues in Drosophila melanogaster, even though fly telomeres are maintained by a telomerase independent pathway. Our analysis shows that Drosophila Rif1 (dRif1) sequence is phylogenetically closer to the one of vertebrates than yeast and has identified a few Rif1 specific motifs conserved through evolution. This includes a Rif1 family specific conserved region within the HEAT repeat domain and a motif involved in protein phosphatase1 docking. We show that dRif1 is nuclear localized with a prominent heterochromatin association and unlike human Rif1, it does not respond to DNA damage by localizing to damaged sites. To test the evolutionary conservation of dRif1 function, we expressed the dRif1 protein in yeast and HeLa cells. In yeast, dRif1 did not perturb yeast Rif1 (yRif1) functions; and in HeLa cells it did not colocalize with DNA damage foci.ConclusionsTelomeres are maintained by retrotransposons in all Drosophila species and consequently, telomerase and many of the telomere associated protein homologues are absent, including Rap1, which is the binding partner of Rif1. We found that a homologue of yRif1 protein is present in fly and dRif1 has evolutionarily conserved motifs. Functional studies show that dRif1 responds differently to DNA damage, implying that dRif1 may have a different function and this may be conserved in other organisms as well.