Summary Genome-scale studies have revealed extensive, cell type-specific co-localization of transcription factors, but the mechanisms underlying this phenomenon remain poorly understood. Here we demonstrate in macrophages and B cells that collaborative interactions of the common factor PU.1 with small sets of macrophage- or B celllineage-determining transcription factors establish cell-specific binding sites that are associated with the majority of promoter-distal H3K4me1-marked genomic regions. PU.1 binding initiates nucleosome remodeling followed by H3K4 monomethylation at large numbers of genomic regions associated with both broadly and specifically expressed genes. These locations serve as beacons for additional factors, exemplified by liver X receptors, which drive both cell-specific gene expression and signal-dependent responses. Together with analyses of transcription factor binding and H3K4me1 patterns in other cell types, these studies suggest that simple combinations of lineage-determining transcription factors can specify the genomic sites ultimately responsible for both cell identity and cell type-specific responses to diverse signaling inputs.

It is now established that the transcription factors E2A, EBF1 and Foxo1 play critical roles in B cell development. Here we show that E2A and EBF1 bound regulatory elements present in the Foxo1 locus. E2A and EBF1 as well as E2A and Foxo1, in turn, were wired together by a vast spectrum of cis-regulatory codes. These associations were dynamic during developmental progression. Occupancy by the E2A isoform, E47, directly elevated the abundance as well as the pattern of histone H3K4 monomethylation across putative enhancer regions. Finally, the pro-B cell epigenome was divided into clusters of loci that show E2A, EBF and Foxo1 occupancy. From this analysis a global network consisting of transcriptional regulators, signaling and survival factors, was constructed that we propose orchestrates the B cell fate.

The genome is folded into domains located in either transcriptionally inert or permissive compartments. Here we used genome-wide strategies to characterize domains during B cell development. Structured Interaction Matrix Analysis revealed that CTCF occupancy was primarily associated with intra-domain interactions, whereas p300, E2A and PU.1 bound sites were associated with intra- and inter-domain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitors the transcriptionally inactive Ebf1 locus was sequestered at the nuclear lamina, thereby preserving multipotency. Upon development into the pro-B cell stage Ebf1 and other genes switched compartments to establish de novo intra- and inter-domain interactions that are associated with a B lineage specific transcription signature.

Compaction and looping of the ∼2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V H genes to be brought in proximity with D H -J H segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ∼60 sites throughout the V H region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5′ of DFL16 and the 3′ regulatory region, and also the intronic enhancer (Eμ), creating a D H -J H -Eμ-C H domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D H region and parts of the V H locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.A ntigen receptors are created through the highly regulated lineage-specific process of V(D)J recombination, creating a diverse repertoire of Ig and T-cell receptors. The generation of the mouse Ig heavy chain in pro-B cells begins with D H -to-J H rearrangement on both alleles, followed by V H -to-D H J H rearrangement. In order for the >100 functional murine V H genes spread across ∼2.5 Mb to gain access to the single D-J rearrangement on that allele, the Igh locus undergoes contraction and looping during the pro-B-cell stage of B-cell differentiation (1-5). By measuring spatial distances between 11 small probes spread throughout the Igh locus, Jhunjhunwala et al. (2) demonstrated that distal and proximal V H genes were approximately equidistant from the D genes specifically at the pro-B-cell stage when the V H genes are rearranging. Computational as well as geometrical approaches have suggested that the locus is organized into rosette-like clusters of loops that compact during rearrangement. Several proteins have been reported to influence Igh locus compaction, including Pax5, YY1, and Ikaros (5-7). These proteins and others, such as Ezh2 (8), are also necessary for the rearrangement of distal V H genes but not proximal V H genes. This is most likely a consequence of the lack of locus compaction in the absence of these proteins. How all these proteins funct...

SummaryT follicular helper (Tfh) cells are essential in the induction of high-affinity, class-switched antibodies. The differentiation of Tfh cells is a multi-step process that depends upon the co-receptor ICOS and the activation of phosphoinositide-3 kinase leading to the expression of key Tfh cell genes. We report that ICOS signaling inactivates the transcription factor FOXO1, and a Foxo1 genetic deletion allowed for generation of Tfh cells with reduced dependence on ICOS ligand. Conversely, enforced nuclear localization of FOXO1 inhibited Tfh cell development even though ICOS was overexpressed. FOXO1 regulated Tfh cell differentiation through a broad program of gene expression exemplified by its negative regulation of Bcl6. Final differentiation to germinal center Tfh cells (GC-Tfh) was instead FOXO1 dependent as the Foxo1−/− GC-Tfh cell population was substantially reduced. We propose that ICOS signaling transiently inactivates FOXO1 to initiate a Tfh cell contingency that is completed in a FOXO1-dependent manner.