Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. ...Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.
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.
The E/ID protein axis is instrumental for defining the developmental progression and functions of hematopoietic cells. The E proteins are dimeric transcription factors that activate gene expression ...programs and coordinate changes in chromatin organization. Id proteins are antagonists of E protein activity. Relative levels of E/Id proteins are modulated throughout hematopoietic development to enable the progression of hematopoietic stem cells into multiple adaptive and innate immune lineages including natural killer cells, B cells and T cells. In early progenitors, the E proteins promote commitment to the T and B cell lineages by orchestrating lineage specific programs of gene expression and regulating VDJ recombination of antigen receptor loci. In mature B cells, the E/Id protein axis functions to promote class switch recombination and somatic hypermutation. E protein activity further regulates differentiation into distinct CD4+ and CD8+ T cells subsets and instructs mature T cell immune responses. In this review, we discuss how the E/Id proteins define the adaptive immune system lineages, focusing on their role in directing developmental gene programs.
The genome is organized into topologically associated domains (TADs) that enclose smaller subTADs. Here, we identify and characterize an enhancer that is located in the middle of the V gene region of ...the immunoglobulin kappa light chain (Igκ) locus that becomes active preceding the stage at which this locus undergoes V(D)J recombination. This enhancer is a hub of long-range chromatin interactions connecting subTADs in the V gene region with the recombination center at the J genes. Deletion of this element results in a highly altered long-range chromatin interaction pattern across the locus and, importantly, affects individual V gene utilization locus-wide. These results indicate the existence of an enhancer-dependent framework in the Igκ locus and further suggest that the composition of the diverse antibody repertoire is regulated in a subTAD-specific manner. This enhancer thus plays a structural role in orchestrating the proper folding of the Igκ locus in preparation for V(D)J recombination.
Display omitted
•An enhancer, E88, in the middle of the Igκ locus is a major hub of interactions•Deletion of E88 alters Vκ gene rearrangement frequencies throughout the locus•Structure of the Igκ locus in pro-B cells influences Vκ rearrangement in pre-B cells•V gene rearrangement is likely mediated in a subTAD-specific manner
Barajas-Mora et al. demonstrate that an enhancer—E88—in the V gene region of the Igκ locus regulates long-range chromatin interactions, shaping its 3D structure, enabling different V-gene-containing subTADs to come in proximity to the J genes to generate a diverse repertoire. Consequently, E88 deletion perturbs the resulting B cell repertoire.
The paths that hematopoietic stem cells take to develop from multipotent, self-renewing cells into committed lymphocytes has been a topic of debate for some time. During early hematopoiesis, multiple ...branchpoints have been described in which progeny cells segregate into cell lineages with distinct developmental potentials. In this issue of
Genes & Development
, Inlay and colleagues (pp. 2376–2381) identify novel intermediate stages through which hematopoietic progenitor cells travel.
The transition from the follicular B to the plasma cell stage is associated with large-scale changes in cell morphology. Here, we examine whether plasma cell development is also associated with ...changes in nuclear architecture. We find that the onset of plasma cell development is concomitant with a decline in remote genomic interactions; a gain in euchromatic character at loci encoding for factors that specify plasma cell fate, including Prdm1 and Atf4; and establishment of de novo inter-chromosomal hubs. We find that, in developing plasma cells and concurrent with transcriptional silencing, the Ebf1 locus repositions from an euchromatic to peri-centromeric heterochromatic environment. Finally, we find that inter-chromosomal hubs are enriched for the deposition of either H3K27Ac or H3K27me3. These data indicate that plasma cell fate is orchestrated by elaborate changes in genome topology and that epigenetic marks, linked with super-enhancers or transcriptionally repressed regions, are enriched at inter-chromosomal hubs.
Display omitted
•Plasma cell fate is orchestrated by elaborate changes in nuclear architecture•The Ebf1 locus relocates to the peri-centromeric heterochromatin in plasma cells•Plasma cell fate is associated with de novo inter-chromosomal hubs•Inter-chromosomal hubs are enriched for H3K27Ac or H3K27me3
Bortnick et al. demonstrate that plasma cell fate is associated with changes in chromatin folding, inter-chromosomal interactions, and nuclear localization involving plasma-cell-specific genes. Inter-chromosomal hubs in maturing B cells are enriched for the deposition of either H3K27Ac or H3K27me3. We propose that deposition of these marks facilitates inter-chromosomal interactions.
Recent studies have identified a number of transcriptional regulators, including E2A, early B-cell factor 1 (EBF1), FOXO1, and paired box gene 5 (PAX5), that promote early B-cell development. ...However, how this ensemble of regulators mechanistically promotes B-cell fate remains poorly understood. Here we demonstrate that B-cell development in FOXO1-deficient mice is arrested in the common lymphoid progenitor (CLP) LY6D ⁺ cell stage. We demonstrate that this phenotype closely resembles the arrest in B-cell development observed in EBF1-deficient mice. Consistent with these observations, we find that the transcription signatures of FOXO1- and EBF1-deficient LY6D ⁺ progenitors are strikingly similar, indicating a common set of target genes. Furthermore, we found that depletion of EBF1 expression in LY6D ⁺ CLPs severely affects FOXO1 mRNA abundance, whereas depletion of FOXO1 activity in LY6D ⁺ CLPs ablates EBF1 transcript levels. We generated a global regulatory network from EBF1 and FOXO1 genome-wide transcription factor occupancy and transcription signatures derived from EBF1- and FOXO1-deficient CLPs. This analysis reveals that EBF1 and FOXO1 act in a positive feedback circuitry to promote and stabilize specification to the B-cell lineage.
The immunoglobulin heavy-chain (
Igh) locus is organized into distinct regions that contain multiple variable (V
H), diversity (D
H), joining (J
H) and constant (C
H) coding elements. How the
Igh ...locus is structured in 3D space is unknown. To probe the topography of the
Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire
Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the
Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V
H, D
H, J
H, and C
H elements, showed compartmentalization and striking conformational changes involving V
H and D
H-J
H elements during early B cell development. In pro-B cells, the entire repertoire of V
H regions (2 Mbp) appeared to have merged and juxtaposed to the D
H elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.
Recent studies have documented genome-wide binding patterns of transcriptional regulators and their associated epigenetic marks in hematopoietic cell lineages. In order to determine how epigenetic ...marks are established and maintained during developmental progression, we have generated long-term cultures of hematopoietic progenitors by enforcing the expression of the E-protein antagonist Id2. Hematopoietic progenitors that express Id2 are multipotent and readily differentiate upon withdrawal of Id2 expression into committed B lineage cells, thus indicating a causative role for E2A (
Tcf3) in promoting the B cell fate. Genome-wide analyses revealed that a substantial fraction of lymphoid and myeloid enhancers are premarked by the poised or active enhancer mark H3K4me1 in multipotent progenitors. Thus, in hematopoietic progenitors, multilineage priming of enhancer elements precedes commitment to the lymphoid or myeloid cell lineages.
► Long -term culture of multipotent hematopoietic progenitors ► The E2A proteins play a causative role in initiating B cell development ► Enhancers are primed for lineage plasticity in multipotent progenitors by H3K4me1
PDF
