Yin C. Lin

Simple Combinations of Lineage-Determining Transcription Factors Prime cis-Regulatory Elements Required for Macrophage and B Cell Identities

Genome-scale studies have revealed extensive, cell type-specific colocalization 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 cell lineage-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.

A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate

It is now established that the transcription factors E2A, EBF1 and Foxo1 have 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 sequences. These associations were dynamic during developmental progression. Occupancy by the E2A isoform E47 directly resulted in greater abundance, as well as a pattern of monomethylation of histone H3 at lysine 4 (H3K4) across putative enhancer regions. Finally, we divided the pro-B cell epigenome into clusters of loci with occupancy by E2A, EBF and Foxo1. From this analysis we constructed a global network consisting of transcriptional regulators, signaling and survival factors that we propose orchestrates B cell fate.

CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all VH genes to be brought in proximity with DH-JH 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 VH 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 DH-JH-Eμ-CH domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the DH region and parts of the VH 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.

Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate

The genome is folded into domains located in compartments that are either transcriptionally inert or transcriptionally permissive. Here we used genome-wide strategies to characterize domains during B cell development. Structured interaction matrix analysis showed that occupancy by the architectural protein CTCF was associated mainly with intradomain interactions, whereas sites bound by the histone acetyltransferase p300 or the transcription factors E2A or PU.1 were associated with intra- and interdomain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitor cells, the transcriptionally inactive locus encoding early B cell factor (Ebf1) was sequestered at the nuclear lamina, which thereby preserved their multipotency. After development into the pro-B cell stage, Ebf1 and other genes switched compartments to establish new intra- and interdomain interactions associated with a B lineage–specific transcription signature.

Multilineage priming of enhancer repertoires precedes commitment to the B and myeloid cell lineages in hematopoietic progenitors.

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.

The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells

It is established that the transcription factor E2A and its antagonist Id3 modulate the checkpoints consisting of the precursor to the T cell antigen receptor (pre-TCR) and the TCR. Here we demonstrate that Id3 expression was higher beyond the pre-TCR checkpoint, remained high in naive T cells and showed a bimodal pattern in the effector-memory population. We show how E2A promoted T lineage specification and how pre-TCR-mediated signaling affected E2A genome-wide occupancy. Thymi in Id3-deficient mice had aberrant development of effector-memory cells, higher expression of the chemokine receptor CXCR5 and the transcriptional repressor Bcl-6 and, unexpectedly, T cell–B cell conjugates and B cell follicles. Collectively, our data show how E2A acted globally to orchestrate development into the T lineage and that Id3 antagonized E2A activity beyond the pre-TCR checkpoint to enforce the naive fate of T cells.It is established that E2A and its antagonist, Id3, modulate developmental progression at the pre-TCR receptor (pre-TCR) and TCR checkpoints. Here we demonstrate that Id3 expression is elevated beyond the pre-TCR checkpoint, remains high in naive T cells and shows a bimodal pattern in the effector/memory population. We show how E2A promotes T-lineage specification and how pre-TCR mediated signaling affects E2A genome-wide occupancy. Thymi in Id3-deficient mice exhibited aberrant development of effector/memory cells, increased CXCR5 and Bcl6 expression, T-B cell conjugates and remarkably B cell follicles. Collectively, these data show how E2A acts globally to orchestrate T-lineage development and that Id3 antagonizes E2A activity beyond the pre-TCR checkpoint to enforce the naïve T cell fate.

Positive intergenic feedback circuitry, involving EBF1 and FOXO1, orchestrates B-cell fate

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.

Factors and networks that underpin early hematopoiesis

Multiple trajectories have recently been described through which hematopoietic progenitor cells travel prior to becoming lineage-committed effectors. A wide spectrum of transcription factors has recently been identified that modulate developmental progression along such trajectories. Here we describe how distinct families of transcription factors act and are linked together to orchestrate early hematopoiesis.

The E-Id Protein Axis Specifies Adaptive Lymphoid Cell Identity and Suppresses Thymic Innate Lymphoid Cell Development

Summary Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early T cell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish T cell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid‐tissue‐inducer‐like cells. E2A and HEB orchestrated T cell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, T cell receptor (TCR) assembly, and TCR‐mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T‐cell‐lineage‐specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E‐Id protein axis specifies innate and adaptive lymphoid cell fate. Graphical Abstract Figure. No Caption available. HighlightsE2A and HEB act in concert to specify T cell fateE protein activity in lymphoid progenitors suppresses aberrant ILC developmentE2A and HEB establish a T‐lineage‐specific program of gene expressionThe E‐Id protein axis specifies the adaptive and innate lymphoid cell fate &NA; Previous studies established that E proteins act at multiple stages to promote T‐cell‐lineage development. Miyazaki et al. demonstrate that E proteins establish T cell identity and suppress the development of thymic ILCs by modulating enhancer repertoires of genes associated with Notch signaling and TCR&bgr; locus assembly.

Nuclear location and the control of developmental progression.

It is now well established that the mammalian genome is highly organized. Chromosomes are structured as territories that only sporadically intermingle. Chromosome territories themselves are segregated into distinct environments, that is, the transcriptionally inert/repressive (heterochromatic) and permissive (euchromatic) compartments. The transcriptionally permissive compartment is organized into domains (∼0.5-3 Mb) that consist of bundles of loops, are gene-rich and closely associated by activating epigenetic marks. During ontogeny and developmental progression chromatin states are highly dynamic. Recent studies have shown that loci and domains readily switch compartments. Switching nuclear neighborhoods is closely associated with changes in transcriptional activity and extensive chromatin reorganization. Here we discuss the implications of a dynamic genome and how it relates to the control of developmental progression.