Alternative Cell Fates Research Articles

The comER Gene Plays an Important Role in Biofilm Formation and Sporulation in both Bacillus subtilis and Bacillus cereus.

Bacteria adopt alternative cell fates during development. In Bacillus subtilis, the transition from planktonic growth to biofilm formation and sporulation is controlled by a complex regulatory circuit, in which the most important event is activation of Spo0A, a transcription factor and a master regulator for genes involved in both biofilm formation and sporulation. In B. cereus, the regulatory pathway controlling biofilm formation and cell differentiation is much less clear. In this study, we show that a novel gene, comER, plays a significant role in biofilm formation as well as sporulation in both B. subtilis and B. cereus. Mutations in the comER gene result in defects in biofilm formation and a delay in spore formation in the two Bacillus species. Our evidence supports the idea that comER may be part of the regulatory circuit that controls Spo0A activation. comER likely acts upstream of sda, a gene encoding a small checkpoint protein for both sporulation and biofilm formation, by blocking the phosphor-relay and thereby Spo0A activation. In summary, our studies outlined a conserved, positive role for comER, a gene whose function was previously uncharacterized, in the regulation of biofilm formation and sporulation in the two Bacillus species.

Wnt Protein Signaling Reduces Nuclear Acetyl-CoA Levels to Suppress Gene Expression during Osteoblast Differentiation

Developmental signals in metazoans play critical roles in inducing cell differentiation from multipotent progenitors. The existing paradigm posits that the signals operate directly through their downstream transcription factors to activate expression of cell type-specific genes, which are the hallmark of cell identity. We have investigated the mechanism through which Wnt signaling induces osteoblast differentiation in an osteoblast-adipocyte bipotent progenitor cell line. Unexpectedly, Wnt3a acutely suppresses the expression of a large number of genes while inducing osteoblast differentiation. The suppressed genes include Pparg and Cebpa, which encode adipocyte-specifying transcription factors and suppression of which is sufficient to induce osteoblast differentiation. The large scale gene suppression induced by Wnt3a corresponds to a global decrease in histone acetylation, an epigenetic modification that is associated with gene activation. Mechanistically, Wnt3a does not alter histone acetyltransferase or deacetylase activities but, rather, decreases the level of acetyl-CoA in the nucleus. The Wnt-induced decrease in histone acetylation is independent of β-catenin signaling but, rather, correlates with suppression of glucose metabolism in the tricarboxylic acid cycle. Functionally, preventing histone deacetylation by increasing nucleocytoplasmic acetyl-CoA levels impairs Wnt3a-induced osteoblast differentiation. Thus, Wnt signaling induces osteoblast differentiation in part through histone deacetylation and epigenetic suppression of an alternative cell fate.

TCR signal strength regulates Akt substrate specificity to induce alternate Th and Treg differentiation programs

Abstract T regulatory (Treg) cells are induced following stimulation of naïve CD4 T cells with low dose antigen to an extent that is negatively correlated with signaling via the Akt/mTOR pathway. Strong TCR signals induce high levels of Akt activity that inhibit development of Treg by poorly understood mechanisms. Here, we show that high dose stimulation of T cells results in the phosphorylation of Akt on two regulatory sites, Serine (S) 473 and Threonine (T) 308, whereas low dose stimulation results in only T308 phosphorylation. Mathematical modeling shows that the phosphorylation of Akt on both S473 and T308 is controlled by a feedback loop involving PTEN, mTORC2 and the transcription factor FoxO1 that creates a sharp activation threshold with respect to antigen dose and stimulus duration. Using mass spectrometry to analyze phosphorylated Akt substrates at different levels of stimulation, we find profound differences in the substrates phosphorylated, suggesting that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates. Proteins differentially phosphorylated by these two states of Akt include RNA splicing factors, and we find changes in the splice variant expression levels of key TCR signaling proteins, such as CD3ζ and CD45 that correlate with the observed differences in cell fate. Knockdown of specific splicing factors impacted the ratios of Th versus Treg cells induced. Together, this work demonstrates that alternative splicing can affect the outcome of T cell fate decisions and identifies alternate Akt-mediated signaling networks that drive CD4+ T cell differentiation.

An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma.

Translocation events are frequent in cancer and may create chimeric fusions or ‘regulatory rearrangements’ that drive oncogene overexpression. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps reveal distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in the alternate ACC lineages.

Escargot controls the sequential specification of two tracheal tip cell types by suppressing FGF signaling in Drosophila.

Extrinsic branching factors promote the elongation and migration of tubular organs. In the Drosophila tracheal system, Branchless (Drosophila FGF) stimulates the branching program by specifying tip cells that acquire motility and lead branch migration to a specific destination. Tip cells have two alternative cell fates: the terminal cell (TC), which produces long cytoplasmic extensions with intracellular lumen, and the fusion cell (FC), which mediates branch connections to form tubular networks. How Branchless controls this specification of cells with distinct shapes and behaviors is unknown. Here we report that this cell type diversification involves the modulation of FGF signaling by the zinc-finger protein Escargot (Esg), which is expressed in the FC and is essential for its specification. The dorsal branch begins elongation with a pair of tip cells with high FGF signaling. When the branch tip reaches its final destination, one of the tip cells becomes an FC and expresses Esg. FCs and TCs differ in their response to FGF: TCs are attracted by FGF, whereas FCs are repelled. Esg suppresses ERK signaling in FCs to control this differential migratory behavior.

Constitutive Expression of MEF2C, LYL1, or LMO2 in CD34+ HSCs Blocks the in Vitro Differentiation before the T-Cell Commitment Point and Could be Oncogenic in Early T-Cell Progenitor ALL

Background:T-cell development in the thymus is a complex process that depends on sequential transcriptional and epigenetic events that induce T-cell lineage commitment and simultaneously suppress alternative cell fates. In T-cell acute lymphoblastic leukemia (T-ALL), aberrantly expressed oncogenes result in the arrest of developing thymocytes, which can lead to the acquisition of secondary mutations, uncontrolled proliferation and disease progression. MEF2C is often expressed as a result of chromosomal rearrangements in immature, early T-cell progenitor ALL (ETP-ALL), but is also expressed in normal thymocyte progenitors before T-cell commitment (in the ETP stage). As the only hematopoietic lineage, thymocytes that have passed the T-cell commitment checkpoint (as well as mature T-cells) do no longer express MEF2C.Aims:We aimed to investigate the effect of constitutive MEF2C expression on early T-cell development. OP9-DL1 co-cultures have been most useful for mimicking in vitro T-cell development starting with hematopoietic stem cells (HSCs) derived from human cord blood or bone marrow. We also aimed to investigate the impact of MEF2C in comparison to LYL1 and LMO2; two T-ALL oncogenes also highly expressed at the ETP stage.Methods:We have utilized the OP9-DL1 in vitro co-culture system to gradually differentiate CD34+ HSCs from umbilical cord blood into the T-cell lineage. HSCs in this co-culture will recapitulate in vivo T-cell development as measured by incremental acquisition of surface markers CD7, CD5, CD1a, and reach the CD4, CD8 double-positive (DP) stage. We generated gene expression profiles of 11 subsequent in vitro stages of differentiation to help us match them to in vivo development stages. We investigated in vitro T-cell differentiation of HSCs after lentiviral transduction with MEF2C or control vectors, as well as with other transcriptional regulators LYL1 and LMO2 that are expressed at the ETP stage.Results:The major change in gene expression of subsequent early T-cell differentiation stages defines two distinct T-cell differentiation clusters that correlate with in vivo pre- and post-T-cell commitment profiles. We found that T-cell commitment occurs in CD7+ CD5+ cells before the acquisition of CD1a surface expression. Expression of control vectors in HSCs does not affect the in vitro T-cell differentiation, but MEF2C expression blocks differentiation into the direction of T-cells as measured by the failure of most cells to acquire CD7 as the first marker. Instead, with increased passage number cells gradually lose CD34 expression and eventually disappear from the co-culture. Similar effects were observed for the expression of LYL1 and LMO2; LYL1 expression arrests the cells at the most immature CD7+ ETP stage and prevents the transition towards CD7+ CD5+ cells, whereas LMO2 expressing cells reach the CD7+ CD5+ stage but fail to acquire CD1a as a marker of T-cell commitment.Summary/Conclusion:The gene expression profiles of 11 human in vitro T-cell differentiation subsets has enabled us to pinpoint T-cell commitment to a stage in which cells have acquired CD7 and CD5, just prior to the acquisition of CD1a. MEF2C, LYL1, and LMO2, expressed in ETP-ALL as well as in normal thymocyte progenitors, do not allow the transition to T-cell commitment when constitutively expressed. These proteins each result in the arrest of in vitro differentiating T-cells at different ETP stages, all before the T-cell commitment as marked by CD1a expression. Constitutive expression of MEF2C, LYL1, or LMO2 in very early thymocyte progenitors is incompatible with development into and beyond the T-cell commitment checkpoint and these proteins could therefore play important roles in the pathogenesis of ETP-ALL. DisclosuresNo relevant conflicts of interest to declare.

An Orthologous Epigenetic Gene Expression Signature Derived from Differentiating Embryonic Stem Cells Identifies Regulators of Cardiogenesis.

Here we used predictive gene expression signatures within a multi-species framework to identify the genes that underlie cardiac cell fate decisions in differentiating embryonic stem cells. We show that the overlapping orthologous mouse and human genes are the most accurate candidate cardiogenic genes as these genes identified the most conserved developmental pathways that characterize the cardiac lineage. An RNAi-based screen of the candidate genes in Drosophila uncovered numerous novel cardiogenic genes. shRNA knockdown combined with transcriptome profiling of the newly-identified transcription factors zinc finger protein 503 and zinc finger E-box binding homeobox 2 and the well-known cardiac regulatory factor NK2 homeobox 5 revealed that zinc finger E-box binding homeobox 2 activates terminal differentiation genes required for cardiomyocyte structure and function whereas zinc finger protein 503 and NK2 homeobox 5 are required for specification of the cardiac lineage. We further demonstrated that an essential role of NK2 homeobox 5 and zinc finger protein 503 in specification of the cardiac lineage is the repression of gene expression programs characteristic of alternative cell fates. Collectively, these results show that orthologous gene expression signatures can be used to identify conserved cardiogenic pathways.

MicroRNAs Provide Feedback Regulation of Epithelial-Mesenchymal Transition Induced by Growth Factors.

As regulators in gene expression, microRNAs take part in most biological processes including cell differentiation, apoptosis, cell cycle, and epithelial-to-mesenchymal transition (EMT). In order to evaluate their roles in EMT process, microRNA expression profile changes induced by EGF or TGF-β treatment on nasopharyngeal carcinoma cell HK-1 were analyzed, and miR-21, miR-148a, miR-505, and miR-1207-5p were found to be upregulated in growth factors-induced EMT process. miR-21 is already known as an oncogenic miRNA to promote metastasis, however, the exact functions of other three miRNAs in EMT are unclear. To our surprise, we found that miR-148a, miR-505, and miR-1207-5p can suppress EMT and metastasis phenotypes in HK-1 cells both in vitro and in vivo, which may relate to their inhibition on EMT and Wnt signaling molecules. MiRNAs confer robustness to biological processes by posttranscriptional repression of key transcriptional programs that are related to previous developmental stages or to alternative cell fates. Our findings indicate that miRNA feedback circuit is tuned to respond to growth factors-induced EMT, and we suggested a new negative feedback loop which may be an important element of the EMT process and confer biological robustness.

BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms (LYM8P.628)

Abstract Generation of long term humoral immunity is a complex process predominantly dependent on germinal centers and CD4 T cell help to B cells. Follicular helper T cells (Tfh) are the specialized CD4 T cells for B cell help. We and others have now resolved many of the stages of Tfh differentiation, and molecules involved. BCL6 is the defining transcription factor of Tfh cells. However, the functions of BCL6 in Tfh have largely remained unclear. We have defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. BCL6 primarily acts as a repressor in Tfh cells, and BCL6 binding was associated with control of Tfh cell migration, Tfh differentiation, and repression of alternative cell fates. Interestingly, although some BCL6 bound genes possessed BCL6 DNA binding motifs, more BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and AP1 and BCL6 co-occupied BCL6 binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology, and provide insight into how this master regulator mediates distinct cell-context dependent phenotypes.

BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms.

Follicular helper T cells (Tfh cells) are required for T cell help to B cells, and BCL6 is the defining transcription factor of Tfh cells. However, the functions of BCL6 in Tfh cells have largely remained unclear. Here we defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. BCL6 primarily acts as a repressor in Tfh cells, and BCL6 binding was associated with control of Tfh cell migration and repression of alternative cell fates. Interestingly, although some BCL6-bound genes possessed BCL6 DNA-binding motifs, many BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and BCL6 depends on AP1 for recruitment to BCL6-binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology and provide insight into how this master regulator mediates distinct cell context-dependent phenotypes.

Bifurcation dynamics and determination of alternate cell fates in bipotent progenitor cells.

The gene regulatory networks in which two lineage-affiliated transcription factors, such as GATA1 and PU.1, inhibit each other but activate themselves so as to regulate the choice between alternative cell fates have been extensively studied. These simple networks can generate bistability and explain the transitions between the alternative cell fates. The commitment of a progenitor cell to a new fate corresponds to the occurrence of different types of bifurcations, depending on if a system is symmetrical and how perturbations affect the system. Here we take a general modeling and analyzing approach and show that the lateral inhibition with symmetry and asymmetry can lead to different bifurcation dynamics. Especially, if cell fate decision-making is initiated with asymmetry or symmetry-breaking perturbations, a progenitor cell pre-patterns itself into a polarized cell, depending on the asymmetry or symmetry-breaking perturbations. This study may help us understand the fundamental features of binary cell fate decisions more clearly and further apply to a wider range of decision-making processes.

FOXF1 inhibits hematopoietic lineage commitment during early mesoderm specification.

The molecular mechanisms orchestrating early mesoderm specification are still poorly understood. In particular, how alternate cell fate decisions are regulated in nascent mesoderm remains mostly unknown. In the present study, we investigated both in vitro in differentiating embryonic stem cells, and in vivo in gastrulating embryos, the lineage specification of early mesodermal precursors expressing or not the Forkhead transcription factor FOXF1. Our data revealed that FOXF1-expressing mesoderm is derived from FLK1(+) progenitors and that in vitro this transcription factor is expressed in smooth muscle and transiently in endothelial lineages, but not in hematopoietic cells. In gastrulating embryos, FOXF1 marks most extra-embryonic mesoderm derivatives including the chorion, the allantois, the amnion and a subset of endothelial cells. Similarly to the in vitro situation, FOXF1 expression is excluded from the blood islands and blood cells. Further analysis revealed an inverse correlation between hematopoietic potential and FOXF1 expression in vivo with increased commitment toward primitive erythropoiesis in Foxf1-deficient embryos, whereas FOXF1-enforced expression in vitro was shown to repress hematopoiesis. Altogether, our data establish that during gastrulation, FOXF1 marks all posterior primitive streak extra-embryonic mesoderm derivatives with the remarkable exception of the blood lineage. Our study further suggests that this transcription factor is implicated in actively restraining the specification of mesodermal progenitors to hematopoiesis.

Epigenetic/Genetic Mismatch: Using Transdifferentiation as a Potential Cancer Therapy to Exploit the Cell Type Specificity of Cancer

Every cell type capable of proliferation can be malignantly transformed. However, there appears to be no naturally occurring universal set of genetic mutations capable of converting every cell type to a malignant state. Any specific cell type is generally resistant to transformation by the cancer mutations accumulated by cells of different lineages, presumably due to epigenetic differences. Evidence for this idea derives from experiments in which the developmental fates of cancer cells are altered to reduce malignancy. Reprogramming cancer cells to more primitive developmental states using pluripotency factors (IPS) or somatic nuclear transfer suppresses the malignant phenotype, as does subsequent directed differentiation to mature cells of lineages distinct from the originating cell. Direct transdifferentiation to an alternative cell fate also reduces tumorigenicity. In contrast, after reprogramming, cells induced to redifferentiate toward the original tumor cell type are tumorigenic. In these types of experiments an epigenetic/genetic mismatch often results in suppression of malignancy or cell death. Elucidating the specific transcription and cell signaling network incompatibilities will identify new targets for cancer therapy. Moreover, novel strategies to induce an incompatible transdifferentiated state, in which expression of thousands of genes are altered, will prove useful in controlling malignancies that otherwise easily evolve resistance to single target-based therapeutics. Engineering small molecules, genetic vectors, cytokines, growth factors, targeted extracellular vesicles, and cell fusion will help realize transdifferentiation-based therapeutics for cancer.

Mirn23a Inhibits B Lymphopoiesis through Antagonism of the B Cell Transcription Factor Network

Transcription factors (TFs) regulate genetic networks to direct the differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature blood lineages. Historically studies have focused on identifying genes activated by TFs that act as master hematopoietic regulators. Recently, it has become appreciated that these regulatory TFs direct one distinct differentiation program while simultaneously directing the repression of genes associated with alternative blood lineages. MicroRNAs (miRNAs) are short (~22nt) non-coding RNA molecules that negatively regulate gene expression at the post-transcriptional level and are hypothesized to act downstream of lineage specific TFs to contribute to the silencing of genes associated with alternative cell fate adoption. MiRNAs are observed to direct cell fate choice in assays utilizing overexpression and knockdown approaches; however, experiments from mouse gene targeting have yet to confirm these results. Specifically no miRNA has been shown to direct the differentiation of multipotent progenitors (MPPs) to granulocyte monocyte progenitors (GMPs) or common lymphoid progenitors (CLPs). We previously observed that overexpression of the mirn23a gene (Codes for three mature miRNAs: miR-23a, miR-24-2, and miR-27a) in hematopoietic progenitors increased myelopoiesis at the expense of B lymphopoiesis both in vitro and in vivo. To follow up this observation we generated a mirn23a germline knockout mouse. Flow cytometry characterization of hematopoiesis in mirn23a-/- mice reveals a significant increase in B220+ B-lymphocytes in both the bone marrow (BM) and the spleen. Furthermore, this increase in B-lymphocytes was shown to be at the expense of myeloid cells, as CD11b+ cells were significantly decreased in both the BM and spleen. Analysis of the BM progenitor populations revealed a significant increase in the CLP population, due to an increase in the B-cell specific lymphoid progenitor (BLP, CLP+ Ly6D+) CLP subset. Consistent with decreased mature myeloid cells, we also observed a decrease in BM GMPs. The phenotype appears to be cell intrinsic as ex vivo culture of mirn23a-/- progenitor cells on OP9 stroma results in a significant increase in B220+ B-cells, and a decrease in CD11b+ myeloid cells compared to wild type cultures. Consistent with mirn23a promoting myeloid development, we observe that expression of mirn23a in 70z/3 pre-B cells increases myeloid specific gene expression programs. The upregulation of the myeloid genes may be due to the insufficient repression by B cell transcription factors Ebf1 and Pax5, which are downregulated in mirn23a expressing cells. Interestingly Ebf1 associates with the mirn23a promoter as shown by chromatin immunoprecipitation (ChIP) assay, and Ebf1 represses mirn23a promoter activity in luciferase reporter assays. Consistent with a role in repressing mirn23a, Ebf1 knockdown in A20 B Lymphoma cells increases miR-23a expression while overexpression of Ebf1 in NIH/3t3 cells decreases miR-23a expression. Previously we observed that the myeloid transcription factor PU.1 binds to the mirn23a promoter to positively regulate transcription. Additionally we observe that myeloid transcription factor C/EBP alpha associates with the mirn23a locus in ChIP assays. Data from our knockout mouse supports a critical role for mirn23a in determining immune cells fates. Our current results support the conclusion that myeloid transcription factors PU.1 and C/EBP alpha activate mirn23a in order to antagonize B cell gene regulation and promote myeloid cell fate adoption, whereas the essential B cell transcription factor Ebf1 represses mirn23a expression to avoid this repression during B cell specification. DisclosuresNo relevant conflicts of interest to declare.

Tumor Suppressors Btg1 and Btg2 Regulate B Lineage Commitment through Modulation of Ebf1 Activity

In B cell neoplasia, many transcription factors known to be involved in B cell differentiation and commitment, like E2A, EBF1 and PAX5, are frequently targeted by focal deletions, mutations or chromosomal aberrations. Recent studies have shown that the human genes BTG1 and BTG2 are commonly affected by gene alterations in different B cell malignancies, but their role in normal B cell development has not been established. BTG1 and BTG2 can act as transcriptional cofactors through recruitment of the protein arginine N-methyltransferase PRMT1, which mediates arginine methylation of transcription factors, like RUNX1, and on histone 4 arginine 3 (H4R3). Here we report that Btg1 and Btg2 display unique and overlapping functions during mouse B cell development. We observed a reduction in the fraction of B220+ progenitor cells in the bone marrow compartment of the different knockout animals, ranging from a 10% decrease in the Btg2-/-, 20% in Btg1-/- , and 40% in the Btg1-/-;Btg2-/- mice relative to wild-type controls. Deficiency for Btg1, but not Btg2, resulted in reduced outgrowth of IL-7 dependent lymphoid progenitors in methylcellulose, which correlated with a higher fraction of apoptotic cells. Btg2-/- mice showed impaired differentiation at the pre-pro-B, pro-B and pre-B cell stage, while Btg1-deficiency mainly affected later stages of B cell differentiation with reduced numbers of immature B cells. Btg1-/-;Btg2-/- mice displayed additive effects with more significant reduction of B220+ cells predominantly at the pre-B and immature B cell stage. Expression analysis revealed no reduction in the mRNA levels of master regulators E2a, Foxo1, Ebf1 and Pax5 in the absence of Btg1 and Btg2. However, higher expression levels of T cell-specific genes were observed in Btg1-/-;Btg2-/- progenitor B cells, e.g. Cd4, Ikzf2 and Tcf7 (Figure 1), some of which are known to be transcriptional repressed by Ebf1, such as Id2, Gata3, Dtx3l and Notch1. Flow cytometric analyses confirmed increased expression of CD3, CD4 and CD8 markers on CD19+ bone marrow cells lacking Btg1 and Btg2 function. Additionally, we detected enhanced levels of DC, NK and myeloid markers on Btg1-/-;Btg2-/- CD19+ BM cells, indicating that Btg1 and Btg2 repress alternative cell fates during B cell lineage specification, and are required for the maintenance of B cell identity. Biochemical studies showed evidence for a physical association between Ebf1, Btg1/Btg2 and PRMT1. We propose a model in which Btg1 and Btg2 affect the function of the critical B cell transcription factor Ebf1 by recruitment of PRMT1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BCL6 Orchestrates Tfh Differentiation Via Multiple Distinct Mechanisms

Follicular Helper CD4 T Cells (Tfh) cells are required for T cell help to B cells and are essential for germinal centers and the development of most high affinity antibody responses that are a hallmark of protective immunity. BCL6 is the defining transcription factor of Tfh cells. However, the functions of Bcl6 in Tfh have largely remained unclear. Intriguingly, Bcl6 is essential in both Tfh cells and germinal center B cells, two cells with very different functions that exist in the same place at the same time and interact extensively. How Bcl6 accomplishes different tasks in these two different cell types has remained unknown. We defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. Bcl6 binds over 3,000 genes in GC Tfh cells and predominantly occupies promoters, but also enhancers. In these cells BCL6 primarily acts as a repressor and BCL6 binding was associated with control of Tfh cell migration, TCR signaling, and repression of alternative cell fates. Interestingly, although some BCL6 bound genes possessed BCL6 DNA binding motifs, many BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and AP1 and BCL6 co-occupied BCL6 binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology, and provide insight into how this master regulator mediates distinct cell-context dependent phenotypes. DisclosuresMelnick:Genentech: Speakers Bureau; Janssen: Research Funding; Calgene: Consultancy; Bioreference: Scientific Advisory Board, Scientific Advisory Board Other.

Somatic transcriptome priming gates lineage-specific differentiation potential of human-induced pluripotent stem cell states.

Human-induced pluripotent stem cells (hiPSCs) provide an invaluable source for regenerative medicine, but are limited by proficient lineage-specific differentiation. Here we reveal that hiPSCs derived from human fibroblasts (Fibs) versus human cord blood (CB) exhibit indistinguishable pluripotency, but harbour biased propensities for differentiation. Genes associated with germ layer specification were identical in Fib- or CB-derived iPSCs, whereas lineage-specific marks emerge upon differentiation induction of hiPSCs that were correlated to the cell of origin. Differentiation propensities come at the expense of other lineages and cannot be overcome with stimuli for alternative cell fates. Although incomplete DNA methylation and distinct histone modifications of lineage-specific loci correlate to lineage-specific transcriptome priming, transitioning hiPSCs into naive state of pluripotency removes iPSC-memorized transcriptome. Upon re-entry to the primed state, transcriptome memory is restored, indicating a human-specific phenomenon whereby lineage gated developmental potential is not permanently erased, but can be modulated by the pluripotent state.

Noncanonical Mode of ERK Action Controls Alternative αβ and γδ T Cell Lineage Fates

Gradations in extracellular regulated kinase (ERK) signaling have been implicated in essentially every developmental checkpoint or differentiation process encountered by lymphocytes. Yet, despite intensive effort, the molecular basis by which differences in ERK activation specify alternative cell fates remains poorly understood. We report here that differential ERK signaling controls lymphoid-fate specification through an alternative mode of action. While ERK phosphorylates most substrates, such as RSK, by targeting them through its D-domain, this well-studied mode of ERK action was dispensable for development of γδ T cells. Instead, development of γδ T cells was dependent upon an alternative mode of action mediated by the DEF-binding pocket (DBP) of ERK. This domain enabled ERK to bind a distinct and select set of proteins required for specification of the γδ fate. These data provide the first in vivo demonstration for the role of DBP-mediated interactions in orchestrating alternate ERK-dependent developmental outcomes.

A Negative Feedback Loop of Transcription Factors Specifies Alternative Dendritic Cell Chromatin States

During hematopoiesis, cells originating from the same stem cell reservoir differentiate into distinct cell types. The mechanisms enabling common progenitors to differentiate into alternative cell fates are not fully understood. Here, we identify cell-fate-determining transcription factors (TFs) governing dendritic cell (DC) development by annotating the enhancer landscapes of the DC lineage. Combining these analyses with detailed overexpression, knockdown, and ChIP-Seq studies, we show that Irf8 functions as a plasmacytoid DC epigenetic and fate-determining TF, regulating massive, cell-specific chromatin changes in thousands of pDC enhancers. Importantly, Irf8 forms a negative feedback loop with Cebpb, a monocyte-derived DC epigenetic fate-determining TF. We show that using this circuit logic, a pulse of TF expression can stably define epigenetic and transcriptional states, regardless of the microenvironment. More broadly, our study proposes a general paradigm that allows closely related cells with a similar set of signal-dependent factors to generate differential and persistent enhancer landscapes.

Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution.

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes.