Cooperation between bHLH transcription factors and histones for DNA access.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

MYC determines lineage commitment in KRAS-driven primary liver cancer development.

BACKGROUND & AIMS: Primary liver cancer (PLC) comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), two frequent and lethal tumour types that differ regarding their tumour biology and responses to cancer therapies. Liver cells harbour a high degree of cellular plasticity and can give rise to either HCC or iCCA. However, little is known about the cell-intrinsic mechanisms directing an oncogenically transformed liver cell to either HCC or iCCA. The scope of this study was to identify cell-intrinsic factors determining lineage commitment in PLC. METHODS: Cross-species transcriptomic and epigenetic profiling was applied to murine HCCs and iCCAs and to two human PLC cohorts. Integrative data analysis comprised epigenetic Landscape In Silico deletion Analysis (LISA) of transcriptomic data and Hypergeometric Optimization of Motif EnRichment (HOMER) analysis of chromatin accessibility data. Identified candidate genes were subjected to functional genetic testing in non-germline genetically engineered PLC mouse models (shRNAmir knockdown or overexpression of full-length cDNAs). RESULTS: Integrative bioinformatic analyses of transcriptomic and epigenetic data pinpointed the Forkhead-family transcription factors FOXA1 and FOXA2 as MYC-dependent determination factors of the HCC lineage. Conversely, the ETS family transcription factor ETS1 was identified as a determinant of the iCCA lineage, which was found to be suppressed by MYC during HCC development. Strikingly, shRNA-mediated suppression of FOXA1 and FOXA2 with concomitant ETS1 expression fully switched HCC to iCCA development in PLC mouse models. CONCLUSIONS: The herein reported data establish MYC as a key determinant of lineage commitment in PLC and provide a molecular explanation why common liver-damaging risk factors such as alcoholic or non-alcoholic steatohepatitis can lead to either HCC or iCCA. IMPACT AND IMPLICATIONS: Liver cancer is a major health problem and comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), two frequent and lethal tumour types that differ regarding their morphology, tumour biology, and responses to cancer therapies. We identified the transcription factor and oncogenic master regulator MYC as a switch between HCC and iCCA development. When MYC levels are high at the time point when a hepatocyte becomes a tumour cell, an HCC is growing out. Conversely, if MYC levels are low at this time point, the result is the outgrowth of an iCCA. Our study provides a molecular explanation why common liver-damaging risk factors such as alcoholic or non-alcoholic steatohepatitis can lead to either HCC or iCCA. Furthermore, our data harbour potential for the development of better PLC therapies.

EVI1 drives leukemogenesis through aberrant ERG activation.

Chromosomal rearrangements involving the MDS1 and EVI1 complex locus (MECOM) on chromosome 3q26 define an aggressive subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and dismal prognosis. Established treatment regimens commonly fail in these patients, therefore, there is an urgent need for new therapeutic concepts that will require a better understanding of the molecular and cellular functions of the ecotropic viral integration site 1 (EVI1) oncogene. To characterize gene regulatory functions of EVI1 and associated dependencies in AML, we developed experimentally tractable human and murine disease models, investigated the transcriptional consequences of EVI1 withdrawal in vitro and in vivo, and performed the first genome-wide CRISPR screens in EVI1-dependent AML. By integrating conserved transcriptional targets with genetic dependency data, we identified and characterized the ETS transcription factor ERG as a direct transcriptional target of EVI1 that is aberrantly expressed and selectively required in both human and murine EVI1-driven AML. EVI1 controls the expression of ERG and occupies a conserved intragenic enhancer region in AML cell lines and samples from patients with primary AML. Suppression of ERG induces terminal differentiation of EVI1-driven AML cells, whereas ectopic expression of ERG abrogates their dependence on EVI1, indicating that the major oncogenic functions of EVI1 are mediated through aberrant transcriptional activation of ERG. Interfering with this regulatory axis may provide entry points for the development of rational targeted therapies.

Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphomas worldwide and is characterized by a high diversity of genetic and molecular alterations. Chromosomal translocations and mutations leading to deregulated expression of the transcriptional repressor BCL6 occur in a significant fraction of DLBCL patients. An oncogenic role of BCL6 in the initiation of DLBCL has been shown as the constitutive expression of BCL6 in mice recapitulates the pathogenesis of human DLBCL. However, the role of BCL6 in tumor maintenance remains poorly investigated due to the absence of suitable genetic models and limitations of pharmacological inhibitors. Here, we have utilized tetracycline-inducible CRISPR/Cas9 mutagenesis to study the consequences of BCL6 deletion in established DLBCL models in culture and in vivo. We show that BCL6 knock-out in SU-DHL-4 cells in vitro results in an anti-proliferative response 4-7 days after Cas9 induction that was characterized by cell cycle (G1) arrest. Conditional BCL6 deletion in established DLBCL tumors in vivo induced a significant tumor growth inhibition with initial tumor stasis followed by slow tumor growth kinetics. Our findings support a role of BCL6 in the maintenance of lymphoma growth and showcase the utility of inducible CRISPR/Cas9 systems for probing oncogene addiction.

Precocious expression of Blimp1 in B cells causes autoimmune disease with increased self-reactive plasma cells.

The transcription factor Blimp1 is not only an essential regulator of plasma cells, but also a risk factor for the development of autoimmune disease in humans. Here, we demonstrate in the mouse that the Prdm1 (Blimp1) gene was partially activated at the chromatin and transcription level in early B cell development, although mature Prdm1 mRNA did not accumulate due to posttranscriptional regulation. By analyzing a mouse model that facilitated ectopic Blimp1 protein expression throughout B lymphopoiesis, we could demonstrate that Blimp1 impaired B cell development by interfering with the B cell gene expression program, while leading to an increased abundance of plasma cells by promoting premature plasmablast differentiation of immature and mature B cells. With progressing age, these mice developed an autoimmune disease characterized by the presence of autoantibodies and glomerulonephritis. Hence, these data identified ectopic Blimp1 expression as a novel mechanism, through which Blimp1 can act as a risk factor in the development of autoimmune disease.

CXCR5(+) follicular cytotoxic T cells control viral infection in B cell follicles.

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes.

Tissue-resident memory T (Trm) cells permanently localize to portals of pathogen entry, where they provide immediate protection against reinfection. To enforce tissue retention, Trm cells up-regulate CD69 and down-regulate molecules associated with tissue egress; however, a Trm-specific transcriptional regulator has not been identified. Here, we show that the transcription factor Hobit is specifically up-regulated in Trm cells and, together with related Blimp1, mediates the development of Trm cells in skin, gut, liver, and kidney in mice. The Hobit-Blimp1 transcriptional module is also required for other populations of tissue-resident lymphocytes, including natural killer T (NKT) cells and liver-resident NK cells, all of which share a common transcriptional program. Our results identify Hobit and Blimp1 as central regulators of this universal program that instructs tissue retention in diverse tissue-resident lymphocyte populations.

The Helix-Loop-Helix Protein ID2 Governs NK Cell Fate by Tuning Their Sensitivity to Interleukin-15.

The inhibitor of DNA binding 2 (Id2) is essential for natural killer (NK) cell development with its canonical role being to antagonize E-protein function and alternate lineage fate. Here we have identified a key role for Id2 in regulating interleukin-15 (IL-15) receptor signaling and homeostasis of NK cells by repressing multiple E-protein target genes including Socs3. Id2 deletion in mature NK cells was incompatible with their homeostasis due to impaired IL-15 receptor signaling and metabolic function and this could be rescued by strong IL-15 receptor stimulation or genetic ablation of Socs3. During NK cell maturation, we observed an inverse correlation between E-protein target genes and Id2. These results shift the current paradigm on the role of ID2, indicating that it is required not only to antagonize E-proteins during NK cell commitment, but constantly required to titrate E-protein activity to regulate NK cell fitness and responsiveness to IL-15.

Blimp-1 controls plasma cell function through the regulation of immunoglobulin secretion and the unfolded protein response.

Plasma cell differentiation requires silencing of B cell transcription, while it establishes antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for the generation of plasma cells; however, their function in mature plasma cells has remained elusive. We found that while IRF4 was essential for the survival of plasma cells, Blimp-1 was dispensable for this. Blimp-1-deficient plasma cells retained their transcriptional identity but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap in the functions of Blimp-1 and XBP-1 was restricted to that response, with Blimp-1 uniquely regulating activity of the kinase mTOR and the size of plasma cells. Thus, Blimp-1 was required for the unique physiological ability of plasma cells that enables the secretion of protective antibody.

Multifunctional role of the transcription factor Blimp-1 in coordinating plasma cell differentiation.

The transcription factor Blimp-1 is necessary for the generation of plasma cells. Here we studied its functions in plasmablast differentiation by identifying regulated Blimp-1 target genes. Blimp-1 promoted the migration and adhesion of plasmablasts. It directly repressed genes encoding several transcription factors and Aicda (which encodes the cytidine deaminase AID) and thus silenced B cell-specific gene expression, antigen presentation and class-switch recombination in plasmablasts. It directly activated genes, which led to increased expression of the plasma cell regulator IRF4 and proteins involved in immunoglobulin secretion. Blimp-1 induced the transcription of immunoglobulin genes by controlling the 3' enhancers of the loci encoding the immunoglobulin heavy chain (Igh) and κ-light chain (Igk) and, furthermore, regulated the post-transcriptional expression switch from the membrane-bound form of the immunoglobulin heavy chain to its secreted form by activating Ell2 (which encodes the transcription-elongation factor ELL2). Notably, Blimp-1 recruited chromatin-remodeling and histone-modifying complexes to regulate its target genes. Hence, many essential functions of plasma cells are under the control of Blimp-1.

The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells.

The unfolded protein response (UPR) is a conserved stress-signaling pathway activated after accumulation of unfolded proteins within the endoplasmic reticulum (ER). Active UPR signaling leads to unconventional, enzymatic splicing of XBP1 mRNA enabling expression of the transcription factor XBP1s to control ER homeostasis. While IRE1 has been identified as the endoribonuclease required for cleavage of this mRNA, the corresponding ligase in mammalian cells has remained elusive. Here, we report that RTCB, the catalytic subunit of the tRNA ligase complex, and its co-factor archease mediate XBP1 mRNA splicing both in vitro and in vivo. Depletion of RTCB in plasma cells of Rtcb(fl/fl) Cd23-Cre mice prevents XBP1s expression, which normally is strongly induced during plasma cell development. RTCB-depleted plasma cells show reduced and disorganized ER structures as well as severe defects in antibody secretion. Targeting RTCB and/or archease thus represents a promising strategy for the treatment of a growing number of diseases associated with elevated expression of XBP1s.

Differential requirement for Nfil3 during NK cell development.

NK cells can be grouped into distinct subsets that are localized to different organs and exhibit a different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor Nfil3 (E4bp4) is essential for bone marrow-derived NK cell development, but it is not clear whether Nfil3 is equally important for all NK cell subsets or how it induces NK lineage commitment. In this article, we show that Nfil3 is required for the formation of Eomes-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL(+) Eomes(-) NK cells develop independently of Nfil3. Loss of Nfil3 during the development of bone marrow-derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil3(-/-) progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cells by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3.

Stage-specific control of early B cell development by the transcription factor Ikaros.

The transcription factor Ikaros is an essential regulator of lymphopoiesis. Here we studied its B cell-specific function by conditional inactivation of the gene encoding Ikaros (Ikzf1) in pro-B cells. B cell development was arrested at an aberrant 'pro-B cell' stage characterized by increased cell adhesion and loss of signaling via the pre-B cell signaling complex (pre-BCR). Ikaros activated genes encoding signal transducers of the pre-BCR and repressed genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of expression of the transcription factor Aiolos did not compensate for the loss of Ikaros in pro-B cells. Ikaros induced or suppressed active chromatin at regulatory elements of activated or repressed target genes. Notably, binding of Ikaros and expression of its target genes were dynamically regulated at distinct stages of early B lymphopoiesis.

Id2-mediated inhibition of E2A represses memory CD8+ T cell differentiation.

The transcription factor inhibitor of DNA binding (Id)2 modulates T cell fate decisions, but the molecular mechanism underpinning this regulation is unclear. In this study we show that loss of Id2 cripples effector differentiation and instead programs CD8(+) T cells to adopt a memory fate with increased Eomesodermin and Tcf7 expression. We demonstrate that Id2 restrains CD8(+) T cell memory differentiation by inhibiting E2A-mediated direct activation of Tcf7 and that Id2 expression level mirrors T cell memory recall capacity. As a result of the defective effector differentiation, Id2-deficient CD8(+) T cells fail to induce sufficient Tbx21 expression to generate short-lived effector CD8(+) T cells. Our findings reveal that the Id2/E2A axis orchestrates T cell differentiation through the induction or repression of downstream transcription factors essential for effector and memory T cell differentiation.

Essential role of EBF1 in the generation and function of distinct mature B cell types.

The transcription factor EBF1 is essential for lineage specification in early B cell development. In this study, we demonstrate by conditional mutagenesis that EBF1 is required for B cell commitment, pro-B cell development, and subsequent transition to the pre-B cell stage. Later in B cell development, EBF1 was essential for the generation and maintenance of several mature B cell types. Marginal zone and B-1 B cells were lost, whereas follicular (FO) and germinal center (GC) B cells were reduced in the absence of EBF1. Activation of the B cell receptor resulted in impaired intracellular signaling, proliferation and survival of EBF1-deficient FO B cells. Immune responses were severely reduced upon Ebf1 inactivation, as GCs were formed but not maintained. ChIP- and RNA-sequencing of FO B cells identified EBF1-activated genes that encode receptors, signal transducers, and transcriptional regulators implicated in B cell signaling. Notably, ectopic expression of EBF1 efficiently induced the development of B-1 cells at the expense of conventional B cells. These gain- and loss-of-function analyses uncovered novel important functions of EBF1 in controlling B cell immunity.

The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells.

Regulatory T cells (T(reg) cells) are required for peripheral tolerance. Evidence indicates that T(reg) cells can adopt specialized differentiation programs in the periphery that are controlled by transcription factors usually associated with helper T cell differentiation. Here we demonstrate that expression of the transcription factor Blimp-1 defined a population of T(reg) cells that localized mainly to mucosal sites and produced IL-10. Blimp-1 was required for IL-10 production by these cells and for their tissue homeostasis. We provide evidence that the transcription factor IRF4, but not the transcription factor T-bet, was essential for Blimp-1 expression and for the differentiation of all effector T(reg) cells. Thus, our study defines a differentiation pathway that leads to the acquisition of T(reg) cell effector functions and requires both IRF4 and Blimp-1.