DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.

Genomic Decoding of Neuronal Depolarization by Stimulus-Specific NPAS4 Heterodimers.

Cells regulate gene expression in response to salient external stimuli. In neurons, depolarization leads to the expression of inducible transcription factors (ITFs) that direct subsequent gene regulation. Depolarization encodes both a neuron's action potential (AP) output and synaptic inputs, via excitatory postsynaptic potentials (EPSPs). However, it is unclear if distinct types of electrical activity can be transformed by an ITF into distinct modes of genomic regulation. Here, we show that APs and EPSPs in mouse hippocampal neurons trigger two spatially segregated and molecularly distinct induction mechanisms that lead to the expression of the ITF NPAS4. These two pathways culminate in the formation of stimulus-specific NPAS4 heterodimers that exhibit distinct DNA binding patterns. Thus, NPAS4 differentially communicates increases in a neuron's spiking output and synaptic inputs to the nucleus, enabling gene regulation to be tailored to the type of depolarizing activity along the somato-dendritic axis of a neuron.

Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping.

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.

IL-2 regulates tumor-reactive CD8+ T cell exhaustion by activating the aryl hydrocarbon receptor.

CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion.

Dynamic Ligand Discrimination in the Notch Signaling Pathway.

The Notch signaling pathway comprises multiple ligands that are used in distinct biological contexts. In principle, different ligands could activate distinct target programs in signal-receiving cells, but it is unclear how such ligand discrimination could occur. Here, we show that cells use dynamics to discriminate signaling by the ligands Dll1 and Dll4 through the Notch1 receptor. Quantitative single-cell imaging revealed that Dll1 activates Notch1 in discrete, frequency-modulated pulses that specifically upregulate the Notch target gene Hes1. By contrast, Dll4 activates Notch1 in a sustained, amplitude-modulated manner that predominantly upregulates Hey1 and HeyL. Ectopic expression of Dll1 or Dll4 in chick neural crest produced opposite effects on myogenic differentiation, showing that ligand discrimination can occur in vivo. Finally, analysis of chimeric ligands suggests that ligand-receptor clustering underlies dynamic encoding of ligand identity. The ability of the pathway to utilize ligands as distinct communication channels has implications for diverse Notch-dependent processes.

Fasting-Mimicking Diet Promotes Ngn3-Driven ß-Cell Regeneration to Reverse Diabetes.

Stem-cell-based therapies can potentially reverse organ dysfunction and diseases, but the removal of impaired tissue and activation of a program leading to organ regeneration pose major challenges. In mice, a 4-day fasting mimicking diet (FMD) induces a stepwise expression of Sox17 and Pdx-1, followed by Ngn3-driven generation of insulin-producing ß cells, resembling that observed during pancreatic development. FMD cycles restore insulin secretion and glucose homeostasis in both type 2 and type 1 diabetes mouse models. In human type 1 diabetes pancreatic islets, fasting conditions reduce PKA and mTOR activity and induce Sox2 and Ngn3 expression and insulin production. The effects of the FMD are reversed by IGF-1 treatment and recapitulated by PKA and mTOR inhibition. These results indicate that a FMD promotes the reprogramming of pancreatic cells to restore insulin generation in islets from T1D patients and reverse both T1D and T2D phenotypes in mouse models. PAPERCLIP.

Bhlhe40 mediates tissue-specific control of macrophage proliferation in homeostasis and type 2 immunity.

Most tissue-resident macrophage populations develop during embryogenesis, self-renew in the steady state and expand during type 2 immunity. Whether shared mechanisms regulate the proliferation of macrophages in homeostasis and disease is unclear. Here we found that the transcription factor Bhlhe40 was required in a cell-intrinsic manner for the self-renewal and maintenance of large peritoneal macrophages (LPMs), but not that of other tissue-resident macrophages. Bhlhe40 was necessary for the proliferation, but not the polarization, of LPMs in response to the cytokine IL-4. During infection with the helminth Heligmosomoides polygyrus bakeri, Bhlhe40 was required for cell cycling of LPMs. Bhlhe40 repressed the expression of genes encoding the transcription factors c-Maf and Mafb and directly promoted expression of transcripts encoding cell cycle-related proteins to enable the proliferation of LPMs. In LPMs, Bhlhe40 bound to genomic sites co-bound by the macrophage lineage-determining factor PU.1 and to unique sites, including Maf and loci encoding cell-cycle-related proteins. Our findings demonstrate a tissue-specific control mechanism that regulates the proliferation of resident macrophages in homeostasis and type 2 immunity.

The Importance of Timing.

Building a nervous system requires a precise sequence of genetic transitions, mediated in part by the temporal and spatial regulation of transcription factors. Quan et al. add to our understanding of this regulation by describing an evolutionarily conserved post-translational mechanism that rapidly extinguishes proneural protein activity in neural precursors.

Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.

Wiring and Molecular Features of Prefrontal Ensembles Representing Distinct Experiences.

A major challenge in understanding the cellular diversity of the brain has been linking activity during behavior with standard cellular typology. For example, it has not been possible to determine whether principal neurons in prefrontal cortex active during distinct experiences represent separable cell types, and it is not known whether these differentially active cells exert distinct causal influences on behavior. Here, we develop quantitative hydrogel-based technologies to connect activity in cells reporting on behavioral experience with measures for both brain-wide wiring and molecular phenotype. We find that positive and negative-valence experiences in prefrontal cortex are represented by cell populations that differ in their causal impact on behavior, long-range wiring, and gene expression profiles, with the major discriminant being expression of the adaptation-linked gene NPAS4. These findings illuminate cellular logic of prefrontal cortex information processing and natural adaptive behavior and may point the way to cell-type-specific understanding and treatment of disease-associated states.

Pioneer factor ASCL1 cooperates with the mSWI/SNF complex at distal regulatory elements to regulate human neural differentiation.

Pioneer transcription factors are thought to play pivotal roles in developmental processes by binding nucleosomal DNA to activate gene expression, though mechanisms through which pioneer transcription factors remodel chromatin remain unclear. Here, using single-cell transcriptomics, we show that endogenous expression of neurogenic transcription factor ASCL1, considered a classical pioneer factor, defines a transient population of progenitors in human neural differentiation. Testing ASCL1's pioneer function using a knockout model to define the unbound state, we found that endogenous expression of ASCL1 drives progenitor differentiation by cis-regulation both as a classical pioneer factor and as a nonpioneer remodeler, where ASCL1 binds permissive chromatin to induce chromatin conformation changes. ASCL1 interacts with BAF SWI/SNF chromatin remodeling complexes, primarily at targets where it acts as a nonpioneer factor, and we provide evidence for codependent DNA binding and remodeling at a subset of ASCL1 and SWI/SNF cotargets. Our findings provide new insights into ASCL1 function regulating activation of long-range regulatory elements in human neurogenesis and uncover a novel mechanism of its chromatin remodeling function codependent on partner ATPase activity.

Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.

Neuronal activity causes the rapid expression of immediate early genes that are crucial for experience-driven changes to synapses, learning, and memory. Here, using both molecular and genome-wide next-generation sequencing methods, we report that neuronal activity stimulation triggers the formation of DNA double strand breaks (DSBs) in the promoters of a subset of early-response genes, including Fos, Npas4, and Egr1. Generation of targeted DNA DSBs within Fos and Npas4 promoters is sufficient to induce their expression even in the absence of an external stimulus. Activity-dependent DSB formation is likely mediated by the type II topoisomerase, Topoisomerase IIß (Topo IIß), and knockdown of Topo IIß attenuates both DSB formation and early-response gene expression following neuronal stimulation. Our results suggest that DSB formation is a physiological event that rapidly resolves topological constraints to early-response gene expression in neurons.

The aryl hydrocarbon receptor AhR links atopic dermatitis and air pollution via induction of the neurotrophic factor artemin.

Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.

Essential role for the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells.

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41-/-Bhlhe40-/- B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12Vκ4 BCR. Expression of a pre-rearranged VH12Vκ4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

Npas4 regulates excitatory-inhibitory balance within neural circuits through cell-type-specific gene programs.

The nervous system adapts to experience by inducing a transcriptional program that controls important aspects of synaptic plasticity. Although the molecular mechanisms of experience-dependent plasticity are well characterized in excitatory neurons, the mechanisms that regulate this process in inhibitory neurons are only poorly understood. Here, we describe a transcriptional program that is induced by neuronal activity in inhibitory neurons. We find that, while neuronal activity induces expression of early-response transcription factors such as Npas4 in both excitatory and inhibitory neurons, Npas4 activates distinct programs of late-response genes in inhibitory and excitatory neurons. These late-response genes differentially regulate synaptic input to these two types of neurons, promoting inhibition onto excitatory neurons while inducing excitation onto inhibitory neurons. These findings suggest that the functional outcomes of activity-induced transcriptional responses are adapted in a cell-type-specific manner to achieve a circuit-wide homeostatic response.

Increased adipocyte O2 consumption triggers HIF-1α, causing inflammation and insulin resistance in obesity.

Adipose tissue hypoxia and inflammation have been causally implicated in obesity-induced insulin resistance. Here, we report that, early in the course of high-fat diet (HFD) feeding and obesity, adipocyte respiration becomes uncoupled, leading to increased oxygen consumption and a state of relative adipocyte hypoxia. These events are sufficient to trigger HIF-1α induction, setting off the chronic adipose tissue inflammatory response characteristic of obesity. At the molecular level, these events involve saturated fatty acid stimulation of the adenine nucleotide translocase 2 (ANT2), an inner mitochondrial membrane protein, which leads to the uncoupled respiratory state. Genetic or pharmacologic inhibition of either ANT2 or HIF-1α can prevent or reverse these pathophysiologic events, restoring a state of insulin sensitivity and glucose tolerance. These results reveal the sequential series of events in obesity-induced inflammation and insulin resistance.

ASCL1 represses a SOX9+ neural crest stem-like state in small cell lung cancer.

ASCL1 is a neuroendocrine lineage-specific oncogenic driver of small cell lung cancer (SCLC), highly expressed in a significant fraction of tumors. However, ∼25% of human SCLC are ASCL1-low and associated with low neuroendocrine fate and high MYC expression. Using genetically engineered mouse models (GEMMs), we show that alterations in Rb1/Trp53/Myc in the mouse lung induce an ASCL1+ state of SCLC in multiple cells of origin. Genetic depletion of ASCL1 in MYC-driven SCLC dramatically inhibits tumor initiation and progression to the NEUROD1+ subtype of SCLC. Surprisingly, ASCL1 loss promotes a SOX9+ mesenchymal/neural crest stem-like state and the emergence of osteosarcoma and chondroid tumors, whose propensity is impacted by cell of origin. ASCL1 is critical for expression of key lineage-related transcription factors NKX2-1, FOXA2, and INSM1 and represses genes involved in the Hippo/Wnt/Notch developmental pathways in vivo. Importantly, ASCL1 represses a SOX9/RUNX1/RUNX2 program in vivo and SOX9 expression in human SCLC cells, suggesting a conserved function for ASCL1. Together, in a MYC-driven SCLC model, ASCL1 promotes neuroendocrine fate and represses the emergence of a SOX9+ nonendodermal stem-like fate that resembles neural crest.

Protein neddylation as a therapeutic target in pulmonary and extrapulmonary small cell carcinomas.

Small cell lung carcinoma (SCLC) is among the most lethal of all solid tumor malignancies. In an effort to identify novel therapeutic approaches for this recalcitrant cancer type, we applied genome-scale CRISPR/Cas9 inactivation screens to cell lines that we derived from a murine model of SCLC. SCLC cells were particularly sensitive to the deletion of NEDD8 and other neddylation pathway genes. Genetic suppression or pharmacological inhibition of this pathway using MLN4924 caused cell death not only in mouse SCLC cell lines but also in patient-derived xenograft (PDX) models of pulmonary and extrapulmonary small cell carcinoma treated ex vivo or in vivo. A subset of PDX models were exceptionally sensitive to neddylation inhibition. Neddylation inhibition suppressed expression of major regulators of neuroendocrine cell state such as INSM1 and ASCL1, which a subset of SCLC rely upon for cell proliferation and survival. To identify potential mechanisms of resistance to neddylation inhibition, we performed a genome-scale CRISPR/Cas9 suppressor screen. Deletion of components of the COP9 signalosome strongly mitigated the effects of neddylation inhibition in small cell carcinoma, including the ability of MLN4924 to suppress neuroendocrine transcriptional program expression. This work identifies neddylation as a regulator of neuroendocrine cell state and potential therapeutic target for small cell carcinomas.

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.

Systematic identification and characterization of repressive domains in Drosophila transcription factors.

All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA-binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains (RDs) are typically unknown and the specific co-repressors (CoRs) they recruit have not been determined. Here, we develop a high-throughput, next-generation sequencing-based screening method, repressive-domain (RD)-seq, to systematically identify RDs in complex DNA-fragment libraries. Screening more than 200,000 fragments covering the coding sequences of all transcription-related proteins in Drosophila melanogaster, we identify 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs, which are conserved between fly and human and are required for RD function, as demonstrated by motif mutagenesis. Moreover, we show that RDs that contain one of five distinct repressive motifs interact with and depend on different CoRs, such as Groucho, CtBP, Sin3A, or Smrter. These findings advance our understanding of repressors, their sequences, and the functional impact of sequence-altering mutations and should provide a valuable resource for further studies.