Transcription is a stochastic process occurring mostly in episodic bursts. Although the local chromatin environment is known to influence the bursting behavior on long timescales, the impact of transcription factors (TFs)--especially in rapidly inducible systems--is largely unknown. Using fluorescence in situ hybridization and computational models, we quantified the transcriptional activity of the proto-oncogene c-Fos with single mRNA accuracy at individual endogenous alleles. We showed that, during MAPK induction, the TF concentration modulates the burst frequency of c-Fos, whereas other bursting parameters remain mostly unchanged. By using synthetic TFs with TALE DNA-binding domains, we systematically altered different aspects of these bursts. Specifically, we linked the polymerase initiation frequency to the strength of the transactivation domain and the burst duration to the TF lifetime on the promoter. Our results show how TFs and promoter binding domains collectively act to regulate different bursting parameters, offering a vast, evolutionarily tunable regulatory range for individual genes.

While squamous transdifferentiation within subpopulations of adenocarcinomas represents an important drug resistance problem, its underlying mechanism remains poorly understood. Here, using surface markers of resistant basal cell carcinomas (BCCs) and patient single-cell and bulk transcriptomic data, we uncover the dynamic roadmap of basal to squamous cell carcinoma transition (BST). Experimentally induced BST identifies activator protein 1 (AP-1) family members in regulating tumor plasticity, and we show that c-FOS plays a central role in BST by regulating the accessibility of distinct AP-1 regulatory elements. Remarkably, despite prominent changes in cell morphology and BST marker expression, we show using inducible model systems that c-FOS-mediated BST demonstrates reversibility. Blocking EGFR pathway activation after c-FOS induction partially reverts BST in vitro and prevents BST features in both mouse models and human tumors. Thus, by identifying the molecular basis of BST, our work reveals a therapeutic opportunity targeting plasticity as a mechanism of tumor resistance.

Muscle satellite cells (SCs) are a quiescent (non-proliferative) stem cell population in uninjured skeletal muscle. Although SCs have been investigated for nearly 60 years, the molecular drivers that transform quiescent SCs into the rapidly dividing (activated) stem/progenitor cells that mediate muscle repair after injury remain largely unknown. Here we identify a prominent FBJ osteosarcoma oncogene (Fos) mRNA and protein signature in recently activated SCs that is rapidly, heterogeneously, and transiently induced by muscle damage. We further reveal a requirement for FOS to efficiently initiate key stem cell functions, including cell cycle entry, proliferative expansion, and muscle regeneration, via induction of "pro-regenerative" target genes that stimulate cell migration, division, and differentiation. Disruption of one of these Fos/AP-1 targets, NAD(+)-consuming mono-ADP-ribosyl-transferase 1 (Art1), in SCs delays cell cycle entry and impedes progenitor cell expansion and muscle regeneration. This work uncovers an early-activated FOS/ART1/mono-ADP-ribosylation (MARylation) pathway that is essential for stem cell-regenerative responses.

Immunological homeostasis in T cells is maintained by a tightly regulated signaling and transcriptional network. Full engagement of effector T cells occurs only when signaling exceeds a critical threshold that enables induction of immune response genes carrying an epigenetic memory of prior activation. Here we investigate the underlying mechanisms causing the suppression of normal immune responses when T cells are rendered anergic by tolerance induction. By performing an integrated analysis of signaling, epigenetic modifications, and gene expression, we demonstrate that immunological tolerance is established when both signaling to and chromatin priming of immune response genes are weakened. In parallel, chromatin priming of immune-repressive genes becomes boosted, rendering them sensitive to low levels of signaling below the threshold needed to activate immune response genes. Our study reveals how repeated exposure to antigens causes an altered epigenetic state leading to T cell anergy and tolerance, representing a basis for treating auto-immune diseases.

Neuronal activity-inducible gene transcription correlates with rapid and transient increases in histone acetylation at promoters and enhancers of activity-regulated genes. Exactly how histone acetylation modulates transcription of these genes has remained unknown. We used single-cell in situ transcriptional analysis to show that Fos and Npas4 are transcribed in stochastic bursts in mouse neurons and that membrane depolarization increases mRNA expression by increasing burst frequency. We then expressed dCas9-p300 or dCas9-HDAC8 fusion proteins to mimic or block activity-induced histone acetylation locally at enhancers. Adding histone acetylation increased Fos transcription by prolonging burst duration and resulted in higher Fos protein levels and an elevation of resting membrane potential. Inhibiting histone acetylation reduced Fos transcription by reducing burst frequency and impaired experience-dependent Fos protein induction in the hippocampus in vivo. Thus, activity-inducible histone acetylation tunes the transcriptional dynamics of experience-regulated genes to affect selective changes in neuronal gene expression and cellular function.

Sensory inputs activate sparse neuronal ensembles in the dentate gyrus of the hippocampus, but how eligibility of individual neurons to recruitment is determined remains elusive. We identify thousands of largely bistable (CpG methylated or unmethylated) regions within neuronal gene bodies, established during mouse dentate gyrus development. Reducing DNA methylation and the proportion of the methylated epialleles at bistable regions compromises novel context-induced neuronal activation. Conversely, increasing methylation and the frequency of the methylated epialleles at bistable regions enhances intrinsic excitability. Single-nucleus profiling reveals enrichment of specific epialleles related to a subset of primarily exonic, bistable regions in activated neurons. Genes displaying both differential methylation and expression in activated neurons define a network of proteins regulating neuronal excitability and structural plasticity. We propose a model in which bistable regions create neuron heterogeneity and constellations of exonic methylation, which may contribute to cell-specific gene expression, excitability, and eligibility to a coding ensemble.

Aversive stimuli can impact motivation and support associative learning as reinforcers. However, the neural circuitry underlying the processing of aversive reinforcers has not been elucidated. Here, we report that a subpopulation of central amygdala (CeA) GABAergic neurons expressing protein kinase C-delta (PKC-δ+) displays robust responses to aversive stimuli during negative reinforcement learning. Importantly, projections from PKC-δ+ neurons of the CeA to the substantia innominata (SI) could bi-directionally modulate negative reinforcement learning. Moreover, consistent with the idea that SI-projecting PKC-δ+ neurons of the CeA encode aversive information, optogenetic activation of this pathway produces conditioned place aversion, a behavior prevented by simultaneous ablating of SI glutamatergic neurons. Taken together, our data define a cell-type-specific neural circuitry modulating associative learning by encoding aversive reinforcement signals.

Anorexia nervosa (AN) is a serious psychiatric disease, but the neural mechanisms underlying its development are unclear. A subpopulation of amygdala neurons, marked by expression of protein kinase C-delta (PKC-δ), has previously been shown to regulate diverse anorexigenic signals. Here, we demonstrate that these neurons regulate development of activity-based anorexia (ABA), a common animal model for AN. PKC-δ neurons are located in two nuclei of the central extended amygdala (EAc): the central nucleus (CeA) and oval region of the bed nucleus of the stria terminalis (ovBNST). Simultaneous ablation of CeAPKC-δ and ovBNSTPKC-δ neurons prevents ABA, but ablating PKC-δ neurons in the CeA or ovBNST alone is not sufficient. Correspondingly, PKC-δ neurons in both nuclei show increased activity with ABA development. Our study shows how neurons in the amygdala regulate ABA by impacting both feeding and wheel activity behaviors and support a complex heterogeneous etiology of AN.

Spatiotemporal regulation of gene expression depends on the cooperation of multiple mechanisms, including the functional interaction of promoters with distally located enhancers. Here, we show that, in cortical neurons, a subset of short interspersed nuclear elements (SINEs) located in the proximity of activity-regulated genes bears features of enhancers. Enhancer SINEs (eSINEs) recruit the Pol III cofactor complex TFIIIC in a stimulus-dependent manner and are transcribed by Pol III in response to neuronal depolarization. Characterization of an eSINE located in proximity to the Fos gene (FosRSINE1) indicated that the FosRSINE1-encoded transcript interacts with Pol II at the Fos promoter and mediates Fos relocation to Pol II factories, providing an unprecedented molecular link between Pol III and Pol II transcription. Strikingly, knockdown of the FosRSINE1 transcript induces defects of both cortical radial migration in vivo and activity-dependent dendritogenesis in vitro, demonstrating that FosRSINE1 acts as a strong enhancer of Fos expression in diverse physiological contexts.

Understanding how brain activation mediates behaviors is a central goal of systems neuroscience. Here, we apply an automated method for mapping brain activation in the mouse in order to probe how sex-specific social behaviors are represented in the male brain. Our method uses the immediate-early-gene c-fos, a marker of neuronal activation, visualized by serial two-photon tomography: the c-fos-GFP+ neurons are computationally detected, their distribution is registered to a reference brain and a brain atlas, and their numbers are analyzed by statistical tests. Our results reveal distinct and shared female and male interaction-evoked patterns of male brain activation representing sex discrimination and social recognition. We also identify brain regions whose degree of activity correlates to specific features of social behaviors and estimate the total numbers and the densities of activated neurons per brain areas. Our study opens the door to automated screening of behavior-evoked brain activation in the mouse.

Here, we use optogenetics and chemogenetics to investigate the contribution of the paraventricular thalamus (PVT) to nucleus accumbens (NAc) pathway in aversion and heroin relapse in two different heroin self-administration models in rats. In one model, rats undergo forced abstinence in the home cage prior to relapse testing, and in the other, they undergo extinction training, a procedure that is likened to cognitive behavioral therapy. We find that the PVT→NAc pathway is both sufficient and necessary to drive aversion and heroin seeking after abstinence, but not extinction. The ability of extinction to reduce this pathway's contribution to heroin relapse is accompanied by a loss of synaptic plasticity in PVT inputs onto a specific subset of NAc neurons. Thus, extinction may exert therapeutic reductions in opioid seeking by altering synaptic plasticity within the PVT→NAc pathway, resulting in reduced aversion during opioid withdrawal as well as reduced relapse propensity.

Sleep regulation involves interdependent signaling among specialized neurons in distributed brain regions. Although acetylcholine promotes wakefulness and rapid eye movement (REM) sleep, it is unclear whether the cholinergic pathway is essential (i.e., absolutely required) for REM sleep because of redundancy from neural circuits to molecules. First, we demonstrate that synaptic inhibition of TrkA+ cholinergic neurons causes a severe short-sleep phenotype and that sleep reduction is mostly attributable to a shortened sleep duration in the dark phase. Subsequent comprehensive knockout of acetylcholine receptor genes by the triple-target CRISPR method reveals that a similar short-sleep phenotype appears in the knockout of two Gq-type acetylcholine receptors Chrm1 and Chrm3. Strikingly, Chrm1 and Chrm3 double knockout chronically diminishes REM sleep to an almost undetectable level. These results suggest that muscarinic acetylcholine receptors, Chrm1 and Chrm3, are essential for REM sleep.

Accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.

Intermittent fasting is one of the most effective dietary restriction regimens that extend life span in C. elegans and mammals. Fasting-stimulus responses are key to the longevity response; however, the mechanisms that sense and transduce the fasting stimulus remain largely unknown. Through a comprehensive transcriptome analysis in C. elegans, we find that along with the FOXO transcription factor DAF-16, AP-1 (JUN-1/FOS-1) plays a central role in fasting-induced transcriptional changes. KGB-1, one of the C. elegans JNKs, acts as an activator of AP-1 and is activated in response to fasting. KGB-1 and AP-1 are involved in intermittent fasting-induced longevity. Fasting-induced upregulation of the components of the SCF E3 ubiquitin ligase complex via AP-1 and DAF-16 enhances protein ubiquitination and reduces protein carbonylation. Our results thus identify a fasting-responsive KGB-1/AP-1 signaling pathway, which, together with DAF-16, causes transcriptional changes that mediate longevity, partly through regulating proteostasis.

Maintaining an appropriate balance between excitation and inhibition is critical for neuronal information processing. Cortical neurons can cell-autonomously adjust the inhibition they receive to individual levels of excitatory input, but the underlying mechanisms are unclear. We describe that Ste20-like kinase (SLK) mediates cell-autonomous regulation of excitation-inhibition balance in the thalamocortical feedforward circuit, but not in the feedback circuit. This effect is due to regulation of inhibition originating from parvalbumin-expressing interneurons, while inhibition via somatostatin-expressing interneurons is unaffected. Computational modeling shows that this mechanism promotes stable excitatory-inhibitory ratios across pyramidal cells and ensures robust and sparse coding. Patch-clamp RNA sequencing yields genes differentially regulated by SLK knockdown, as well as genes associated with excitation-inhibition balance participating in transsynaptic communication and cytoskeletal dynamics. These data identify a mechanism for cell-autonomous regulation of a specific inhibitory circuit that is critical to ensure that a majority of cortical pyramidal cells participate in information coding.

Schlafen 11 (SLFN11) was recently discovered as a cellular restriction factor against replication stress. Here, we show that SLFN11 increases chromatin accessibility genome wide, prominently at active promoters in response to replication stress induced by the checkpoint kinase 1 (CHK1) inhibitor prexasertib or the topoisomerase I (TOP1) inhibitor camptothecin. Concomitantly, SLFN11 selectively activates cellular stress response pathways by inducing the transcription of the immediate early genes (IEGs), including JUN, FOS, EGR1, NFKB2, and ATF3, together with the cell cycle arrest genes CDKN1A (p21WAF1) and GADD45. Both chromatin remodeling and IEG activation require the putative ATPase and helicase activity of SLFN11, whereas canonical extrinsic IEG activation is SLFN11 independent. SLFN11-dependent IEG activation by camptothecin is also observed across 55 non-isogenic NCI-60 cell lines. We conclude that SLFN11 acts as a global regulator of chromatin structure and an intrinsic IEG activator with the potential to engage the innate immune activation in response to replicative stress.

Genome-wide association studies (GWASs) have identified hundreds of loci associated with psychiatric diseases, yet there is a lack of understanding of disease pathophysiology. Common risk variants can shed light on the underlying molecular mechanisms; however, identifying causal variants remains challenging. We map cis-regulatory elements in human neurons derived from pluripotent stem cells. This system allows us to determine enhancers that activate the transcription of neuronal activity-regulated gene programs, which are thought to be critical for synaptic plasticity and are not possible to identify from postmortem tissues. Using the activity-by-contact model, we create variant-to-gene maps to interpret the function of GWAS variants. Our work nominates a subset of variants to elucidate the molecular mechanisms involving GWAS-significant loci. It also highlights that in vitro human cellular models are a powerful platform for identifying and mechanistic studies of human trait-associated genetic variants in cell states that are inaccessible from other types of human samples.

Bisphenol S (BPS) exposure has been implied epidemiologically to increase obesity risk, but the underlying mechanism is unclear. Here, we propose that BPS exposure at an environmentally relevant dose aggravates diet-induced obesity in female mice by inducing brown adipose tissue (BAT) whitening. We explored the underlying mechanism by which KDM5A-associated demethylation of the trimethylation of lysine 4 on histone H3 (H3K4me3) in thermogenic genes is overactivated in BAT upon BPS exposure, leading to the reduced expression of thermogenic genes. Further studies have suggested that BPS activates KDM5A transcription in BAT by binding to glucocorticoid receptor (GR) in an estrogen-dependent manner. Estrogen-estrogen receptors facilitate the accessibility of the KDM5A gene promoter to BPS-activated GR by recruiting the activator protein 1 (AP-1) complex. These results indicate that BAT is another important target of BPS and that targeting KDM5A-related signals may serve as an approach to counteract the BPS-induced susceptivity to obesity.

Regulatory T cells (Tregs) represent a major population in the control of immune homeostasis and autoimmunity. Here we show that Fos-like 2 (Fosl2), a TCR-induced AP1 transcription factor, represses Treg development and controls autoimmunity. Mice overexpressing Fosl2 (Fosl2tg) indeed show a systemic inflammatory phenotype, with immune infiltrates in multiple organs. This phenotype is absent in Fosl2tg × Rag2-/- mice lacking T and B cells, and Fosl2 induces T cell-intrinsic reduction of Treg development that is responsible for the inflammatory phenotype. Fosl2tg T cells can transfer inflammation, which is suppressed by the co-delivery of Tregs, while Fosl2 deficiency in T cells reduces the severity of autoimmunity in the EAE model. We find that Fosl2 could affect expression of FoxP3 and other Treg development genes. Our data highlight the importance of AP1 transcription factors, in particular Fosl2, during T cell development to determine Treg differentiation and control autoimmunity.

Caloric restriction has anti-inflammatory effects. However, the coordinated physiological actions that lead to reduced inflammation in a state of caloric deficit (hunger) are largely unknown. Using a mouse model of injury-induced peripheral inflammation, we find that food deprivation reduces edema, temperature, and cytokine responses that occur after injury. The magnitude of the anti-inflammatory effect that occurs during hunger is more robust than that of non-steroidal anti-inflammatory drugs. The effects of hunger are recapitulated centrally by activity in nutrient-sensing hypothalamic agouti-related protein (AgRP)-expressing neurons. We find that AgRP neurons projecting to the paraventricular nucleus of the hypothalamus rapidly and robustly reduce inflammation and mediate the majority of hunger's anti-inflammatory effects. Intact vagal efferent signaling is required for the anti-inflammatory action of hunger, revealing a brain-to-periphery pathway for this reduction in inflammation. Taken together, these data begin to unravel a potent anti-inflammatory pathway engaged by hypothalamic AgRP neurons to reduce inflammation.