Endothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

RNase H2 has two distinct functions: initiation of the ribonucleotide excision repair (RER) pathway by cleaving ribonucleotides (rNMPs) incorporated during DNA replication and processing the RNA portion of an R-loop formed during transcription. An RNase H2 mutant lacking RER activity but supporting R-loop removal revealed that rNMPs in DNA initiate p53-dependent DNA damage response and early embryonic arrest in mouse. However, an RNase H2 AGS-related mutant with residual RER activity develops to birth. Estimations of the number of rNMPs in DNA in these two mutants define a ribonucleotide threshold above which p53 induces apoptosis. Below the threshold, rNMPs in DNA trigger an innate immune response. Compound heterozygous cells, containing both defective enzymes, retain rNMPs above the threshold, indicative of competition for RER substrates between active and inactive enzymes, suggesting that patients with compound heterozygous mutations in RNASEH2 genes may not reflect the properties of recombinantly expressed proteins.

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.

Misregulation of alternative splicing is a hallmark of human tumors, yet to what extent and how it contributes to malignancy are only beginning to be unraveled. Here, we define which members of the splicing factor SR and SR-like families contribute to breast cancer and uncover differences and redundancies in their targets and biological functions. We identify splicing factors frequently altered in human breast tumors and assay their oncogenic functions using breast organoid models. We demonstrate that not all splicing factors affect mammary tumorigenesis in MCF-10A cells. Specifically, the upregulation of SRSF4, SRSF6, or TRA2β disrupts acinar morphogenesis and promotes cell proliferation and invasion in MCF-10A cells. By characterizing the targets of these oncogenic splicing factors, we identify shared spliced isoforms associated with well-established cancer hallmarks. Finally, we demonstrate that TRA2β is regulated by the MYC oncogene, plays a role in metastasis maintenance in vivo, and its levels correlate with breast cancer patient survival.

Nuclear factor κB (NF-κB) RelA is the potent transcriptional activator of inflammatory response genes. We stringently defined a list of direct RelA target genes by integrating physical (chromatin immunoprecipitation sequencing [ChIP-seq]) and functional (RNA sequencing [RNA-seq] in knockouts) datasets. We then dissected each gene's regulatory strategy by testing RelA variants in a primary-cell genetic-complementation assay. All endogenous target genes require RelA to make DNA-base-specific contacts, and none are activatable by the DNA binding domain alone. However, endogenous target genes differ widely in how they employ the two transactivation domains. Through model-aided analysis of the dynamic time-course data, we reveal the gene-specific synergy and redundancy of TA1 and TA2. Given that post-translational modifications control TA1 activity and intrinsic affinity for coactivators determines TA2 activity, the differential TA logics suggests context-dependent versus context-independent control of endogenous RelA-target genes. Although some inflammatory initiators appear to require co-stimulatory TA1 activation, inflammatory resolvers are a part of the NF-κB RelA core response.

Breast cancer progression, treatment resistance, and relapse are thought to originate from a small population of tumor cells, breast cancer stem cells (BCSCs). Identification of factors critical for BCSC function is therefore vital for the development of therapies. Here, we identify the arginine methyltransferase PRMT5 as a key in vitro and in vivo regulator of BCSC proliferation and self-renewal and establish FOXP1, a winged helix/forkhead transcription factor, as a critical effector of PRMT5-induced BCSC function. Mechanistically, PRMT5 recruitment to the FOXP1 promoter facilitates H3R2me2s, SET1 recruitment, H3K4me3, and gene expression. Our findings are clinically significant, as PRMT5 depletion within established tumor xenografts or treatment of patient-derived BCSCs with a pre-clinical PRMT5 inhibitor substantially reduces BCSC numbers. Together, our findings highlight the importance of PRMT5 in BCSC maintenance and suggest that small-molecule inhibitors of PRMT5 or downstream targets could be an effective strategy eliminating this cancer-causing population.

Septal parvalbumin-expressing (PV+) and calbindin-expressing (CB+) projections inhibit low-threshold and fast-spiking interneurons, respectively, in the medial entorhinal cortex (MEC). We investigate how the two inputs control neuronal activity in the MEC in freely moving mice. Stimulation of PV+ and CB+ terminals causes disinhibition of spatially tuned MEC neurons, but exerts differential effects on temporal coding and burst firing. Thus, recruitment of PV+ projections disrupts theta-rhythmic firing of MEC neurons, while stimulation of CB+ projections increases burst firing of grid cells and enhances phase precession in a cell-type-specific manner. Inactivation of septal PV+ or CB+ neurons differentially affects context, reference, and working memory. Together, our results reveal how specific connectivity of septal GABAergic projections with MEC interneurons translates into differential modulation of MEC neuronal coding.

Timing uncertainty is a critical component of temporal decision-making, as it determines the decision strategies that maximize reward rate. However, little is known about the biological substrates of timing uncertainty. In this study, we report that the CA3 subregion of the ventral hippocampus (vCA3), a relatively unexplored area in timing, is critical in regulating timing uncertainty that informs temporal decision making. Using a variant of the differential reinforcement of low rates of responding (DRL) task that incorporates differential levels of approach-avoidance conflict, rats were trained to wait a minimum of 6 s to earn a reward that was paired with varying durations of foot shock. Post-training chemogenetic inhibition of the vCA3 reduced timing uncertainty without affecting mean wait times, irrespective of the level of conflict experienced. Simulations based on the information-processing variant of scalar expectancy theory (SET) revealed that the vCA3 may be important in modulating decision threshold or switch closure latency variability.

C-LTMRs are known to convey affective aspects of touch and to modulate injury-induced pain in humans and mice. However, a role for these neurons in temperature sensation has been suggested, but not fully demonstrated. Here, we report that deletion of C-low-threshold mechanoreceptor (C-LTMR)-expressed bhlha9 causes impaired thermotaxis behavior and exacerbated formalin-evoked pain in male, but not female, mice. Positive modulators of GABAA receptors failed to relieve inflammatory formalin pain and failed to decrease the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) selectively in bhlha9 knockout (KO) males. This could be explained by a drastic change in the GABA content of lamina II inner inhibitory interneurons contacting C-LTMR central terminals. Finally, C-LTMR-specific deep RNA sequencing revealed more genes differentially expressed in male than in female bhlha9 KO C-LTMRs. Our data consolidate the role of C-LTMRs in modulation of formalin pain and provide in vivo evidence of their role in the discriminative aspects of temperature sensation.

Bats asymptomatically harbor many viruses that can cause severe human diseases. The Egyptian rousette bat (ERB) is the only known reservoir for Marburgviruses and Sosuga virus, making it an exceptional animal model to study antiviral mechanisms in an asymptomatic host. With this goal in mind, we constructed and annotated the immunoglobulin heavy chain locus, finding an expansion on immunoglobulin variable genes associated with protective human antibodies to different viruses. We also annotated two functional and distinct immunoglobulin epsilon genes and four distinctive functional immunoglobulin gamma genes. We described the Fc receptor repertoire in ERBs, including features that may affect activation potential, and discovered the lack of evolutionary conserved short pentraxins. These findings reinforce the hypothesis that a differential threshold of regulation and/or absence of key immune mediators may promote tolerance and decrease inflammation in ERBs.

Diabetogenic ablation of beta cells in mice triggers a regenerative response whereby surviving beta cells proliferate and euglycemia is regained. Here, we identify and characterize heterogeneity in response to beta cell ablation. Efficient beta cell elimination leading to severe hyperglycemia (>28 mmol/L), causes permanent diabetes with failed regeneration despite cell cycle engagement of surviving beta cells. Strikingly, correction of glycemia via insulin, SGLT2 inhibition, or a ketogenic diet for about 3 weeks allows partial regeneration of beta cell mass and recovery from diabetes, demonstrating regenerative potential masked by extreme glucotoxicity. We identify gene expression changes in beta cells exposed to extremely high glucose levels, pointing to metabolic stress and downregulation of key cell cycle genes, suggesting failure of cell cycle completion. These findings reconcile conflicting data on the impact of glucose on beta cell regeneration and identify a glucose threshold converting glycemic load from pro-regenerative to anti-regenerative.

The thalamus transmits sensory information to the neocortex and receives neocortical, subcortical, and neuromodulatory inputs. Despite its obvious importance, surprisingly little is known about thalamic function in awake animals. Here, using intracellular and extracellular recordings in awake head-restrained mice, we investigate membrane potential dynamics and action potential firing in the two major thalamic nuclei related to whisker sensation, the ventral posterior medial nucleus (VPM) and the posterior medial group (Pom), which receive distinct inputs from brainstem and neocortex. We find heterogeneous state-dependent dynamics in both nuclei, with an overall increase in action potential firing during active states. Whisking increased putative lemniscal and corticothalamic excitatory inputs onto VPM and Pom neurons, respectively. A subpopulation of VPM cells fired spikes phase-locked to the whisking cycle during free whisking, and these cells may therefore signal whisker position. Our results suggest differential processing of whisking comparing thalamic nuclei at both sub- and supra-threshold levels.

Transcription factor XBP1s, activated by endoplasmic reticulum (ER) stress in a dose-dependent manner, plays a central role in adaptive unfolded protein response (UPR) via direct activation of multiple genes controlling protein refolding. Here, we report that elevation of ER stress above a critical threshold causes accumulation of XBP1s protein sufficient for binding to the promoter and activation of a gene encoding a transcription factor KLF9. In comparison to other XBP1s targets, KLF9 promoter contains an evolutionary conserved lower-affinity binding site that requires higher amounts of XBP1s for activation. In turn, KLF9 induces expression of two regulators of ER calcium storage, TMEM38B and ITPR1, facilitating additional calcium release from ER, exacerbation of ER stress, and cell death. Accordingly, Klf9 deficiency attenuates tunicamycin-induced ER stress in mouse liver. These data reveal a role for XBP1s in cytotoxic UPR and provide insights into mechanisms of life-or-death decisions in cells under ER stress.

Mutations of the transcriptional regulator PHF6 cause the X-linked intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS), but the pathogenesis of BFLS remains poorly understood. Here, we report a mouse model of BFLS, generated using a CRISPR-Cas9 approach, in which cysteine 99 within the PHD domain of PHF6 is replaced with phenylalanine (C99F). Mice harboring the patient-specific C99F mutation display deficits in cognitive functions, emotionality, and social behavior, as well as reduced threshold to seizures. Electrophysiological studies reveal that the intrinsic excitability of entorhinal cortical stellate neurons is increased in PHF6 C99F mice. Transcriptomic analysis of the cerebral cortex in C99F knockin mice and PHF6 knockout mice show that PHF6 promotes the expression of neurogenic genes and represses synaptic genes. PHF6-regulated genes are also overrepresented in gene signatures and modules that are deregulated in neurodevelopmental disorders of cognition. Our findings advance our understanding of the mechanisms underlying BFLS pathogenesis.

DC-SIGN+ monocyte-derived dendritic cells (mo-DCs) play important roles in bacterial infections and inflammatory diseases, but the factors regulating their differentiation and proinflammatory status remain poorly defined. Here, we identify a microRNA, miR-181a, and a molecular mechanism that simultaneously regulate the acquisition of DC-SIGN expression and the activation state of DC-SIGN+ mo-DCs. Specifically, we show that miR-181a promotes DC-SIGN expression during terminal mo-DC differentiation and limits its sensitivity and responsiveness to TLR triggering and CD40 ligation. Mechanistically, miR-181a sustains ERK-MAPK signaling in mo-DCs, thereby enabling the maintenance of high levels of DC-SIGN and a high activation threshold. Low miR-181a levels during mo-DC differentiation, induced by inflammatory signals, do not support the high phospho-ERK signal transduction required for DC-SIGNhi mo-DCs and lead to development of proinflammatory DC-SIGNlo/- mo-DCs. Collectively, our study demonstrates that high DC-SIGN expression levels and a high activation threshold in mo-DCs are linked and simultaneously maintained by miR-181a.

T cell differentiation in the thymus proceeds in an ordered sequence of developmental events characterized by variable expression of CD4 and CD8 coreceptors. Here, we report that immature single-positive (ISP) thymocytes are molecularly distinct from all other T cell populations in the thymus in their expression of a gene profile that is dependent on the transcription factor BRD4. Conditional deletion of BRD4 at various stages of thymic differentiation reveals that BRD4 selectively regulates the further differentiation of ISPs by targeting cell cycle and metabolic pathways, but it does not affect the extensive proliferation that results in the generation of ISPs. These studies lead to the conclusion that the ISP subpopulation is not a hybrid transitional state but a molecularly distinct subpopulation that is selectively dependent on BRD4.

Comprehensive sequencing approaches have allowed for the identification of the most frequent contributors to cancer, known as drivers. They have also revealed a class of mutations in understudied, infrequently altered genes, referred to as "long tail" (LT) drivers. A key challenge has been to find clinically relevant LT drivers and to understand how they cooperate to drive disease. Here, we identified far upstream binding protein 1 (FUBP1) as an LT driver using an in vivo CRISPR screen. FUBP1 cooperates with other tumor suppressor genes to transform mammary epithelial cells by disrupting cellular differentiation and tissue architecture. Mechanistically, FUBP1 participates in regulating N6-methyladenosine (m6A) RNA methylation, and its loss leads to global changes in RNA splicing and widespread expression of aberrant driver isoforms. These findings suggest that somatic alteration of a single gene involved in RNA splicing and m6A methylation can produce the necessary panoply of contributors for neoplastic transformation.

RNA-binding proteins play important roles in X-linked intellectual disability (XLID). In this study, we investigate the contribution of the XLID-associated RBMX in neuronal differentiation. We show that RBMX-depleted cells exhibit aberrant activation of the p53 pathway. Moreover, we identify that the RBMX RGG/RG motif is methylated by protein arginine methyltransferase 5 (PRMT5), and this regulates assembly with the SRSF1 splicing factor into higher-order complexes. Depletion of RBMX or disruption of the RBMX/SRSF1 complex in PRMT5-depleted cells reduces SRSF1 binding to the MDM4 precursor (pre-)mRNA, leading to exon 6 exclusion and lower MDM4 protein levels. Transcriptomic analysis of isogenic Shashi-XLID human-induced pluripotent stem cells (hiPSCs) generated using CRISPR-Cas9 reveals a dysregulation of MDM4 splicing and aberrant p53 upregulation. Shashi-XLID neural progenitor cells (NPCs) display differentiation and morphological abnormalities accompanied with excessive apoptosis. Our findings identify RBMX as a regulator of SRSF1 and the p53 pathway, suggesting that the loss of function of the RBMX RGG/RG motif is the cause of Shashi-XLID syndrome.

The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39 deletion reduces the pre-malignant B cells in Eμ-Myc transgenic mice and significantly improves their survival.

Cutaneous C-unmyelinated MRGPRD+ free nerve endings and C-LTMRs innervating hair follicles convey two opposite aspects of touch sensation: a sensation of pain and a sensation of pleasant touch. The molecular mechanisms underlying these diametrically opposite functions are unknown. Here, we used a mouse model that genetically marks C-LTMRs and MRGPRD+ neurons in combination with fluorescent cell surface labeling, flow cytometry, and RNA deep-sequencing technology (RNA-seq). Cluster analysis of RNA-seq profiles of the purified neuronal subsets revealed 486 and 549 genes differentially expressed in MRGPRD-expressing neurons and C-LTMRs, respectively. We validated 48 MRGPD- and 68 C-LTMRs-enriched genes using a triple-staining approach, and the Cav3.3 channel, found to be exclusively expressed in C-LTMRs, was validated using electrophysiology. Our study greatly expands the molecular characterization of C-LTMRs and suggests that this particular population of neurons shares some molecular features with Aβ and Aδ low-threshold mechanoreceptors.