Genetic Adaptation and Neandertal Admixture Shaped the Immune System of Human Populations.

Humans differ in the outcome that follows exposure to life-threatening pathogens, yet the extent of population differences in immune responses and their genetic and evolutionary determinants remain undefined. Here, we characterized, using RNA sequencing, the transcriptional response of primary monocytes from Africans and Europeans to bacterial and viral stimuli-ligands activating Toll-like receptor pathways (TLR1/2, TLR4, and TLR7/8) and influenza virus-and mapped expression quantitative trait loci (eQTLs). We identify numerous cis-eQTLs that contribute to the marked differences in immune responses detected within and between populations and a strong trans-eQTL hotspot at TLR1 that decreases expression of pro-inflammatory genes in Europeans only. We find that immune-responsive regulatory variants are enriched in population-specific signals of natural selection and show that admixture with Neandertals introduced regulatory variants into European genomes, affecting preferentially responses to viral challenges. Together, our study uncovers evolutionarily important determinants of differences in host immune responsiveness between human populations.

Single-Cell RNA-Seq Mapping of Human Thymopoiesis Reveals Lineage Specification Trajectories and a Commitment Spectrum in T Cell Development.

The challenges in recapitulating in vivo human T cell development in laboratory models have posed a barrier to understanding human thymopoiesis. Here, we used single-cell RNA sequencing (sRNA-seq) to interrogate the rare CD34+ progenitor and the more differentiated CD34- fractions in the human postnatal thymus. CD34+ thymic progenitors were comprised of a spectrum of specification and commitment states characterized by multilineage priming followed by gradual T cell commitment. The earliest progenitors in the differentiation trajectory were CD7- and expressed a stem-cell-like transcriptional profile, but had also initiated T cell priming. Clustering analysis identified a CD34+ subpopulation primed for the plasmacytoid dendritic lineage, suggesting an intrathymic dendritic specification pathway. CD2 expression defined T cell commitment stages where loss of B cell potential preceded that of myeloid potential. These datasets delineate gene expression profiles spanning key differentiation events in human thymopoiesis and provide a resource for the further study of human T cell development.

CRISPRi screen of long non-coding RNAs identifies LINC03045 regulating glioblastoma invasion.

INTRODUCTION: Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. METHODS: We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. RESULTS: Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. CONCLUSION: CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.

Dynamic changes in cis-regulatory occupancy by Six1 and its cooperative interactions with distinct cofactors drive lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

The transcription factor Six1 is essential for induction of sensory cell fate and formation of auditory sensory epithelium, but how it activates gene expression programs to generate distinct cell-types remains unknown. Here, we perform genome-wide characterization of Six1 binding at different stages of auditory sensory epithelium development and find that Six1-binding to cis-regulatory elements changes dramatically at cell-state transitions. Intriguingly, Six1 pre-occupies enhancers of cell-type-specific regulators and effectors before their expression. We demonstrate in-vivo cell-type-specific activity of Six1-bound novel enhancers of Pbx1, Fgf8, Dusp6, Vangl2, the hair-cell master regulator Atoh1 and a cascade of Atoh1's downstream factors, including Pou4f3 and Gfi1. A subset of Six1-bound sites carry consensus-sequences for its downstream factors, including Atoh1, Gfi1, Pou4f3, Gata3 and Pbx1, all of which physically interact with Six1. Motif analysis identifies RFX/X-box as one of the most significantly enriched motifs in Six1-bound sites, and we demonstrate that Six1-RFX proteins cooperatively regulate gene expression through binding to SIX:RFX-motifs. Six1 targets a wide range of hair-bundle regulators and late Six1 deletion disrupts hair-bundle polarity. This study provides a mechanistic understanding of how Six1 cooperates with distinct cofactors in feedforward loops to control lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

In vivo CRISPR screening for novel noncoding RNA functional targets in glioblastoma models.

CRISPR (clustered regularly interspaced short palindromic repeat)-based genetic screens offer unbiased and powerful tools for systematic and specific evaluation of phenotypes associated with specific target genes. CRISPR screens have been utilized heavily in vitro to identify functional coding and noncoding genes in a large number of cell types, including glioblastoma (GB), though no prior study has described the evaluation of CRISPR screening in GB in vivo. Here, we describe a protocol for targeting and transcriptionally repressing GB-specific long noncoding RNAs (lncRNAs) by CRISPR interference (CRISPRi) system in vivo, with tumor growth in the mouse cerebral cortex. Given the target-specific parameters of each individual screen, we list general steps involved in transducing guide RNA libraries into GB tumor lines, maintaining sufficient coverage, as well as cortically injecting and subsequently isolating transduced screen tumor cell populations for analysis. Finally, in order to demonstrate the use of this technique to discern an essential lncRNA, HOTAIR, from a nonessential lncRNA, we injected a 1:1 (HOTAIR:control nonessential lncRNA knockdown) mixture of fluorescently tagged U87 GB cells into the cortex of eight mice, evaluating selective depletion of HOTAIR-tagged cells at 2 weeks of growth. Fluorescently tagged populations were analyzed via flow cytometry for hiBFP (control knockdown) and green fluorescent protein (HOTAIR knockdown), revealing 17% (p = 0.007) decrease in fluorescence associated with HOTAIR knockdown relative to control. The described in vivo CRISPR screening methodology thus appears to be an effective option for identifying noncoding (and coding) genes affecting GB growth within the mouse cortex.

bacterial infection drives the expression dynamics of microRNAs and their isomiRs.

The optimal coordination of the transcriptional response of host cells to infection is essential for establishing appropriate immunological outcomes. In this context, the role of microRNAs (miRNAs)--important epigenetic regulators of gene expression--in regulating mammalian immune systems is increasingly well recognised. However, the expression dynamics of miRNAs, and that of their isoforms, in response to infection remains largely unexplored. Here, we characterized the genome-wide miRNA transcriptional responses of human dendritic cells, over time, to various mycobacteria differing in their virulence as well as to other bacteria outside the genus Mycobacterium, using small RNA-sequencing. We detected the presence of a core temporal response to infection, shared across bacteria, comprising 49 miRNAs, highlighting a set of miRNAs that may play an essential role in the regulation of basic cellular responses to stress. Despite such broadly shared expression dynamics, we identified specific elements of variation in the miRNA response to infection across bacteria, including a virulence-dependent induction of the miR-132/212 family in response to mycobacterial infections. We also found that infection has a strong impact on both the relative abundance of the miRNA hairpin arms and the expression dynamics of miRNA isoforms. That we observed broadly consistent changes in relative arm expression and isomiR distribution across bacteria suggests that this additional, internal layer of variability in miRNA responses represents an additional source of subtle miRNA-mediated regulation upon infection. Collectively, this study increases our understanding of the dynamism and role of miRNAs in response to bacterial infection, revealing novel features of their internal variability and identifying candidate miRNAs that may contribute to differences in the pathogenicity of mycobacterial infections.

Decoding the IGF1 signaling gene regulatory network behind alveologenesis from a mouse model of bronchopulmonary dysplasia.

Lung development is precisely controlled by underlying gene regulatory networks (GRN). Disruption of genes in the network can interrupt normal development and cause diseases such as bronchopulmonary dysplasia (BPD) - a chronic lung disease in preterm infants with morbid and sometimes lethal consequences characterized by lung immaturity and reduced alveolarization. Here, we generated a transgenic mouse exhibiting a moderate severity BPD phenotype by blocking IGF1 signaling in secondary crest myofibroblasts (SCMF) at the onset of alveologenesis. Using approaches mirroring the construction of the model GRN in sea urchin's development, we constructed the IGF1 signaling network underlying alveologenesis using this mouse model that phenocopies BPD. The constructed GRN, consisting of 43 genes, provides a bird's eye view of how the genes downstream of IGF1 are regulatorily connected. The GRN also reveals a mechanistic interpretation of how the effects of IGF1 signaling are transduced within SCMF from its specification genes to its effector genes and then from SCMF to its neighboring alveolar epithelial cells with WNT5A and FGF10 signaling as the bridge. Consistently, blocking WNT5A signaling in mice phenocopies BPD as inferred by the network. A comparative study on human samples suggests that a GRN of similar components and wiring underlies human BPD. Our network view of alveologenesis is transforming our perspective to understand and treat BPD. This new perspective calls for the construction of the full signaling GRN underlying alveologenesis, upon which targeted therapies for this neonatal chronic lung disease can be viably developed.

Mechanistic basis for chromosomal translocations at the E2A gene and its broader relevance to human B cell malignancies.

Analysis of translocation breakpoints in human B cell malignancies reveals that DNA double-strand breaks at oncogenes most frequently occur at CpG sites located within 20-600 bp fragile zones and depend on activation-induced deaminase (AID). AID requires single-stranded DNA (ssDNA) to act, but it has been unclear why or how this region transiently acquires a ssDNA state. Here, we demonstrate the ssDNA state in the 23 bp E2A fragile zone using several methods, including native bisulfite DNA structural analysis in live human pre-B cells. AID deamination within the E2A fragile zone does not require but is increased upon transcription. High C-string density, nascent RNA tails, and direct DNA sequence repeats prolong the ssDNA state of the E2A fragile zone and increase AID deamination at overlapping AID hotspots that contain the CpG sites at which breaks occur in patients. These features provide key insights into lymphoid fragile zones generally.

CD200-CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma.

The prevalence of asthma and airway hyperreactivity (AHR) is increasing at an alarming rate. Group 2 innate lymphoid cells (ILC2s) are copious producers of type 2 cytokines, which leads to AHR and lung inflammation. Here, we show that mouse ILC2s express CD200 receptor (CD200R) and this expression is inducible. CD200R engagement inhibits activation, proliferation and type 2 cytokine production, indicating an immunoregulatory function for the CD200-CD200R axis on ILC2s. Furthermore, CD200R engagement inhibits both canonical and non-canonical NF-κB signaling pathways in activated ILC2s. Additionally, we demonstrate both preventative and therapeutic approaches utilizing CD200R engagement on ILC2s, which lead to improved airway resistance, dynamic compliance and eosinophilia. These results show CD200R is expressed on human ILC2s, and its engagement ameliorates AHR in humanized mouse models, emphasizing the translational applications for treatment of ILC2-related diseases such as allergic asthma.

Creation of a Single Cell RNASeq Meta-Atlas to Define Human Liver Immune Homeostasis.

The liver is unique in both its ability to maintain immune homeostasis and in its potential for immune tolerance following solid organ transplantation. Single-cell RNA sequencing (scRNA seq) is a powerful approach to generate highly dimensional transcriptome data to understand cellular phenotypes. However, when scRNA data is produced by different groups, with different data models, different standards, and samples processed in different ways, it can be challenging to draw meaningful conclusions from the aggregated data. The goal of this study was to establish a method to combine 'human liver' scRNA seq datasets by 1) characterizing the heterogeneity between studies and 2) using the meta-atlas to define the dominant phenotypes across immune cell subpopulations in healthy human liver. Publicly available scRNA seq data generated from liver samples obtained from a combined total of 17 patients and ~32,000 cells were analyzed. Liver-specific immune cells (CD45+) were extracted from each dataset, and immune cell subpopulations (myeloid cells, NK and T cells, plasma cells, and B cells) were examined using dimensionality reduction (UMAP), differential gene expression, and ingenuity pathway analysis. All datasets co-clustered, but cell proportions differed between studies. Gene expression correlation demonstrated similarity across all studies, and canonical pathways that differed between datasets were related to cell stress and oxidative phosphorylation rather than immune-related function. Next, a meta-atlas was generated via data integration and compared against PBMC data to define gene signatures for each hepatic immune subpopulation. This analysis defined key features of hepatic immune homeostasis, with decreased expression across immunologic pathways and enhancement of pathways involved with cell death. This method for meta-analysis of scRNA seq data provides a novel approach to broadly define the features of human liver immune homeostasis. Specific pathways and cellular phenotypes described in this human liver immune meta-atlas provide a critical reference point for further study of immune mediated disease processes within the liver.

Runx2-Twist1 interaction coordinates cranial neural crest guidance of soft palate myogenesis.

Cranial neural crest (CNC) cells give rise to bone, cartilage, tendons, and ligaments of the vertebrate craniofacial musculoskeletal complex, as well as regulate mesoderm-derived craniofacial muscle development through cell-cell interactions. Using the mouse soft palate as a model, we performed an unbiased single-cell RNA-seq analysis to investigate the heterogeneity and lineage commitment of CNC derivatives during craniofacial muscle development. We show that Runx2, a known osteogenic regulator, is expressed in the CNC-derived perimysial and progenitor populations. Loss of Runx2 in CNC-derivatives results in reduced expression of perimysial markers (Aldh1a2 and Hic1) as well as soft palate muscle defects in Osr2-Cre;Runx2fl/fl mice. We further reveal that Runx2 maintains perimysial marker expression through suppressing Twist1, and that myogenesis is restored in Osr2-Cre;Runx2fl/fl;Twist1fl/+ mice. Collectively, our findings highlight the roles of Runx2, Twist1, and their interaction in regulating the fate of CNC-derived cells as they guide craniofacial muscle development through cell-cell interactions.

Neuron-specific chromosomal megadomain organization is adaptive to recent retrotransposon expansions.

Regulatory mechanisms associated with repeat-rich sequences and chromosomal conformations in mature neurons remain unexplored. Here, we map cell-type specific chromatin domain organization in adult mouse cerebral cortex and report strong enrichment of Endogenous Retrovirus 2 (ERV2) repeat sequences in the neuron-specific heterochromatic B2NeuN+ megabase-scaling subcompartment. Single molecule long-read sequencing and comparative Hi-C chromosomal contact mapping in wild-derived SPRET/EiJ (Mus spretus) and laboratory inbred C57BL/6J (Mus musculus) reveal neuronal reconfigurations tracking recent ERV2 expansions in the murine germline, with significantly higher B2NeuN+ contact frequencies at sites with ongoing insertions in Mus musculus. Neuronal ablation of the retrotransposon silencer Kmt1e/Setdb1 triggers B2NeuN+ disintegration and rewiring with open chromatin domains enriched for cellular stress response genes, along with severe neuroinflammation and proviral assembly with infiltration of dendrites . We conclude that neuronal megabase-scale chromosomal architectures include an evolutionarily adaptive heterochromatic organization which, upon perturbation, results in transcriptional dysregulation and unleashes ERV2 proviruses with strong neuronal tropism.

Preclinical Evaluation of a Novel Dual Targeting PI3Kδ/BRD4 Inhibitor, SF2535, in B-Cell Acute Lymphoblastic Leukemia.

The PI3K/Akt pathway-and in particular PI3Kδ-is known for its role in drug resistant B-cell acute lymphoblastic leukemia (B-ALL) and it is often upregulated in refractory or relapsed B-ALL. Myc proteins are transcription factors responsible for transcribing pro-proliferative genes and c-Myc is often overexpressed in cancers. The chromatin regulator BRD4 is required for expression of c-Myc in hematologic malignancies including B-ALL. Previously, combination of BRD4 and PI3K inhibition with SF2523 was shown to successfully decrease Myc expression. However, the underlying mechanism and effect of dual inhibition of PI3Kδ/BRD4 in B-ALL remains unknown. To study this, we utilized SF2535, a novel small molecule dual inhibitor which can specifically target the PI3Kδ isoform and BRD4. We treated primary B-ALL cells with various concentrations of SF2535 and studied its effect on specific pharmacological on-target mechanisms such as apoptosis, cell cycle, cell proliferation, and adhesion molecules expression usingin vitro and in vivo models. SF2535 significantly downregulates both c-Myc mRNA and protein expression through inhibition of BRD4 at the c-Myc promoter site and decreases p-AKT expression through inhibition of the PI3Kδ/AKT pathway. SF2535 induced apoptosis in B-ALL by downregulation of BCL-2 and increased cleavage of caspase-3, caspase-7, and PARP. Moreover, SF2535 induced cell cycle arrest and decreased cell counts in B-ALL. Interestingly, SF2535 decreased the mean fluorescence intensity (MFI) of integrin α4, α5, α6, and ß1 while increasing MFI of CXCR4, indicating that SF2535 may work through inside-out signaling of integrins. Taken together, our data provide a rationale for the clinical evaluation of targeting PI3Kδ/BRD4 in refractory or relapsed B-ALL using SF2535.

Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis.

Cranial neural crest (CNC) cells contribute to different cell types during embryonic development. It is unknown whether postmigratory CNC cells undergo dynamic cellular movement and how the process of cell fate decision occurs within the first pharyngeal arch (FPA). Our investigations demonstrate notable heterogeneity within the CNC cells, refine the patterning domains, and identify progenitor cells within the FPA. These progenitor cells undergo fate bifurcation that separates them into common progenitors and mesenchymal cells, which are characterized by Cdk1 and Spry2/Notch2 expression, respectively. The common progenitors undergo further bifurcations to restrict them into osteogenic/odontogenic and chondrogenic/fibroblast lineages. Disruption of a patterning domain leads to specific mandible and tooth defects, validating the binary cell fate restriction process. Different from the compartment model of mandibular morphogenesis, our data redefine heterogeneous cellular domains within the FPA, reveal dynamic cellular movement in time, and describe a sequential series of binary cell fate decision-making process.

Stress resilience is promoted by a Zfp189-driven transcriptional network in prefrontal cortex.

Understanding the transcriptional changes that are engaged in stress resilience may reveal novel antidepressant targets. Here we use gene co-expression analysis of RNA-sequencing data from brains of resilient mice to identify a gene network that is unique to resilience. Zfp189, which encodes a previously unstudied zinc finger protein, is the highest-ranked key driver gene in the network, and overexpression of Zfp189 in prefrontal cortical neurons preferentially activates this network and promotes behavioral resilience. The transcription factor CREB is a predicted upstream regulator of this network and binds to the Zfp189 promoter. To probe CREB-Zfp189 interactions, we employ CRISPR-mediated locus-specific transcriptional reprogramming to direct CREB or G9a (a repressive histone methyltransferase) to the Zfp189 promoter in prefrontal cortex neurons. Induction of Zfp189 with site-specific CREB is pro-resilient, whereas suppressing Zfp189 expression with G9a increases susceptibility. These findings reveal an essential role for Zfp189 and CREB-Zfp189 interactions in mediating a central transcriptional network of resilience.

Estrogen receptor α drives pro-resilient transcription in mouse models of depression.

Most people exposed to stress do not develop depression. Animal models have shown that stress resilience is an active state that requires broad transcriptional adaptations, but how this homeostatic process is regulated remains poorly understood. In this study, we analyze upstream regulators of genes differentially expressed after chronic social defeat stress. We identify estrogen receptor α (ERα) as the top regulator of pro-resilient transcriptional changes in the nucleus accumbens (NAc), a key brain reward region implicated in depression. In accordance with these findings, nuclear ERα protein levels are altered by stress in male and female mice. Further, overexpression of ERα in the NAc promotes stress resilience in both sexes. Subsequent RNA-sequencing reveals that ERα overexpression in NAc reproduces the transcriptional signature of resilience in male, but not female, mice. These results indicate that NAc ERα is an important regulator of pro-resilient transcriptional changes, but with sex-specific downstream targets.

Bioinformatic Analysis for Profiling Drug-induced Chromatin Modification Landscapes in Mouse Brain Using ChlP-seq Data.

Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is a powerful technology to profile genome-wide chromatin modification patterns and is increasingly being used to study the molecular mechanisms of brain diseases such as drug addiction. This protocol discusses the typical procedures involved in ChIP-seq data generation, bioinformatic analysis, and interpretation of results, using a chronic cocaine treatment study as a template. We describe an experimental design that induces significant chromatin modifications in mouse brain, and the use of ChIP-seq to derive novel information about the chromatin regulatory mechanisms involved. We describe the bioinformatic methods used to preprocess the sequencing data, generate global enrichment profiles for specific histone modifications, identify enriched genomic loci, find differential modification sites, and perform functional analyses. These ChIP-seq analyses provide many details into the chromatin changes that are induced in brain by chronic exposure to cocaine, and generates an invaluable source of information to understand the molecular mechanisms underlying drug addiction. Our protocol provides a standardized procedure for data analysis and can serve as a starting point for any other ChIP-seq projects.

Comprehensive mapping of 5-hydroxymethylcytosine epigenetic dynamics in axon regeneration.

In contrast to central nervous system neurons, dorsal root ganglia (DRG) neurons can switch to a regenerative state after peripheral axotomy. In a screen for chromatin regulators of the regenerative responses in this conditioning lesion paradigm, we identified Tet methylcytosine dioxygenase 3 (Tet3) as upregulated in DRG neurons, along with increased 5-hydroxymethylcytosine (5hmC). We generated genome-wide 5hmC maps in adult DRG, which revealed that peripheral and central axotomy (leading to no regenerative effect) triggered differential 5hmC changes that are associated with distinct signaling pathways. 5hmC was altered in a large set of regeneration-associated genes (RAGs), including well-known RAGs, such as Atf3, Bdnf, and Smad1, that regulate axon growth potential of DRG neurons, thus supporting its role for RAG regulation. Our analyses also predicted HIF-1, STAT, and IRF as potential transcription factors that may collaborate with Tet3 for 5hmC modifications. Intriguingly, central axotomy resulted in widespread 5hmC modifications that had little overlap with those of peripheral axotomy, thus potentially constituting a roadblock for regeneration. Our study revealed 5hmC dynamics as a previously unrecognized epigenetic mechanism underlying the divergent responses after axonal injury.

Neuropathic pain promotes adaptive changes in gene expression in brain networks involved in stress and depression.

Neuropathic pain is a complex chronic condition characterized by various sensory, cognitive, and affective symptoms. A large percentage of patients with neuropathic pain are also afflicted with depression and anxiety disorders, a pattern that is also seen in animal models. Furthermore, clinical and preclinical studies indicate that chronic pain corresponds with adaptations in several brain networks involved in mood, motivation, and reward. Chronic stress is also a major risk factor for depression. We investigated whether chronic pain and stress affect similar molecular mechanisms and whether chronic pain can affect gene expression patterns that are involved in depression. Using two mouse models of neuropathic pain and depression [spared nerve injury (SNI) and chronic unpredictable stress (CUS)], we performed next-generation RNA sequencing and pathway analysis to monitor changes in gene expression in the nucleus accumbens (NAc), the medial prefrontal cortex (mPFC), and the periaqueductal gray (PAG). In addition to finding unique transcriptome profiles across these regions, we identified a substantial number of signaling pathway-associated genes with similar changes in expression in both SNI and CUS mice. Many of these genes have been implicated in depression, anxiety, and chronic pain in patients. Our study provides a resource of the changes in gene expression induced by long-term neuropathic pain in three distinct brain regions and reveals molecular connections between pain and chronic stress.

Early life stress confers lifelong stress susceptibility in mice via ventral tegmental area OTX2.

Early life stress increases risk for depression. Here we establish a "two-hit" stress model in mice wherein stress at a specific postnatal period increases susceptibility to adult social defeat stress and causes long-lasting transcriptional alterations that prime the ventral tegmental area (VTA)-a brain reward region-to be in a depression-like state. We identify a role for the developmental transcription factor orthodenticle homeobox 2 (Otx2) as an upstream mediator of these enduring effects. Transient juvenile-but not adult-knockdown of Otx2 in VTA mimics early life stress by increasing stress susceptibility, whereas its overexpression reverses the effects of early life stress. This work establishes a mechanism by which early life stress encodes lifelong susceptibility to stress via long-lasting transcriptional programming in VTA mediated by Otx2.