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Striatal dopamine (DA) neurotransmission is critical for an array of reward-related behaviors and goal-directed motor control. In rodents, 95% of striatal neurons are GABAergic medium spiny neurons (MSNs) that have been traditionally segregated into two subpopulations based on the expression of stimulatory DA D1-like receptors versus inhibitory D2-like receptors. However, emerging evidence suggests that striatal cell composition is anatomically and functionally more heterogenous than previously appreciated. The presence of MSNs that co-express multiple DA receptors offers a means to more accurately understand this heterogeneity. To dissect the precise nature of MSN heterogeneity, here we used multiplex RNAscope to identify expression of three predominantly expressed DA receptors in the striatum: DA D1(D1R), D2(D2R), and D3(D3R) receptors. We report heterogenous subpopulations of MSNs that are distinctly distributed across the dorsal-ventral and rostral-caudal axes of the adult mouse striatum. These subpopulations include MSNs that co-express D1R and D2R (D1/2R), D1R and D3R (D1/3R), and D2R and D3R (D2/3R). Overall, our characterization of distinct MSN subpopulations informs our understanding of region-specific striatal cell heterogeneity.

DNA double-strand breaks (DSBs) are linked to neurodegeneration and senescence. However, it is not clear how DSB-bearing neurons influence neuroinflammation associated with neurodegeneration. Here, we characterize DSB-bearing neurons from the CK-p25 mouse model of neurodegeneration using single-nucleus, bulk, and spatial transcriptomic techniques. DSB-bearing neurons enter a late-stage DNA damage response marked by nuclear factor κB (NFκB)–activated senescent and antiviral immune pathways. In humans, Alzheimer’s disease pathology is closely associated with immune activation in excitatory neurons. Spatial transcriptomics reveal that regions of CK-p25 brain tissue dense with DSB-bearing neurons harbor signatures of inflammatory microglia, which is ameliorated by NFκB knockdown in neurons. Inhibition of NFκB in DSB-bearing neurons also reduces microglia activation in organotypic mouse brain slice culture. In conclusion, DSBs activate immune pathways in neurons, which in turn adopt a senescence-associated secretory phenotype to elicit microglia activation. These findings highlight a previously unidentified role for neurons in the mechanism of disease-associated neuroinflammation.

Recent large genome-wide association studies (GWAS) have identified multiple confident risk loci linked to addiction-associated behavioral traits. Genetic variants linked to addiction-associated traits lie largely in non-coding regions of the genome, likely disrupting cis-regulatory element (CRE) function. CREs tend to be highly cell type-specific and may contribute to the functional development of the neural circuits underlying addiction. Yet, a systematic approach for predicting the impact of risk variants on the CREs of specific cell populations is lacking. To dissect the cell types and brain regions underlying addiction-associated traits, we applied LD score regression to compare GWAS to genomic regions collected from human and mouse assays for open chromatin, which is associated with CRE activity. We found enrichment of addiction-associated variants in putativeCREsmarked by open chromatin in neuronal (NeuN+) nuclei collected from multiple prefrontal cortical areas and striatal regions known to play major roles in reward and addiction. To further dissect the cell type-specific basis of addiction-associated traits, we also identified enrichments in human orthologs of open chromatin regions of mouseneuronal subtypes: cortical excitatory, D1, D2, and PV. Lastly, we developed machine learning models from mouse cell type-specific regions of open chromatin to further dissect human NeuN+ open chromatin regions into cortical excitatory or striatal D1 and D2 neurons and predict the functional impact of addiction-associated genetic variants. Our results suggest that differentneuronal subtypeswithin the reward system play distinct roles in the variety of traits that contribute to addiction.Significance StatementWe combine statistical genetic and machine learning techniques to find that the predisposition to for nicotine, alcohol, and cannabis use behaviors can be partially explained by genetic variants in conserved regulatory elements within specific brain regions and neuronal subtypes of the reward system.This computational framework can flexibly integrateopen chromatindata across species to screen for putative causal variants in a cell type-and tissue-specific manner across numerous complex traits.

Background: Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Methods: Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to simultaneously quantify and rank efficiency of competing AAV vectors across all cell types in the same animal. Results: To demonstrate proof-of-concept for the scAAVengr workflow, we quantified – with cell-type resolution – the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant identified using this pipeline, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. scAAVengr was then used to identify top-performing AAV variants in mouse brain, heart, and liver following systemic injection. Conclusions: These results validate scAAVengr as a powerful method for development of AAV vectors. Funding: This work was supported by funding from the Ford Foundation, NEI/NIH, Research to Prevent Blindness, Foundation Fighting Blindness, UPMC Immune Transplant and Therapy Center, and the Van Sloun fund for canine genetic research., eLife digest Gene therapy is an experimental approach to treating disease that involves altering faulty genes or replacing them with new, working copies. Most often, the new genetic material is delivered into cells using a modified virus that no longer causes disease, called a viral vector. Virus-mediated gene therapies are currently being explored for degenerative eye diseases, such as retinitis pigmentosa, and neurological disorders, like Alzheimer’s and Parkinson’s disease. A number of gene therapies have also been approved for treating some rare cancers, blood disorders and a childhood form of motor neuron disease. Despite the promise of virus-mediated gene therapy, there are significant hurdles to its widespread success. Viral vectors need to deliver enough genetic material to the right cells without triggering an immune response or causing serious side effects. Selecting an optimal vector is key to achieving this. A type of viruses called adeno-associated viruses (AAV) are prime candidates, partly because they can be easily engineered. However, accurately comparing the safety and efficacy of newly engineered AAVs is difficult, due to variation between test subjects and the labor and cost involved in careful testing. Öztürk et al. addressed this issue by developing an experimental pipeline called scAAVengr for comparing gene therapy vectors head-to-head. The process involves tagging potential AAV vectors with unique genetic barcodes, which can then be detected and quantified in individual cells using a technique called single-cell RNA sequencing. This means that when several vectors are used to infect lab-grown cells or a test animal at the same time, they can be tracked. The vectors can then be ranked on their ability to infect specific cell types and deliver useful genetic material. Using scAAVengr, Öztürk et al. compared viral vectors designed to target the light-sensitive cells of the retina, which allow animals to see. First, a set of promising viral vectors were evaluated using the scAAVengr pipeline in the eyes of marmosets and macaques, two small primates. Precise levels and locations of gene delivery were quantified. The top-performing vector was then identified and used to deliver Cas9, a genome editing tool, to primate retinas. Öztürk et al. also used scAAVengr to compare viral vectors in mice, analysing the vectors’ ability to deliver their genetic cargo to the brain, heart, and liver. These experiments demonstrated that scAAVengr can be used to evaluate vectors in multiple tissues and in different organisms. In summary, this work outlines a method for identifying and precisely quantifying the performance of top-performing viral vectors for gene therapy. By aiding the selection of optimal viral vectors, the scAAVengr pipeline could help to improve the success of preclinical studies and early clinical trials testing gene therapies.

Severe and persistent disruptions to sleep and circadian rhythms are common in people with opioid use disorder (OUD). Preclinical evidence suggests altered molecular rhythms in the brain modulate opioid reward and relapse. However, whether molecular rhythms are disrupted in the brains of people with OUD remained an open question, critical to understanding the role of circadian rhythms in opioid addiction. Using subjects' times of death as a marker of time of day, we investigated transcriptional rhythms in the brains of subjects with OUD compared to unaffected comparison subjects. We discovered rhythmic transcripts in both the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc), key brain areas involved in OUD, that were largely distinct between OUD and unaffected subjects. Fewer rhythmic transcripts were identified in DLPFC of subjects with OUD compared to unaffected subjects, whereas in the NAc, nearly double the number of rhythmic transcripts was identified in subjects with OUD. In NAc of subjects with OUD, rhythmic transcripts peaked either in the evening or near sunrise, and were associated with an opioid, dopamine, and GABAergic neurotransmission. Associations with altered neurotransmission in NAc were further supported by co-expression network analysis which identified OUD-specific modules enriched for transcripts involved in dopamine, GABA, and glutamatergic synaptic functions. Additionally, rhythmic transcripts in DLPFC and NAc of subjects with OUD were enriched for genomic loci associated with sleep-related GWAS traits, including sleep duration and insomnia. Collectively, our findings connect transcriptional rhythm changes in opioidergic, dopaminergic, GABAergic signaling in the human brain to sleep-related traits in opioid addiction.

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations within heterogeneous tissue. Available approaches for engineering-targeted technologies for new neuron subtypes are low yield, involving intensive transgenic strain or virus screening. Here, we present Specific Nuclear-Anchored Independent Labeling (SNAIL), an improved virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and then make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV+) neurons. Nuclear isolation using SNAIL in wild-type mice is sufficient to capture characteristic open chromatin features of PV+ neurons in the cortex, striatum, and external globus pallidus. The SNAIL framework also has high utility for multispecies cell probe engineering; expression from a mouse PV+ SNAIL enhancer sequence was enriched in PV+ neurons of the macaque cortex. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations, enabling characterization of their roles in behavior and in disease states. Available approaches for engineering targeted technologies for new neuron subtypes are low-yield, involving intensive transgenic strain or virus screening. Here, we introduce SNAIL (Specific Nuclear-Anchored Independent Labeling), a new virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and using them to make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV) neurons. Furthermore, we show that nuclear isolation using SNAIL in wild type mice is sufficient to capture characteristic open chromatin features of PV neurons in the cortex, striatum, and external globus pallidus. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

Background Prevalence rates of opioid use disorder (OUD) have increased dramatically, accompanied by a surge of overdose deaths. While opioid dependence has been extensively studied in preclinical models, an understanding of the biological alterations that occur in the brains of people who chronically use opioids and who are diagnosed with OUD remains limited. To address this limitation, RNA sequencing was conducted on the dorsolateral prefrontal cortex and nucleus accumbens, regions heavily implicated in OUD, from postmortem brains in subjects with OUD. Methods We performed RNA sequencing on the dorsolateral prefrontal cortex and nucleus accumbens from unaffected comparison subjects (n = 20) and subjects diagnosed with OUD (n = 20). Our transcriptomic analyses identified differentially expressed transcripts and investigated the transcriptional coherence between brain regions using rank-rank hypergeometric orderlap. Weighted gene coexpression analyses identified OUD-specific modules and gene networks. Integrative analyses between differentially expressed transcripts and genome-wide association study datasets using linkage disequilibrium scores assessed the genetic liability of psychiatric-related phenotypes in OUD. Results Rank-rank hypergeometric overlap analyses revealed extensive overlap in transcripts between the dorsolateral prefrontal cortex and nucleus accumbens in OUD, related to synaptic remodeling and neuroinflammation. Identified transcripts were enriched for factors that control proinflammatory cytokine, chondroitin sulfate, and extracellular matrix signaling. Cell-type deconvolution implicated a role for microglia as a potential driver for opioid-induced neuroplasticity. Linkage disequilibrium score analysis suggested genetic liabilities for risky behavior, attention-deficit/hyperactivity disorder, and depression in subjects with OUD. Conclusions Overall, our findings suggest connections between the brain’s immune system and opioid dependence in the human brain.

Mammalian nephrons originate from a population of nephron progenitor cells (NPCs), and it is known that NPCs’ transcriptomes change throughout nephrogenesis during healthy kidney development. To characterize chromatin accessibility and microRNA (miRNA) expression throughout this process, we collected NPCs from mouse kidneys at embryonic day 14.5 (E14.5) and postnatal day zero (P0) and assayed cells for transposase-accessible chromatin and small RNA expression. We observe 46,374 genomic regions of accessible chromatin, with 2,103 showing significant changes in accessibility between E14.5 and P0. In addition, we detect 1,104 known microRNAs, with 114 showing significant changes in expression. Genome-wide, changes in DNA accessibility and microRNA expression highlight biological processes like cellular differentiation, cell migration, extracellular matrix interactions, and developmental signaling pathways such as Notch. Furthermore, our data identify novel candidate cis-regulatory elements for Eya1 and Pax8, both genes with a role in NPC differentiation; we also associate expression-changing microRNAs, including let-7-5p, miR-125b-5p, miR-181a-2-3p, and miR-9-3p, with candidate cis-regulatory elements. Overall, our data characterize NPCs during kidney development and point out new candidate regulatory elements for genes and microRNA with key roles in nephrogenesis.

Medium spiny neurons (MSNs) constitute the vast majority of striatal neurons and the principal interface between dopamine reward signals and functionally diverse cortico-basal ganglia circuits. Information processing in these circuits is dependent on distinct MSN types – cell types that are traditionally defined according to their projection targets or dopamine receptor expression. Single cell transcriptional studies have revealed greater MSN heterogeneity than predicted by traditional circuit models, but the transcriptional landscape in the primate striatum remains unknown. Here, we set out to establish molecular definitions for MSN subtypes in Rhesus monkeys, and to explore the relationships between transcriptionally defined subtypes and anatomical subdivisions of the striatum. Our results suggest at least nine MSN subtypes, including dorsal striatum subtypes associated with striosome and matrix compartments, ventral striatum subtypes associated with the nucleus accumbens shell and olfactory tubercle, and an MSN-like cell type restricted to μ-opioid receptor rich islands in the ventral striatum. Although each subtype was demarcated by discontinuities in gene expression, continuous variation within subtypes defined gradients corresponding to anatomical locations and, potentially, functional specializations. These results lay the foundation for achieving cell-type-specific transgenesis in the primate striatum and provide a blueprint for investigating circuit specific information processing.

Background Evolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation of many enhancers, distal regulatory elements that control spatial and temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high. Results We present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that can predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Next, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss of regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity have a stronger tendency than the general population to have predicted lineage-specific open chromatin. Conclusion The framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements.

Single amino acid variants (SAVs) may provide clues to understanding evolution of traits. A complex trait that has evolved convergently among species is vocal learning, the rare ability to imitate sounds heard and an important component of spoken-language. Here we assessed whether convergent vocal learning bird species have convergent SAVs (CSAVs) that could be associated with their specialized trait. We analyzed avian genomes and identified CSAVs in vocal learners, but also in most species combinations tested. The number of CSAVs among species was proportional to the product of the most recent common ancestor (MRCA; origin) branch lengths of the species in question, and vocal learning birds did not exceed the overall proportion in most test. However, genes with identical CSAVs (iCSAVs) in vocal learning species were uniquely enriched in ‘learning’ functions, and a subset of iCSAV genes were under positive selection and had enriched specialized regulation in vocal learning and their adjacent brain subdivisions. Several top candidate genes converge on the cAMP signaling pathway, including DRD1B and PRKAR2B. Our findings suggest a complex mechanism of amino acid convergences and specialized gene regulation upon which selection acts for specialized convergent traits.

In mammals, fine motor control is essential for skilled behavior, and is subserved by specialized subdivisions of the primary motor cortex (M1) and other components of the brain’s motor circuitry. We profiled the epigenomic state of several components of the Rhesus macaque motor system, including subdivisions of M1 corresponding to hand and orofacial control. We compared this to open chromatin data from M1 in rat, mouse, and human. We found broad similarities as well as unique specializations in open chromatin regions (OCRs) between M1 subdivisions and other brain regions, as well as species- and lineage-specific differences reflecting their evolutionary histories. By distinguishing shared mammalian M1 OCRs from primate- and human-specific specializations, we highlight gene regulatory programs that could subserve the evolution of skilled motor behaviors such as speech and tool use.

The striatum is the neural interface between dopamine reward signals and cortico-basal ganglia circuits responsible for value assignments, decisions, and actions. Medium spiny neurons (MSNs) make up the vast majority of striatal neurons and are traditionally classified as two distinct types: direct- and indirect-pathway MSNs. The direct- and indirect-pathway model has been useful for understanding some aspects of striatal functions, but it accounts for neither the anatomical heterogeneity, nor the functional diversity of the striatum. Here, we use single nucleus RNA-sequencing and Fluorescent In-Situ Hybridization to explore MSN diversity in the Rhesus macaque striatum. We identified MSN subtypes that correspond to the major subdivisions of the striatum. These include dorsal striatum subtypes associated with striosome and matrix compartments, as well as ventral striatum subtypes associated with the shell of the nucleus accumbens. We also describe a cell type that is anatomically restricted to "Neurochemically Unique Domains in the Accumbens and Putamen (NUDAPs)". Together, these results help to advance nonhuman primate studies into the genomics era. The identified cell types provide a comprehensive blueprint for investigating cell type-specific information processing, and the differentially expressed genes lay a foundation for achieving cell type-specific transgenesis in the primate striatum.

Adeno-associated virus (AAV)-mediated gene therapies are rapidly advancing to the clinic, and AAV engineering has resulted in vectors with increased ability to deliver therapeutic genes. Although the choice of vector is critical, quantitative comparison of AAVs, especially in large animals, remains challenging. Here, we developed an efficient single-cell AAV engineering pipeline (scAAVengr) to quantify efficiency of AAV-mediated gene expression across all cell types. scAAVengr allows for definitive, head-to-head comparison of vectors in the same animal. To demonstrate proof-of-concept for the scAAVengr workflow, we quantified – with cell-type resolution – the abilities of naturally occurring and newly engineered AAVs to mediate gene expression in primate retina following intravitreal injection. A top performing variant, K912, was used to deliver SaCas9 and edit the rhodopsin gene in macaque retina, resulting in editing efficiency similar to infection rates detected by the scAAVengr workflow. These results validate scAAVengr as a powerful method for development of AAV vectors.

BackgroundPrevalence rates of opioid use disorder (OUD) have increased dramatically, accompanied by a surge of overdose deaths. While opioid dependence has been extensively studied in preclinical models, an understanding of the biological alterations that occur in the brains of people who chronically use opioids and who are diagnosed with OUD remains limited. To address this limitation, RNA-sequencing (RNA-seq) was conducted on the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc), regions heavily implicated in OUD, from postmortem brains in subjects with OUD.MethodsWe performed RNA-seq on the DLPFC and NAc from unaffected comparison subjects (n=20) and subjects diagnosed with OUD (n=20). Our transcriptomic analyses identified differentially expressed (DE) transcripts and investigated the transcriptional coherence between brain regions using rank-rank hypergeometric ordering (RRHO). Weighted gene co-expression analyses (WGCNA) also identified OUD-specific modules and gene networks. Integrative analyses between DE transcripts and GWAS datasets using linkage disequilibrium score (LDSC) assessed the genetic liability psychiatric-related phenotypes.ResultsRRHO analyses revealed extensive overlap in transcripts between DLPFC and NAc in OUD, primarily relating to synaptic remodeling and neuroinflammation. Identified transcripts were enriched for factors that control pro-inflammatory cytokine-mediated, chondroitin sulfate, and extracellular matrix signaling. Cell-type deconvolution implicated a role for microglia as a critical driver for opioid-induced neuroplasticity. Using LDSC, we discovered genetic liabilities for risky behavior, attention deficit hyperactivity disorder, and depression.ConclusionsOverall, our findings reveal new connections between the brain’s immune system and opioid dependence in the human brain.

Significance The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19, a major pandemic that threatens millions of human lives and the global economy. We identified a large number of mammals that can potentially be infected by SARS-CoV-2 via their ACE2 proteins. This can assist the identification of intermediate hosts for SARS-CoV-2 and hence reduce the opportunity for a future outbreak of COVID-19. Among the species we found with the highest risk for SARS-CoV-2 infection are wildlife and endangered species. These species represent an opportunity for spillover of SARS-CoV-2 from humans to other susceptible animals. Given the limited infectivity data for the species studied, we urge caution not to overinterpret the predictions of the present study., The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of COVID-19. The main receptor of SARS-CoV-2, angiotensin I converting enzyme 2 (ACE2), is now undergoing extensive scrutiny to understand the routes of transmission and sensitivity in different species. Here, we utilized a unique dataset of ACE2 sequences from 410 vertebrate species, including 252 mammals, to study the conservation of ACE2 and its potential to be used as a receptor by SARS-CoV-2. We designed a five-category binding score based on the conservation properties of 25 amino acids important for the binding between ACE2 and the SARS-CoV-2 spike protein. Only mammals fell into the medium to very high categories and only catarrhine primates into the very high category, suggesting that they are at high risk for SARS-CoV-2 infection. We employed a protein structural analysis to qualitatively assess whether amino acid changes at variable residues would be likely to disrupt ACE2/SARS-CoV-2 spike protein binding and found the number of predicted unfavorable changes significantly correlated with the binding score. Extending this analysis to human population data, we found only rare (frequency

Accumulation of tau and amyloid-β are two pathologic hallmarks of Alzheimer’s disease (AD). We conducted an epigenome-wide association study using the H3K9 acetylation (H3K9ac) mark in 669 aged human prefrontal cortices: in contrast to amyloid-β, tau protein burden had a broad effect on the epigenome, affecting 5,990 out of 26,384 H3K9ac domains. Tau-related alterations aggregated in large genomic segments reflecting spatial chromatin organization, and the magnitude of these effects correlated with the segment’s nuclear lamina association. Functional relevance of these chromatin changes was demonstrated by (1) consistent transcriptional changes in three independent datasets and (2) similar findings in two AD mouse models. Finally, we found that tau overexpression in iPSC-derived neurons altered chromatin structure and that these effects could be blocked by a small molecule predicted to reverse the tau effect. Thus, we report broad tau-driven chromatin rearrangements in the aging human brain that may be reversible with Hsp90 inhibitors., Reporting Summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Summary Diverse traits have evolved through cis-regulatory changes in genome sequence that influence the magnitude, timing and cell type-specificity of gene expression. Advances in high-throughput sequencing and regulatory genomics have led to the identification of regulatory elements in individual species, but these genomic regions remain difficult to align across taxonomic orders due to their lack of sequence conservation relative to protein coding genes. The groundwork for tracing the evolution of regulatory elements is provided by the recent assembly of hundreds of genomes, the generation of reference-free Cactus multiple sequence alignments of these genomes, and the development of the halLiftover tool for mapping regions across these alignments. We present halLiftover Post-processing for the Evolution of Regulatory Elements (HALPER), a tool for constructing contiguous regulatory element orthologs from the outputs of halLiftover. We anticipate that this tool will enable users to efficiently identify orthologs of regulatory elements across hundreds of species, providing novel insights into the evolution of traits that have evolved through gene expression. Availability and implementation HALPER is implemented in python and available on github: https://github.com/pfenninglab/halLiftover-postprocessing. Supplementary information Supplementary data are available at Bioinformatics online.

The Zoonomia Project is investigating the genomics of shared and specialized traits in eutherian mammals. Here we provide genome assemblies for 131 species, of which all but 9 are previously uncharacterized, and describe a whole-genome alignment of 240 species of considerable phylogenetic diversity, comprising representatives from more than 80% of mammalian families. We find that regions of reduced genetic diversity are more abundant in species at a high risk of extinction, discern signals of evolutionary selection at high resolution and provide insights from individual reference genomes. By prioritizing phylogenetic diversity and making data available quickly and without restriction, the Zoonomia Project aims to support biological discovery, medical research and the conservation of biodiversity., This project was funded by NIH NHGRI R01HG008742 (E.K.K., B.B., D.P.G., R.S., J.T.-M., J.J., H.J.N., B.P. and J. Armstrong), Swedish Research Council Distinguished Professor Award (K.L.-T., V.D.M., E.M. and J.R.S.M.), Swedish Research Council grant 2018-05973 (K.L.-T.), Knut and Alice Wallenberg Foundation (K.L.-T., V.D.M., E.M. and J.R.S.M.), Uppsala University (K.L.-T., V.D.M., E.M., J.R.S.M., J.J., J. Alfoldi and L.G.), Broad Institute Next10 (L.G.), Gladstone Institutes (K.S.P.), NIH NHGRI 5R01HG002939 (A.F.A.S. and R.H.), NIH NHGRI 5U24HG010136 (A.F.A.S. and R.H.), NIH NHGRI 5R01HG010485 (B.P. and M.D.), NIH NHGRI 2U41HG007234 (B.P., M.D. and J. Armstrong), NIH NIA 5PO1AG047200 (V.N.G.), NIH NIA 1UH2AG064706 (V.N.G.), BFU2017-86471-P MINECO/FEDER, UE (T.M.-B.), Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya GRC 2017 SGR 880 (T.M.-B.), Howard Hughes International Early Career (T.M.-B.), European Research Council Horizon 2020 no. 864203 (T.M.-B.), Obra Social ‘La Caixa’ (T.M.-B.), BBSRC BBS/E/T/000PR9818, BBS/E/T/ 000PR9783 (W.H. and W.N.), BBSRC Core Strategic Programme Grant BB/P016774/1 (W.H., W.N. and F.D.), Sir Henry Dale Fellowship 200517/Z/16/Z jointly funded by the Wellcome Trust and the Royal Society (N.R.C.), FJCI-2016-29558 MICINN (D.J.), Prince Albert II Foundation of Monaco and Canada, Global Genome Initiative, Smithsonian Institution (M.N.), European Research Council Research Grant ERC-2012-StG311000 (E.C.T.), Irish Research Council Laureate Award (E.C.T.), UK Medical Research Council MR/P026028/1 (W.H. and W.N.), National Science Foundation DEB-1457735 (M.S.S.), National Science Foundation DEB-1753760 (W.J.M.), National Science Foundation IOS-2029774 (E.K.K. and D.P.G.), Robert and Rosabel Osborne Endowment (H.A.L. and J.D.), Swedish Research Council, FORMAS 221-2012-1531 (J.R.S.M.), NSF RoL: FELS: EAGER: DEB 1838283 (D.A.R.) and Academy of Finland grant to Center of Excellence in Tumor Genetics Research no. 312042 (T.K. and J.T.).

Vocal learning is a behavioral trait in which the social and acoustic environment shapes the vocal repertoire of individuals. Over the past century, the study of vocal learning has progressed at the intersection of ecology, physiology, neuroscience, molecular biology, genomics, and evolution. Yet, despite the complexity of this trait, vocal learning is frequently described as a binary trait, with species being classified as either vocal learners or vocal non-learners. As a result, studies have largely focused on a handful of species for which strong evidence for vocal learning exists. Recent studies, however, suggest a continuum in vocal learning capacity across taxa. Here, we further suggest that vocal learning is a multi-component behavioral phenotype comprised of distinct yet interconnected modules. Discretizing the vocal learning phenotype into its constituent modules would facilitate integration of findings across a wider diversity of species, taking advantage of the ways in which each excels in a particular module, or in a specific combination of features. Such comparative studies can improve understanding of the mechanisms and evolutionary origins of vocal learning. We propose an initial set of vocal learning modules supported by behavioral and neurobiological data and highlight the need for diversifying the field in order to disentangle the complexity of the vocal learning phenotype.

Promoters initiate RNA synthesis, and enhancers stimulate promoter activity. Whether promoter and enhancer activities are encoded distinctly in DNA sequences is unknown. We measured the enhancer and promoter activities of thousands of DNA fragments transduced into mouse neurons. We focused on genomic loci bound by the neuronal activity-regulated coactivator CREBBP, and we measured enhancer and promoter activities both before and after neuronal activation. We find that the same sequences typically encode both enhancer and promoter activities. However, gene promoters generate more promoter activity than distal enhancers, despite generating similar enhancer activity. Surprisingly, the greater promoter activity of gene promoters is not due to conventional core promoter elements or splicing signals. Instead, we find that particular transcription factor binding motifs are intrinsically biased toward the generation of promoter activity, whereas others are not. Although the specific biases we observe may be dependent on experimental or cellular context, our results suggest that gene promoters are distinguished from distal enhancers by specific complements of transcriptional activators., National Institute of Mental Health (U.S.) (Grant R01 MH101528-01)

This study describes a gauge for brain aging predictive of cognitive decline and Alzheimer's disease synergistic with the APOE ε4 allele., Advanced age and the APOE ε4 allele are the two biggest risk factors for Alzheimer’s disease (AD) and declining cognitive function. We describe a universal gauge to measure molecular brain age using transcriptome analysis of four human postmortem cohorts (n = 673, ages 25–97) free of neurological disease. In a fifth cohort of older subjects with or without neurological disease (n = 438, ages 67–108), we show that subjects with brains deviating in the older direction from what would be expected based on chronological age show an increase in AD, Parkinson’s disease, and cognitive decline. Strikingly, a younger molecular age (−5 yr than chronological age) protects against AD even in the presence of APOE ε4. An established DNA methylation gauge for age correlates well with the transcriptome gauge for determination of molecular age and assigning deviations from the expected. Our results suggest that rapid brain aging and APOE ε4 are synergistic risk factors, and interventions that slow aging may substantially reduce risk of neurological disease and decline even in the presence of APOE ε4.

SUMMARYAll living things experience entropy, manifested as a loss of inherited genetic and epigenetic information over time. As budding yeast cells age, epigenetic changes result in a loss of cell identity and sterility, both hallmarks of yeast aging. In mammals, epigenetic information is also lost over time, but what causes it to be lost and whether it is a cause or a consequence of aging is not known. Here we show that the transient induction of genomic instability, in the form of a low number of non-mutagenic DNA breaks, accelerates many of the chromatin and tissue changes seen during aging, including the erosion of the epigenetic landscape, a loss of cellular identity, advancement of the DNA methylation clock and cellular senescence. These data support a model in which a loss of epigenetic information is a cause of aging in mammals.One Sentence SummaryThe act of repairing DNA breaks induces chromatin reorganization and a loss of cell identity that may contribute to mammalian aging

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 IIβ (Topo IIβ), and knockdown of Topo IIβ 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., National Institutes of Health (U.S.) (R01HG004037-07), National Human Genome Research Institute (U.S.) (R01HG004037-07), National Institutes of Health (U.S.) (RC1HG005334), National Human Genome Research Institute (U.S.) (RC1HG005334), National Institutes of Health (U.S.) (R01AG046174)

SummaryAccumulation of tau and amyloid-β are two pathologic hallmarks of Alzheimer’s disease (AD). Here, we conducted an epigenome-wide association study using the H3K9 acetylation (H3K9Ac) mark in 669 aged human prefrontal cortices: in contrast to amyloid-β, tau protein burden had a broad effect on the epigenome, affecting 5,590 out of 26,384 H3K9Ac domains. Tau-related alterations aggregated in large genomic segments reflecting spatial chromatin organization, and the magnitude of these effects correlated with the segment’s nuclear lamina association. We confirmed the functional relevance of these chromatin changes by demonstrating (1) consistent transcriptional changes in three independent datasets and (2) similar findings in two AD mouse models. Finally, we found that tau overexpression in iPSC-derived neurons disrupted chromatin organization and that these effects could be blocked by a small molecule predicted to reverse the tau effect. Thus, we report large-scale tau-driven chromatin rearrangements in the aging human brain that may be reversible with HSP90 inhibitors.

Alzheimer’s disease (AD) is a severe age-related neurodegenerative disorder characterized by accumulation of amyloid-β plaques and neurofibrillary tangles, synaptic and neuronal loss, and cognitive decline. Several genes have been implicated in AD, but chromatin state alterations during neurodegeneration remain uncharacterized. Here we profile transcriptional and chromatin state dynamics across early and late pathology in the hippocampus of an inducible mouse model of AD-like neurodegeneration. We find a coordinated downregulation of synaptic plasticity genes and regulatory regions, and upregulation of immune response genes and regulatory regions, which are targeted by factors that belong to the ETS family of transcriptional regulators, including PU.1. Human regions orthologous to increasing-level enhancers show immune-cell-specific enhancer signatures as well as immune cell expression quantitative trait loci, while decreasing-level enhancer orthologues show fetal-brain-specific enhancer activity. Notably, AD-associated genetic variants are specifically enriched in increasing-level enhancer orthologues, implicating immune processes in AD predisposition. Indeed, increasing enhancers overlap known AD loci lacking protein-altering variants, and implicate additional loci that do not reach genome-wide significance. Our results reveal new insights into the mechanisms of neurodegeneration and establish the mouse as a useful model for functional studies of AD regulatory regions., Belfer Neurodegeneration Consortium, National Institutes of Health (U.S.) (National Institute of Neurological Disorders and Stroke (U.S.)/National Institute on Aging RO1NS078839), Swiss National Science Foundation (Early Postdoc Mobility Fellowship P2BSP3_151885), National Institutes of Health (U.S.) (National Human Genome Research Institute (U.S.) R01HG004037-07), National Institutes of Health (U.S.) (National Human Genome Research Institute (U.S.) RC1HG005334)

Neuron subtype dysfunction is a key contributor to neurologic disease circuits, but identifying associated gene regulatory pathways is complicated by the molecular complexity of the brain. For example, parvalbumin-expressing (PV+) neurons in the external globus pallidus (GPe) are critically involved in the motor deficits of dopamine-depleted mouse models of Parkinson's disease, where cell type-specific optogenetic stimulation of PV+neurons over other neuron populations rescues locomotion. Despite the distinct roles these cell types play in the neural circuit, the molecular correlates remain unknown because of the difficulty of isolating rare neuron subtypes. To address this issue, we developed a new viral affinity purification strategy, Cre-Specific Nuclear Anchored Independent Labeling, to isolate Cre recombinase-expressing (Cre+) nuclei from the adult mouse brain. Applying this technology, we performed targeted assessments of the cell type-specific transcriptomic and epigenetic effects of dopamine depletion on PV+and PV–cells within three brain regions of male and female mice: GPe, striatum, and cortex. We found GPe PV+neuron-specific gene expression changes that suggested increased hypoxia-inducible factor 2α signaling. Consistent with transcriptomic data, regions of open chromatin affected by dopamine depletion within GPe PV+neurons were enriched for hypoxia-inducible factor family binding motifs. The gene expression and epigenomic experiments performed on PV+neurons isolated by Cre-Specific Nuclear Anchored Independent Labeling identified a transcriptional regulatory network mediated by the neuroprotective factor Hif2a as underlying neural circuit differences in response to dopamine depletion.SIGNIFICANCE STATEMENTCre-Specific Nuclear Anchored Independent Labeling is an enhanced, virus-based approach to isolate nuclei of a specific cell type for transcriptome and epigenome interrogation that decreases dependency on transgenic animals. Applying this technology to GPe parvalbumin-expressing neurons in a mouse model of Parkinson's disease, we discovered evidence for an upregulation of the oxygen homeostasis maintaining pathway involving Hypoxia-inducible factor 2α. These results provide new insight into how neuron subtypes outside the substantia nigra pars compacta may be compensating at a molecular level for differences in the motor production neural circuit during the progression of Parkinson's disease. Furthermore, they emphasize the utility of cell type-specific technologies, such as Cre-Specific Nuclear Anchored Independent Labeling, for isolated assessment of specific neuron subtypes in complex systems.

Advanced age and the APOE ε4 allele are the two biggest risk factors for Alzheimer’s disease (AD) and declining cognitive function. We describe a universal gauge to measure molecular brain age using transcriptome analysis of four human postmortem cohorts (n = 673, ages 25–97) free of neurological disease. In a fifth cohort of older subjects with or without neurological disease (n = 438, ages 67–108), we show that subjects with brains deviating in the older direction from what would be expected based on chronological age show an increase in AD, Parkinson’s disease, and cognitive decline. Strikingly, a younger molecular age (−5 yr than chronological age) protects against AD even in the presence of APOE ε4. An established DNA methylation gauge for age correlates well with the transcriptome gauge for determination of molecular age and assigning deviations from the expected. Our results suggest that rapid brain aging and APOE ε4 are synergistic risk factors, and interventions that slow aging may substantially reduce risk of neurological disease and decline even in the presence of APOE ε4.

In our companion study (Jarvis et al. [2013] J Comp Neurol. doi: 10.1002/cne.23404) we used quantitative brain molecular profiling to discover that distinct subdivisions in the avian pallium above and below the ventricle and the associated mesopallium lamina have similar molecular profiles, leading to a hypothesis that they may form as continuous subdivisions around the lateral ventricle. To explore this hypothesis, here we profiled the expression of 16 genes at eight developmental stages. The genes included those that define brain subdivisions in the adult and some that are also involved in brain development. We found that phyletic hierarchical cluster and linear regression network analyses of gene expression profiles implicated single and mixed ancestry of these brain regions at early embryonic stages. Most gene expression-defined pallial subdivisions began as one ventral or dorsal domain that later formed specific folds around the lateral ventricle. Subsequently a clear ventricle boundary formed, partitioning them into dorsal and ventral pallial subdivisions surrounding the mesopallium lamina. These subdivisions each included two parts of the mesopallium, the nidopallium and hyperpallium, and the arcopallium and hippocampus, respectively. Each subdivision expression profile had a different temporal order of appearance, similar in timing to the order of analogous cell types of the mammalian cortex. Furthermore, like the mammalian pallium, expression in the ventral pallial subdivisions became distinct during prehatch development, whereas the dorsal portions did so during posthatch development. These findings support the continuum hypothesis of avian brain subdivision development around the ventricle and influence hypotheses on homologies of the avian pallium with other vertebrates.

Neuronal activity sculpts brain development by inducing the transcription of genes such as brain-derived neurotrophic factor (Bdnf) that modulate the function of synapses. Sensory experience is transduced into changes in gene transcription via the activation of calcium signaling pathways downstream of both L-type voltage-gated calcium channels (L-VGCCs) and NMDA-type glutamate receptors (NMDARs). These signaling pathways converge on the regulation of transcription factors including calcium-response factor (CaRF). Although CaRF is dispensable for the transcriptional induction of Bdnf following the activation of L-VGCCs, here we show that the loss of CaRF leads to enhanced NMDAR-dependent transcription of Bdnf as well as Arc. We identify the NMDAR subunit-encoding gene Grin3a as a regulatory target of CaRF, and we show that expression of both Carf and Grin3a is depressed by the elevation of intracellular calcium, linking the function of this transcriptional regulatory pathway to neuronal activity. We find that light-dependent activation of Bdnf and Arc transcription is enhanced in the visual cortex of young CaRF knockout mice, suggesting a role for CaRF-dependent dampening of NMDAR-dependent transcription in the developing brain. Finally, we demonstrate that enhanced Bdnf expression in CaRF-lacking neurons increases inhibitory synapse formation. Taken together, these data reveal a novel role for CaRF as an upstream regulator of NMDAR-dependent gene transcription and synapse formation in the developing brain. NMDARs promote brain development by inducing the transcription of genes, including brain-derived neurotrophic factor (BDNF). We show that the transcription factor calcium-response factor (CaRF) limits NMDAR-dependent BDNF induction by regulating expression of the NMDAR subunit GluN3A. Loss of CaRF leads to enhanced BDNF-dependent GABAergic synapse formation indicating the importance of this process for brain development. Our observation that both CaRF and GluN3A are down-regulated by intracellular calcium suggests that this may be a mechanism for experience-dependent modulation of synapse formation.

INTRODUCTION Brain activity drives both behavior and regulated gene expression in neurons. Although past studies have identified activity-induced signaling and gene regulation cascades in cultured neurons, much less is known about how activity- dependent transcriptional networks are affected by the variations in cell-type composition, network interconnections, and firing patterns that comprise behaviorally active brain circuits in vivo. Dual mechanism model for behaviorally regulated gene expression diversity. ( Left ) Song brain circuit and zebra finch song motif (transcribed using https://my.scorecloud.com). ( Middle ) Song nucleus–specific (RA, red; Area X, blue) singing- regulated genes (gene A and gene B) in response to neural f ring (yellow). ( Right ) Region-general EATF and region-specific TF only bind to genomic DNA (lines) with region-specific acetylated histone 3 (H3K27ac peaks) and then transcribe their mRNAs (green arrow). RATIONALE We tested the hypothesis that behaviorally regulated gene expression is anatomically and temporally diverse and that the key determinants of this diversity are networks of transcription factors, their genomic binding sites, and epigenetic chromatin states. We analyzed genome-wide, singing-regulated gene expression across time in the four major forebrain regions of the song control system in songbirds, a model of speech production in humans. We then performed a transcription factor motif analysis to identify gene regulatory networks enriched in each song nucleus and measured acetylation of histone 3 at lysine 27 (H3K27ac) to identify chromatin regions that were transcriptionally active in the genomes of song nuclei before and after singing. RESULTS We found that singing was associated with differential regulation of about 10% of all genes in the avian genome that came in several waves across time. Less than 1% of these genes were comparably regulated in all song nuclei tested, and these comprised a core set dominated by immediate-early gene (IEG) transcription factors. By contrast, the vast majority of singing-regulated genes were regulated in only one or a subset of song nuclei, such that each song nucleus had its own dominant subset of genes regulated with defined temporal profiles, controlling a variety of functions. The promoters of many of the singing-regulated genes contained binding motifs for known early-activated transcription factors (EATFs) that become active in response to neural firing, some of which were expressed differentially between song nuclei at baseline. One EATF, calcium-response factor ( CaRF ), was tested with RNA interference knockdown in cultured neurons and found to regulate the predicted genes in response to neural activity, but was also found to modulate their expression even at baseline. More strikingly, we found with H3K27ac analysis that many song nucleus–specific singing-regulated genes did not show increased chromatin regulatory element activity after singing but rather already had primed region-specific regulatory activity before singing began. CONCLUSIONS We propose a dual mechanism for the diversity of behaviorally regulated genes across different brain regions in vivo (see the figure). First, the neural activity associated with singing activates EATFs, and some TFs differentially expressed in brain regions at baseline, to trigger region-specific expression of their target genes. Second, brain region–specific enhancers near activity- regulated genes are waiting in an epigenetically primed state, ready to modulate transcription of general and song nucleus–specific genes at a moment’s notice when the neurons fire. The combination of these two mechanisms underlies a great diversity of behaviorally regulated gene expression, permitting each nucleus to perform its particular function in this complex behavior.

Song-learning birds and humans share independently evolved similarities in brain pathways for vocal learning that are essential for song and speech and are not found in most other species. Comparisons of brain transcriptomes of song-learning birds and humans relative to vocal nonlearners identified convergent gene expression specializations in specific song and speech brain regions of avian vocal learners and humans. The strongest shared profiles relate bird motor and striatal song-learning nuclei, respectively, with human laryngeal motor cortex and parts of the striatum that control speech production and learning. Most of the associated genes function in motor control and brain connectivity. Thus, convergent behavior and neural connectivity for a complex trait are associated with convergent specialized expression of multiple genes.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease., National Human Genome Research Institute (U.S.) (RC1HG005334), National Human Genome Research Institute (U.S.) (R01HG004037), National Human Genome Research Institute (U.S.) (R01HG004037-S1), National Human Genome Research Institute (U.S.) (RO1NS078839), National Science Foundation (U.S.) (CAREER Award 1254200)

Based on quantitative cluster analyses of 52 constitutively expressed or behaviorally regulated genes in 23 brain regions, we present a global view of telencephalic organization of birds. The patterns of constitutively expressed genes revealed a partial mirror image organization of three major cell populations that wrap above, around, and below the ventricle and adjacent lamina through the mesopallium. The patterns of behaviorally regulated genes revealed functional columns of activation across boundaries of these cell populations, reminiscent of columns through layers of the mammalian cortex. The avian functionally regulated columns were of two types: those above the ventricle and associated mesopallial lamina, formed by our revised dorsal mesopallium, hyperpallium, and intercalated hyperpallium; and those below the ventricle, formed by our revised ventral mesopallium, nidopallium, and intercalated nidopallium. Based on these findings and known connectivity, we propose that the avian pallium has four major cell populations similar to those in mammalian cortex and some parts of the amygdala: 1) a primary sensory input population (intercalated pallium); 2) a secondary intrapallial population (nidopallium/hyperpallium); 3) a tertiary intrapallial population (mesopallium); and 4) a quaternary output population (the arcopallium). Each population contributes portions to columns that control different sensory or motor systems. We suggest that this organization of cell groups forms by expansion of contiguous developmental cell domains that wrap around the lateral ventricle and its extension through the middle of the mesopallium. We believe that the position of the lateral ventricle and its associated mesopallium lamina has resulted in a conceptual barrier to recognizing related cell groups across its border, thereby confounding our understanding of homologies with mammals. J. Comp. Neurol. 521:3614–3665, 2013. © 2013 Wiley Periodicals, Inc.

Sodium appetite is an instinct that involves avid specific intention. It is elicited by sodium deficiency, stress-evoked adrenocorticotropic hormone (ACTH), and reproduction. Genome-wide microarrays in sodium-deficient mice or after ACTH infusion showed up-regulation of hypothalamic genes, including dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa (DARPP-32), dopamine receptors-1 and -2, α-2C- adrenoceptor, and striatally enriched protein tyrosine phosphatase (STEP). Both DARPP-32 and neural plasticity regulator activity-regulated cytoskeleton associated protein (ARC) were up-regulated in lateral hypothalamic orexinergic neurons by sodium deficiency. Administration of dopamine D1 (SCH23390) and D2 receptor (raclopride) antagonists reduced gratification of sodium appetite triggered by sodium deficiency. SCH23390 was specific, having no effect on osmotic-induced water drinking, whereas raclopride also reduced water intake. D1 receptor KO mice had normal sodium appetite, indicating compensatory regulation. Appetite was insensitive to SCH23390, confirming the absence of off-target effects. Bilateral microinjection of SCH23390 (100 nM in 200 nL) into rats’ lateral hypothalamus greatly reduced sodium appetite. Gene set enrichment analysis in hypothalami of mice with sodium appetite showed significant enrichment of gene sets previously linked to addiction (opiates and cocaine). This finding of concerted gene regulation was attenuated on gratification with perplexingly rapid kinetics of only 10 min, anteceding significant absorption of salt from the gut. Salt appetite and hedonic liking of salt taste have evolved over >100 million y (e.g., being present in Metatheria). Drugs causing pleasure and addiction are comparatively recent and likely reflect usurping of evolutionary ancient systems with high survival value by the gratification of contemporary hedonic indulgences. Our findings outline a molecular logic for instinctive behavior encoded by the brain with possible important translational–medical implications.

Calcium-Response Factor (CaRF) was first identified as a transcription factor based on its affinity for a neuronal-selective calcium-response element (CaRE1) in the gene encoding Brain-Derived Neurotrophic Factor (BDNF). However, because CaRF shares no homology with other transcription factors, its properties and gene targets have remained unknown. Here we show that the DNA binding domain of CaRF has been highly conserved across evolution and that CaRF binds DNA directly in a sequence-specific manner in the absence of other eukaryotic cofactors. Using a binding site selection screen we identify a high-affinity consensus CaRF response element (cCaRE) that shares significant homology with the CaRE1 element of Bdnf. In a genome-wide chromatin immunoprecipitation analysis (ChIP-Seq), we identified 176 sites of CaRF-specific binding (peaks) in neuronal genomic DNA. 128 of these peaks are within 10kB of an annotated gene, and 60 are within 1kB of an annotated transcriptional start site. At least 138 of the CaRF peaks contain a common 10-bp motif with strong statistical similarity to the cCaRE, and we provide evidence predicting that CaRF can bind independently to at least 64.5% of these motifs in vitro. Analysis of this set of putative CaRF targets suggests the enrichment of genes that regulate intracellular signaling cascades. Finally we demonstrate that expression of a subset of these target genes is altered in the cortex of Carf knockout (KO) mice. Together these data strongly support the characterization of CaRF as a unique transcription factor and provide the first insight into the program of CaRF-regulated transcription in neurons.

AIM To identify differences between primed mouse embryonic stem cells (ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state. METHODS We have cultured 3 lines of cells from different mouse strains that have been shown to be naive or primed as determined by generating germline-transmitting chimeras. Cells were put through a battery of tests to measure the different features. RNA from cells was analyzed using microarrays, to determine a priority list of the differentially expressed genes. These were later validated by quantificational real-time polymerase chain reaction. Viral cassettes were created to induce expression of differentially expressed genes in the primed cells through lentiviral transduction. Primed reprogrammed cells were subjected to in-vivo incorporation studies. RESULTS Most results show that both primed and naive cells have similar features (morphology, proliferation rates, stem cell genes expressed). However, there were some genes that were differentially expressed in the naïve cells relative to the primed cells. Key upregulated genes in naïve cells include ESRRB, ERAS, ATRX, RNF17, KLF-5, and MYC. After over-expressing some of these genes the primed cells were able to incorporate into embryos in-vivo, re-acquiring a feature previously absent in these cells. CONCLUSION Although there are no notable phenotypic differences, there are key differences in gene expression between these naïve and primed stem cells. These differences can be overcome through overexpression.

Background Neuronal activity regulates gene expression to control learning and memory, homeostasis of neuronal function, and pathological disease states such as epilepsy. A great deal of experimental evidence supports the involvement of two particular transcription factors in shaping the genomic response to neuronal activity and mediating plasticity: CREB and zif268 (egr-1, krox24, NGFI-A). The gene targets of these two transcription factors are of considerable interest, since they may help develop hypotheses about how neural activity is coupled to changes in neural function. Results We have developed a computational approach for identifying binding sites for these transcription factors within the promoter regions of annotated genes in the mouse, rat, and human genomes. By combining a robust search algorithm to identify discrete binding sites, a comparison of targets across species, and an analysis of binding site locations within promoter regions, we have defined a group of candidate genes that are strong CREB- or zif268 targets and are thus regulated by neural activity. Our analysis revealed that CREB and zif268 share a disproportionate number of targets in common and that these common targets are dominated by transcription factors. Conclusion These observations may enable a more detailed understanding of the regulatory networks that are induced by neural activity and contribute to the plasticity transcriptome. The target genes identified in this study will be a valuable resource for investigators who hope to define the functions of specific genes that underlie activity-dependent changes in neuronal properties.