Fan Gao

We present an organism-wide, transcriptomic cell atlas of the hydrozoan medusa Clytia hemisphaerica and describe how its component cell types respond to perturbation. Using multiplexed single-cell RNA sequencing, in which individual animals were indexed and pooled from control and perturbation conditions into a single sequencing run, we avoid artifacts from batch effects and are able to discern shifts in cell state in response to organismal perturbations. This work serves as a foundation for… Show more

We present an organism-wide, transcriptomic cell atlas of the hydrozoan medusa Clytia hemisphaerica and describe how its component cell types respond to perturbation. Using multiplexed single-cell RNA sequencing, in which individual animals were indexed and pooled from control and perturbation conditions into a single sequencing run, we avoid artifacts from batch effects and are able to discern shifts in cell state in response to organismal perturbations. This work serves as a foundation for future studies of development, function, and regeneration in a genetically tractable jellyfish species. Moreover, we introduce a powerful workflow for high-resolution, whole-animal, multiplexed single-cell genomics that is readily adaptable to other traditional or nontraditional model organisms. Show less

The immature and dysfunctional vascular network within solid tumors poses a substantial obstacle to immunotherapy because it creates a hypoxic tumor microenvironment that actively limits immune cell infiltration. The molecular basis underpinning this vascular dysfunction is not fully understood. Using genome-scale receptor array technology, we showed here that insulin-like growth factor binding protein 7 (IGFBP7) interacts with its receptor CD93, and we subsequently demonstrated that this… Show more

The immature and dysfunctional vascular network within solid tumors poses a substantial obstacle to immunotherapy because it creates a hypoxic tumor microenvironment that actively limits immune cell infiltration. The molecular basis underpinning this vascular dysfunction is not fully understood. Using genome-scale receptor array technology, we showed here that insulin-like growth factor binding protein 7 (IGFBP7) interacts with its receptor CD93, and we subsequently demonstrated that this interaction contributes to abnormal tumor vasculature. Both CD93 and IGFBP7 were up-regulated in tumor-associated endothelial cells. IGFBP7 interacted with CD93 via a domain different from multimerin-2, the known ligand for CD93. In two mouse tumor models, blockade of the CD93/IGFBP7 interaction by monoclonal antibodies promoted vascular maturation to reduce leakage, leading to reduced tumor hypoxia and increased tumor perfusion. CD93 blockade in mice increased drug delivery, resulting in an improved antitumor response to gemcitabine or fluorouracil. Blockade of the CD93 pathway triggered a substantial increase in intratumoral effector T cells, thereby sensitizing mouse tumors to immune checkpoint therapy. Last, analysis of samples from patients with cancer under anti–programmed death 1/programmed death-ligand 1 treatment revealed that overexpression of the IGFBP7/CD93 pathway was associated with poor response to therapy. Thus, our study identified a molecular interaction involved in tumor vascular dysfunction and revealed an approach to promote a favorable tumor microenvironment for therapeutic intervention. Show less

We describe a workflow for preprocessing of single-cell RNA-sequencing data that balances efficiency and accuracy. Our workflow is based on the kallisto and bustools programs, and is near optimal in speed with a constant memory requirement providing scalability for arbitrarily large datasets. The workflow is modular, and we demonstrate its flexibility by showing how it can be used for RNA velocity analyses.

Altered Ng levels affect the phosphorylation landscape of neuronal proteins. PP2B activity is required for mediating the deficit in synaptic plasticity caused by decreasing Ng levels, revealing a novel mechanistic link of a schizophrenia risk gene to cognitive deficits.

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression… Show more

Cerebrospinal fluid (CSF) provides vital support for the brain. Abnormal CSF accumulation, such as hydrocephalus, can negatively affect perinatal neurodevelopment. The mechanisms regulating CSF clearance during the postnatal critical period are unclear. Here, we show that CSF K+, accompanied by water, is cleared through the choroid plexus (ChP) during mouse early postnatal development. We report that, at this developmental stage, the ChP showed increased ATP production and increased expression of ATP-dependent K+ transporters, particularly the Na+, K+, Cl−, and water cotransporter NKCC1. Overexpression of NKCC1 in the ChP resulted in increased CSF K+ clearance, increased cerebral compliance, and reduced circulating CSF in the brain without changes in intracranial pressure in mice. Moreover, ChP-specific NKCC1 overexpression in an obstructive hydrocephalus mouse model resulted in reduced ventriculomegaly. Collectively, our results implicate NKCC1 in regulating CSF K+ clearance through the ChP in the critical period during postnatal neurodevelopment in mice. Show less

The epigenome and three-dimensional (3D) genomic architecture are emerging as key factors in the dynamic regulation of different transcriptional programs required for neuronal functions. In this study, we used an activity-dependent tagging system in mice to determine the epigenetic state, 3D genome architecture and transcriptional landscape of engram cells over the lifespan of memory formation and recall. Our findings reveal that memory encoding leads to an epigenetic priming event, marked by… Show more

The epigenome and three-dimensional (3D) genomic architecture are emerging as key factors in the dynamic regulation of different transcriptional programs required for neuronal functions. In this study, we used an activity-dependent tagging system in mice to determine the epigenetic state, 3D genome architecture and transcriptional landscape of engram cells over the lifespan of memory formation and recall. Our findings reveal that memory encoding leads to an epigenetic priming event, marked by increased accessibility of enhancers without the corresponding transcriptional changes. Memory consolidation subsequently results in spatial reorganization of large chromatin segments and promoter–enhancer interactions. Finally, with reactivation, engram neurons use a subset of de novo long-range interactions, where primed enhancers are brought in contact with their respective promoters to upregulate genes involved in local protein translation in synaptic compartments. Collectively, our work elucidates the comprehensive transcriptional and epigenomic landscape across the lifespan of memory formation and recall in the hippocampal engram ensemble. Show less

The mechanisms by which mutant huntingtin (mHTT) leads to neuronal cell death in Huntington’s disease (HD) are not fully understood. To gain new molecular insights, we used single nuclear RNA sequencing (snRNA-seq) and translating ribosome affinity purification (TRAP) to conduct transcriptomic analyses of caudate/putamen (striatal) cell type-specific gene expression changes in human HD and mouse models of HD. In striatal spiny projection neurons, the most vulnerable cell type in HD, we observe… Show more

The mechanisms by which mutant huntingtin (mHTT) leads to neuronal cell death in Huntington’s disease (HD) are not fully understood. To gain new molecular insights, we used single nuclear RNA sequencing (snRNA-seq) and translating ribosome affinity purification (TRAP) to conduct transcriptomic analyses of caudate/putamen (striatal) cell type-specific gene expression changes in human HD and mouse models of HD. In striatal spiny projection neurons, the most vulnerable cell type in HD, we observe a release of mitochondrial RNA (mtRNA) (a potent mitochondrial-derived innate immunogen) and a concomitant upregulation of innate immune signaling in spiny projection neurons. Further, we observe that the released mtRNAs can directly bind to the innate immune sensor protein kinase R (PKR). We highlight the importance of studying cell type-specific gene expression dysregulation in HD pathogenesis and reveal that the activation of innate immune signaling in the most vulnerable HD neurons provides a novel framework to understand the basis of mHTT toxicity and raises new therapeutic opportunities. Show less

The glycoprotein Reelin maintains neuronal positioning and regulates neuronal plasticity in the adult brain. Reelin deficiency has been associated with neurological diseases. We recently showed that Reelin is depleted in mice with a targeted disruption of the Ndel1 gene in forebrain postnatal excitatory neurons (Ndel1 conditional knockout (CKO)). Ndel1 CKO mice exhibit fragmented microtubules in CA1 pyramidal neurons, profound deterioration of the CA1 hippocampus and a shortened lifespan (~10… Show more

The glycoprotein Reelin maintains neuronal positioning and regulates neuronal plasticity in the adult brain. Reelin deficiency has been associated with neurological diseases. We recently showed that Reelin is depleted in mice with a targeted disruption of the Ndel1 gene in forebrain postnatal excitatory neurons (Ndel1 conditional knockout (CKO)). Ndel1 CKO mice exhibit fragmented microtubules in CA1 pyramidal neurons, profound deterioration of the CA1 hippocampus and a shortened lifespan (~10 weeks). Here we report that Ndel1 CKO mice (of both sexes) experience spatial learning and memory deficits that are associated with deregulation of neuronal cell adhesion, plasticity and neurotransmission genes, as assessed by genome-wide transcriptome analysis of the hippocampus. Importantly, a single injection of Reelin protein in the hippocampus of Ndel1 CKO mice improves spatial learning and memory function and this is correlated with reduced intrinsic hyperexcitability of CA1 pyramidal neurons, and normalized gene deregulation in the hippocampus. Strikingly, when treated with Reelin, Ndel1 CKO animals that die from an epileptic phenotype, live twice as long as nontreated, or vehicle-treated CKO animals. Thus, Reelin confers striking beneficial effects in the CA1 hippocampus, and at both behavioral and organismal levels. Show less

Pioneer factors such as Zelda (Zld) help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls the transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates expression through enhancer sog_Distal along the dorso-ventral axis, we hypothesized Opa’s role is more… Show more

Pioneer factors such as Zelda (Zld) help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls the transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates expression through enhancer sog_Distal along the dorso-ventral axis, we hypothesized Opa’s role is more general. Chromatin-immunoprecipitation (ChIP-seq) confirmed its in vivo binding to sog_Distal but also identified widespread binding throughout the genome, comparable to Zld. Furthermore, chromatin assays (ATAC-seq) demonstrate that Opa, like Zld, influences chromatin accessibility genome-wide at cellularization, suggesting both are pioneer factors with common as well as distinct targets. Lastly, embryos lacking opa exhibit widespread, late patterning defects spanning both axes. Collectively, these data suggest Opa is a general timing factor and likely late-acting pioneer factor that drives a secondary wave of zygotic gene expression. Show less

DNA damage contributes to brain aging and neurodegenerative diseases. However, the factors stimulating DNA repair to stave off functional decline remain obscure. We show that HDAC1 modulates OGG1-initated 8-oxoguanine (8-oxoG) repair in the brain. HDAC1-deficient mice display age-associated DNA damage accumulation and cognitive impairment. HDAC1 stimulates OGG1, a DNA glycosylase known to remove 8-oxoG lesions that are associated with transcriptional repression. HDAC1 deficiency causes impaired… Show more

DNA damage contributes to brain aging and neurodegenerative diseases. However, the factors stimulating DNA repair to stave off functional decline remain obscure. We show that HDAC1 modulates OGG1-initated 8-oxoguanine (8-oxoG) repair in the brain. HDAC1-deficient mice display age-associated DNA damage accumulation and cognitive impairment. HDAC1 stimulates OGG1, a DNA glycosylase known to remove 8-oxoG lesions that are associated with transcriptional repression. HDAC1 deficiency causes impaired OGG1 activity, 8-oxoG accumulation at the promoters of genes critical for brain function, and transcriptional repression. Moreover, we observe elevated 8-oxoG along with reduced HDAC1 activity and downregulation of a similar gene set in the 5XFAD mouse model of Alzheimer’s disease. Notably, pharmacological activation of HDAC1 alleviates the deleterious effects of 8-oxoG in aged wild-type and 5XFAD mice. Our work uncovers important roles for HDAC1 in 8-oxoG repair and highlights the therapeutic potential of HDAC1 activation to counter functional decline in brain aging and neurodegeneration. Show less

Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations… Show more

Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington’s disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways. Show less

The membrane-anchored CX3CL1 is best known to exert its signaling function through binding its receptor CX3CR1. This study demonstrates a novel function that CX3CL1 exerts. CX3CL1 is sequentially cleaved by α-, β-, and γ-secretase, and the released CX3CL1 intracellular domain (CX3CL1-ICD) would translocate into the cell nucleus to alter gene expression due to this back-signaling function. Amyloid deposition and neuronal loss were significantly reduced when membrane-anchored CX3CL1 C-terminal… Show more

The membrane-anchored CX3CL1 is best known to exert its signaling function through binding its receptor CX3CR1. This study demonstrates a novel function that CX3CL1 exerts. CX3CL1 is sequentially cleaved by α-, β-, and γ-secretase, and the released CX3CL1 intracellular domain (CX3CL1-ICD) would translocate into the cell nucleus to alter gene expression due to this back-signaling function. Amyloid deposition and neuronal loss were significantly reduced when membrane-anchored CX3CL1 C-terminal fragment (CX3CL1-ct) was overexpressed in Alzheimer’s 5xFAD mouse model. The reversal of neuronal loss in 5xFAD can be attributed to increased neurogenesis by CX3CL1-ICD, as revealed by morphological and unbiased RNA-sequencing analyses. Mechanistically, this CX3CL1 back-signal likely enhances developmental and adult neurogenesis through the TGFβ2/3-Smad2/3 pathway and other genes important for neurogenesis. Induction of CX3CL1 back-signaling may not only be a promising novel mechanism to replenish neuronal loss but also for reducing amyloid deposition for Alzheimer’s treatment. Show less

Neuronal and synaptic loss is characteristic in many neurodegenerative diseases, such as frontotemporal dementia and Alzheimer’s disease. Recently, we showed that inducing gamma oscillations with visual stimulation (gamma entrainment using sensory stimuli, or GENUS) reduced amyloid plaques and phosphorylated tau in multiple mouse models. Whether GENUS can affect neurodegeneration or cognitive performance remains unknown. Here, we demonstrate that GENUS can entrain gamma oscillations in the… Show more

Neuronal and synaptic loss is characteristic in many neurodegenerative diseases, such as frontotemporal dementia and Alzheimer’s disease. Recently, we showed that inducing gamma oscillations with visual stimulation (gamma entrainment using sensory stimuli, or GENUS) reduced amyloid plaques and phosphorylated tau in multiple mouse models. Whether GENUS can affect neurodegeneration or cognitive performance remains unknown. Here, we demonstrate that GENUS can entrain gamma oscillations in the visual cortex, hippocampus, and prefrontal cortex in Tau P301S and CK-p25 mouse models of neurodegeneration. Tau P301S and CK-p25 mice subjected to chronic, daily GENUS from the early stages of neurodegeneration showed a preservation of neuronal and synaptic density across multiple brain areas and modified cognitive performance. Our transcriptomic and phosphoproteomic data suggest that chronic GENUS shifts neurons to a less degenerative state, improving synaptic function, enhancing neuroprotective factors, and reducing DNA damage in neurons while also reducing inflammatory response in microglia. Show less

Alzheimer’s disease is a pervasive neurodegenerative disorder, the molecular complexity of which remains poorly understood. Here, we analysed 80,660 single-nucleus transcriptomes from the prefrontal cortex of 48 individuals with varying degrees of Alzheimer’s disease pathology. Across six major brain cell types, we identified transcriptionally distinct subpopulations, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival. The… Show more

Alzheimer’s disease is a pervasive neurodegenerative disorder, the molecular complexity of which remains poorly understood. Here, we analysed 80,660 single-nucleus transcriptomes from the prefrontal cortex of 48 individuals with varying degrees of Alzheimer’s disease pathology. Across six major brain cell types, we identified transcriptionally distinct subpopulations, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival. The strongest disease-associated changes appeared early in pathological progression and were highly cell-type specific, whereas genes upregulated at late stages were common across cell types and primarily involved in the global stress response. Notably, we found that female cells were overrepresented in disease-associated subpopulations, and that transcriptional responses were substantially different between sexes in several cell types, including oligodendrocytes. Overall, myelination-related processes were recurrently perturbed in multiple cell types, suggesting that myelination has a key role in Alzheimer’s disease pathophysiology. Our single-cell transcriptomic resource provides a blueprint for interrogating the molecular and cellular basis of Alzheimer’s disease. Show less

The CD28H/B7-H5 pathway is a newly identified pathway of the B7 family. In human peripheral blood, the receptor CD28H is preferentially expressed on naïve T cells and repetitive stimulation of T cells leads to the loss of CD28H expression. Here we examined the expression of the CD28H/B7-H5 pathway in human peripheral tissues, as well as in human cancers. We found that CD28H is preferentially expressed on T cells with tissue-resident phenotypes (TRM). Supporting that, stimulation via IL-15 and… Show more

The CD28H/B7-H5 pathway is a newly identified pathway of the B7 family. In human peripheral blood, the receptor CD28H is preferentially expressed on naïve T cells and repetitive stimulation of T cells leads to the loss of CD28H expression. Here we examined the expression of the CD28H/B7-H5 pathway in human peripheral tissues, as well as in human cancers. We found that CD28H is preferentially expressed on T cells with tissue-resident phenotypes (TRM). Supporting that, stimulation via IL-15 and TGF-β, presumably major cytokines essential for TRM cell homeostasis, sustains CD28H expression on T cells. The ligand B7-H5 is constitutively expressed on normal epithelium of human oral-gastrointestinal tracts. In human cancers, CD28H is preferentially present on tumor infiltrating lymphocytes (TILs) with TRM features and identifies a TRM subset with less cytotoxicity. Taken together, our studies suggest that the CD28H/B7-H5 pathway involves the interactions between TRM cells and epithelium, and could be important for human TRM homeostasis and function. Show less

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer’s disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function… Show more

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer’s disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aβ42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aβ uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aβ phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. Show less

Alteration of corticostriatal glutamatergic function is an early pathophysiological change associated with Huntington’s disease (HD). The factors that regulate the maintenance of corticostriatal glutamatergic synapses post-developmentally are not well understood. Recently, the striatum-enriched transcription factor Foxp2 was implicated in the development of these synapses. Here, we show that, in mice, overexpression of Foxp2 in the adult striatum of two models of HD leads to rescue of… Show more

Alteration of corticostriatal glutamatergic function is an early pathophysiological change associated with Huntington’s disease (HD). The factors that regulate the maintenance of corticostriatal glutamatergic synapses post-developmentally are not well understood. Recently, the striatum-enriched transcription factor Foxp2 was implicated in the development of these synapses. Here, we show that, in mice, overexpression of Foxp2 in the adult striatum of two models of HD leads to rescue of HD-associated behaviors, while knockdown of Foxp2 in wild-type mice leads to development of HD-associated behaviors. We note that Foxp2 encodes the longest polyglutamine repeat protein in the human reference genome, and we show that it can be sequestered into aggregates with polyglutamine-expanded mutant Huntingtin protein (mHTT). Foxp2 overexpression in HD model mice leads to altered expression of several genes associated with synaptic function, genes that present additional targets for normalization of corticostriatal dysfunction in HD. Show less

Receptor-like tyrosine kinase (Ryk) is a Wnt receptor and is important for many developmental processes, including cranial facial development, neurogenesis, and axon guidance. However, little is known about the role of the intracellular domain, Ryk-ICD, in signal transduction. Its downstream targets are also unknown. We have previously shown that Ryk-ICD is located in the cytoplasm of neural stem cells whereas it moves into the nucleus upon neuronal differentiation. In this study, we discovered… Show more

Receptor-like tyrosine kinase (Ryk) is a Wnt receptor and is important for many developmental processes, including cranial facial development, neurogenesis, and axon guidance. However, little is known about the role of the intracellular domain, Ryk-ICD, in signal transduction. Its downstream targets are also unknown. We have previously shown that Ryk-ICD is located in the cytoplasm of neural stem cells whereas it moves into the nucleus upon neuronal differentiation. In this study, we discovered that Smek1/2 function as a chaperone for Ryk-ICD during its nuclear localization and that both Smek and Ryk-ICD associate with chromatin to regulate the transcription of downstream target genes and neural differentiation. Show less

Microglia, the tissue-resident macrophages in the
brain, are damage sensors that react to nearly any
perturbation, including neurodegenerative diseases
such as Alzheimer’s disease (AD). Here, using single-cell
RNA sequencing, we determined the transcriptome
of more than 1,600 individual microglia
cells isolated from the hippocampus of a mouse
model of severe neurodegeneration with AD-like
phenotypes and of control mice at multiple time
points during progression of… Show more

Microglia, the tissue-resident macrophages in the
brain, are damage sensors that react to nearly any
perturbation, including neurodegenerative diseases
such as Alzheimer’s disease (AD). Here, using single-cell
RNA sequencing, we determined the transcriptome
of more than 1,600 individual microglia
cells isolated from the hippocampus of a mouse
model of severe neurodegeneration with AD-like
phenotypes and of control mice at multiple time
points during progression of neurodegeneration. In
this neurodegeneration model, we discovered two
molecularly distinct reactive microglia phenotypes
that are typified by modules of co-regulated type I
and type II interferon response genes, respectively.
Furthermore, our work identified previously unobserved
heterogeneity in the response of microglia
to neurodegeneration, discovered disease stagespecific
microglia cell states, revealed the trajectory
of cellular reprogramming of microglia in response to
neurodegeneration, and uncovered the underlying
transcriptional programs. Show less

The histone deacetylase HDAC2, which negatively regulates synaptic gene expression and neuronal plasticity, is upregulated in Alzheimer’s disease (AD) patients and mouse models. Therapeutics targeting HDAC2 hold promise for ameliorating AD-related cognitive impairment; however, attempts to generate HDAC2-specific inhibitors have failed. Here, we take an integrative genomics approach to identify proteins that mediate HDAC2 recruitment to synaptic plasticity genes. Functional screening revealed… Show more

The histone deacetylase HDAC2, which negatively regulates synaptic gene expression and neuronal plasticity, is upregulated in Alzheimer’s disease (AD) patients and mouse models. Therapeutics targeting HDAC2 hold promise for ameliorating AD-related cognitive impairment; however, attempts to generate HDAC2-specific inhibitors have failed. Here, we take an integrative genomics approach to identify proteins that mediate HDAC2 recruitment to synaptic plasticity genes. Functional screening revealed that knockdown of the transcription factor Sp3 phenocopied HDAC2 knockdown and that Sp3 facilitated recruitment of HDAC2 to synaptic genes. Importantly, like HDAC2, Sp3 expression was elevated in AD patients and mouse models, where Sp3 knockdown ameliorated synaptic dysfunction. Furthermore, exogenous expression of an HDAC2 fragment containing the Sp3-binding domain restored synaptic plasticity and memory in a mouse model with severe neurodegeneration. Our findings indicate that targeting the HDAC2-Sp3 complex could enhance cognitive function without affecting HDAC2 function in other processes. Show less

Diverse molecular mechanisms regulate synaptic composition and function in the mammalian nervous system. The multifunctional protein arginine methyltransferase 8 (PRMT8) possesses both methyltransferase and phospholipase activities. Here we examine the role of this neuron-specific protein in hippocampal plasticity and cognitive function. PRMT8 protein localizes to synaptic sites and conditional whole brain Prmt8 deletion results in altered levels of multiple synaptic proteins in the… Show more

Diverse molecular mechanisms regulate synaptic composition and function in the mammalian nervous system. The multifunctional protein arginine methyltransferase 8 (PRMT8) possesses both methyltransferase and phospholipase activities. Here we examine the role of this neuron-specific protein in hippocampal plasticity and cognitive function. PRMT8 protein localizes to synaptic sites and conditional whole brain Prmt8 deletion results in altered levels of multiple synaptic proteins in the hippocampus, using both male and female mice. Interestingly these altered protein levels are due to post-transcriptional mechanisms as the corresponding mRNA levels are unaffected. Strikingly, electrophysiological recordings from hippocampal slices of mice lacking PRMT8 reveal multiple defects in excitatory synaptic function and plasticity. Furthermore, behavioral analyses show that PRMT8 conditional knockout mice exhibit impaired hippocampal-dependent fear learning. Together these findings establish PRMT8 as an important component of the molecular machinery required for hippocampal neuronal function. Show less

Changes in gamma oscillations (20–50 Hz) have been observed in several neurological disorders. However, the relationship between gamma oscillations and cellular pathologies is unclear. Here we show reduced, behaviourally driven gamma oscillations before the onset of plaque formation or cognitive decline in a mouse model of Alzheimer’s disease. Optogenetically driving fast-spiking parvalbumin-positive (FS-PV)-interneurons at gamma (40 Hz), but not other frequencies, reduces levels of amyloid-β… Show more

Changes in gamma oscillations (20–50 Hz) have been observed in several neurological disorders. However, the relationship between gamma oscillations and cellular pathologies is unclear. Here we show reduced, behaviourally driven gamma oscillations before the onset of plaque formation or cognitive decline in a mouse model of Alzheimer’s disease. Optogenetically driving fast-spiking parvalbumin-positive (FS-PV)-interneurons at gamma (40 Hz), but not other frequencies, reduces levels of amyloid-β (Aβ)1–40 and Aβ 1–42 isoforms. Gene expression profiling revealed induction of genes associated with morphological transformation of microglia, and histological analysis confirmed increased microglia co-localization with Aβ. Subsequently, we designed a non-invasive 40 Hz light-flickering regime that reduced Aβ1–40 and Aβ1–42 levels in the visual cortex of pre-depositing mice and mitigated plaque load in aged, depositing mice. Our findings uncover a previously unappreciated function of gamma rhythms in recruiting both neuronal and glial responses to attenuate Alzheimer’s-disease-associated pathology. Show less

De novo mutations in CHD8 are strongly associated with autism spectrum disorder, but the basic biology of CHD8 remains poorly understood. Here we report that Chd8 knockdown during cortical development results in defective neural progenitor proliferation and differentiation that ultimately manifests in abnormal neuronal morphology and behaviors in adult mice. Transcriptome analysis revealed that while Chd8 stimulates the transcription of cell cycle genes, it also precludes the induction of… Show more

De novo mutations in CHD8 are strongly associated with autism spectrum disorder, but the basic biology of CHD8 remains poorly understood. Here we report that Chd8 knockdown during cortical development results in defective neural progenitor proliferation and differentiation that ultimately manifests in abnormal neuronal morphology and behaviors in adult mice. Transcriptome analysis revealed that while Chd8 stimulates the transcription of cell cycle genes, it also precludes the induction of neural-specific genes by regulating the expression of PRC2 complex components. Furthermore, knockdown of Chd8 disrupts the expression of key transducers of Wnt signaling, and enhancing Wnt signaling rescues the transcriptional and behavioral deficits caused by Chd8 knockdown. We propose that these roles of Chd8 and the dynamics of Chd8 expression during development help negotiate the fine balance between neural progenitor proliferation and differentiation. Together, these observations provide new insights into the neurodevelopmental role of Chd8. Show less

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). The RTT missense MECP2R306C mutation prevents MeCP2 from interacting with the NCoR/histone deacetylase 3 (HDAC3) complex; however, the neuronal function of HDAC3 is incompletely understood. We found that neuronal deletion of Hdac3 in mice elicited abnormal locomotor coordination, sociability and cognition. Transcriptional and chromatin profiling revealed that HDAC3 positively regulated a subset of genes and was… Show more

Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). The RTT missense MECP2R306C mutation prevents MeCP2 from interacting with the NCoR/histone deacetylase 3 (HDAC3) complex; however, the neuronal function of HDAC3 is incompletely understood. We found that neuronal deletion of Hdac3 in mice elicited abnormal locomotor coordination, sociability and cognition. Transcriptional and chromatin profiling revealed that HDAC3 positively regulated a subset of genes and was recruited to active gene promoters via MeCP2. HDAC3-associated promoters were enriched for the FOXO transcription factors, and FOXO acetylation was elevated in Hdac3 knockout (KO) and Mecp2 KO neurons. Human RTT-patient-derived MECP2R306C neural progenitor cells had deficits in HDAC3 and FOXO recruitment and gene expression. Gene editing of MECP2R306C cells to generate isogenic controls rescued HDAC3-FOXO-mediated impairments in gene expression. Our data suggest that HDAC3 interaction with MeCP2 positively regulates a subset of neuronal genes through FOXO deacetylation, and disruption of HDAC3 contributes to cognitive and social impairment. Show less

Circularized Chromosome Conformation Capture followed by deep sequencing (4C-Seq) is a powerful technique to identify genome-wide partners interacting with a pre-specified genomic locus. Here, we present a computational and statistical approach to analyze 4C-Seq data generated from both enzyme digestion and sonication fragmentation-based methods. We implemented a command line software tool and a web interface called w4CSeq, which takes in the raw 4C sequencing data (FASTQ files) as input… Show more

Circularized Chromosome Conformation Capture followed by deep sequencing (4C-Seq) is a powerful technique to identify genome-wide partners interacting with a pre-specified genomic locus. Here, we present a computational and statistical approach to analyze 4C-Seq data generated from both enzyme digestion and sonication fragmentation-based methods. We implemented a command line software tool and a web interface called w4CSeq, which takes in the raw 4C sequencing data (FASTQ files) as input, performs automated statistical analysis and presents results in a user-friendly manner. Besides providing users with the list of candidate interacting sites/regions, w4CSeq generates figures showing genome-wide distribution of interacting regions, and sketches the enrichment of key features such as TSSs, TTSs, CpG sites and DNA replication timing around 4C sites. Show less

Genome structure or nuclear organization has fascinated researchers investigating genome function. Recently, much effort has gone into defining relationships between specific genome structures and gene expression in pluripotent cells. We previously analyzed chromosomal interactions of the endogenous Oct4 distal enhancer in pluripotent cells. Here, we derive ES and iPS cells from a transgenic Oct4 distal enhancer reporter mouse. Using sonication-based Circularized Chromosome Conformation Capture… Show more

Genome structure or nuclear organization has fascinated researchers investigating genome function. Recently, much effort has gone into defining relationships between specific genome structures and gene expression in pluripotent cells. We previously analyzed chromosomal interactions of the endogenous Oct4 distal enhancer in pluripotent cells. Here, we derive ES and iPS cells from a transgenic Oct4 distal enhancer reporter mouse. Using sonication-based Circularized Chromosome Conformation Capture (4C) coupled with next generation sequencing, we determined and compared the genome-wide interactome of the endogenous and transgenic Oct4 distal enhancers. Integrative genomic analysis indicated that the transgenic enhancer binds to a similar set of loci and shares similar key enrichment profiles with its endogenous counterpart. Both the endogenous and transgenic Oct4 enhancer interacting loci were enriched in the open nucleus compartment, which is associated with active histone marks (H3K4me1, H3K27ac, H3K4me3 and H3K9ac), active cis-regulatory sequences (DNA hypersensitivity sites (DHS)), 5-hydroxymethylcytosine (5-hmc), and early DNA replication domains. In addition, binding of some pluripotency-related transcription factors was consistently enriched in our 4C sites, and genes in those sites were generally more highly expressed. Overall, our work reveals critical features that may function in gene expression regulation in mouse pluripotent cells. Show less

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… Show more

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 IIb (Topo IIb), and knockdown of Topo IIb attenuates both DSB formation and earlyresponse 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. Show less

Background
As one of the genetic mechanisms for adaptive immunity, V(D)J recombination generates an enormous repertoire of T-cell receptors (TCRs). With the development of high-throughput sequencing techniques, systematic exploration of V(D)J recombination becomes possible. Multiplex PCR has been previously developed to assay immune repertoire; however, the use of primer pools leads to inherent biases in target amplification. In our study, we developed a “single-primer" ligation-anchored PCR… Show more

Background
As one of the genetic mechanisms for adaptive immunity, V(D)J recombination generates an enormous repertoire of T-cell receptors (TCRs). With the development of high-throughput sequencing techniques, systematic exploration of V(D)J recombination becomes possible. Multiplex PCR has been previously developed to assay immune repertoire; however, the use of primer pools leads to inherent biases in target amplification. In our study, we developed a “single-primer" ligation-anchored PCR method that may amplify the repertoire with much less biases.

Results
By utilizing a universal primer paired with a single primer targeting the conserved constant region, we amplified TCR-beta (TRB) variable regions from total RNA extracted from blood. Next-generation sequencing libraries were then prepared for Illumina HiSeq 2500 sequencer, which generates 151-bp read length to cover the entire V(D)J recombination region. We evaluated this approach on blood samples from healthy donors and from patients with malignant and benign meningiomas. Mapping of sequencing data showed that 64% to 96% of mapped TCRV-containing reads belong to TRB subtype. An increased usage of specific V segments and V-J pairing were observed in malignant meningiomas samples. The CDR3 sequences of the expanded V-J pairs were distinct in each malignant individual, even for pairing of TRBV7-3 with TRBJ2-2 that showed increased usage in both cases.

Conclusions
We demonstrated the technical feasibility and effectiveness of ligation-anchored PCR approach in capturing the TCR-beta landscapes. Further development of this technology may enable a comprehensive delineation of immune repertoire, including other forms of TCRs as well as immunoglobulins. Show less

Wnt signaling has been implicated in promoting somatic cell reprogramming. However, its molecular mechanisms remain unknown. Here we report that Wnt/β-catenin enhances iPSCs induction at the early stage of reprogramming. The augmented reprogramming induced by β-catenin is not due to increased total cell population or activation of c-Myc. In addition, β-catenin interacts with reprogramming factors Klf4, Oct4, and Sox2, further enhancing expression of pluripotency circuitry genes. These studies… Show more

Wnt signaling has been implicated in promoting somatic cell reprogramming. However, its molecular mechanisms remain unknown. Here we report that Wnt/β-catenin enhances iPSCs induction at the early stage of reprogramming. The augmented reprogramming induced by β-catenin is not due to increased total cell population or activation of c-Myc. In addition, β-catenin interacts with reprogramming factors Klf4, Oct4, and Sox2, further enhancing expression of pluripotency circuitry genes. These studies reveal novel mechanisms underlying the regulation of reprogramming somatic cells to pluripotency by Wnt/β-catenin signaling. Show less

In human immune system, V(D)J recombination produces an enormously large repertoire of immunoglobulins (Ig) so that they can tackle different antigens from bacteria, viruses and tumor cells. Several studies have demonstrated the utility of next-generation sequencers such as Roche 454 and Illumina Genome Analyzer to characterize the repertoire of immunoglobulins. However, these techniques typically require separation of B cell population from whole blood and require a few weeks for running the… Show more

In human immune system, V(D)J recombination produces an enormously large repertoire of immunoglobulins (Ig) so that they can tackle different antigens from bacteria, viruses and tumor cells. Several studies have demonstrated the utility of next-generation sequencers such as Roche 454 and Illumina Genome Analyzer to characterize the repertoire of immunoglobulins. However, these techniques typically require separation of B cell population from whole blood and require a few weeks for running the sequencers, so it may not be practical to implement them in clinical settings. Recently, the Ion Torrent personal genome sequencer has emerged as a tabletop personal genome sequencer that can be operated in a time-efficient and cost-effective manner. In this study, we explored the technical feasibility to use multiplex PCR for amplifying V(D)J recombination for IgH, directly from whole blood, then sequence the amplicons by the Ion Torrent sequencer. The whole process including data generation and analysis can be completed in one day. We tested the method in a pilot study on patients with benign, atypical and malignant meningiomas. Despite the noisy data, we were able to compare the samples by their usage frequencies of the V segment, as well as their somatic hypermutation rates. In summary, our study suggested that it is technically feasible to perform clinical monitoring of V(D)J recombination within a day by personal genome sequencers. Show less

BACKGROUND:
Meningiomas are the most common primary intracranial tumor, with ~3% meeting current histopathologic criteria for malignancy.
METHODS:
In this study, we explored the transcriptome of meningiomas using RNA-Seq.
RESULTS:
Inversion-mediated fusions between two adjacent genes, NAB2 and STAT6, were detected in one malignant tumor, creating two novel in-frame transcripts that were validated by RT-PCR and Sanger sequencing.
CONCLUSIONS:
Gene fusions of NAB2-STAT6 were… Show more

BACKGROUND:
Meningiomas are the most common primary intracranial tumor, with ~3% meeting current histopathologic criteria for malignancy.
METHODS:
In this study, we explored the transcriptome of meningiomas using RNA-Seq.
RESULTS:
Inversion-mediated fusions between two adjacent genes, NAB2 and STAT6, were detected in one malignant tumor, creating two novel in-frame transcripts that were validated by RT-PCR and Sanger sequencing.
CONCLUSIONS:
Gene fusions of NAB2-STAT6 were recently implicated in the pathogenesis of solitary fibrous tumors; our study suggested that similar fusions may also play a role in a malignant meningioma with unusual histopathologic features. Show less

Development of high-throughput sequencing-based methods has enabled us to examine nuclear architecture at unprece- dented resolution, allowing further examination of the function of long-range chromosomal interactions. Here, we review meth- ods used to investigate novel long-range chromosomal interac- tions and genome-wide organization of chromatin. We further discuss transcriptional activation and silencing in relation to organization and positioning of gene loci and regulation of chromatin… Show more

Development of high-throughput sequencing-based methods has enabled us to examine nuclear architecture at unprece- dented resolution, allowing further examination of the function of long-range chromosomal interactions. Here, we review meth- ods used to investigate novel long-range chromosomal interac- tions and genome-wide organization of chromatin. We further discuss transcriptional activation and silencing in relation to organization and positioning of gene loci and regulation of chromatin organization through protein complexes and non- coding RNAs.
Show less

Epigenetic mechanisms underlying somatic reprogramming have been extensively studied, but little is known about the nuclear architecture of pluripotent stem cells (PSCs). Using circular chromosome conformation capture with high-throughput sequencing (4C-seq) and fluorescence in situ hybridization (FISH), we identified chromosomal regions that colocalize frequently with the Oct4 locus in PSCs. These PSC-specific long-range interactions are established prior to transcriptional activation of… Show more

Epigenetic mechanisms underlying somatic reprogramming have been extensively studied, but little is known about the nuclear architecture of pluripotent stem cells (PSCs). Using circular chromosome conformation capture with high-throughput sequencing (4C-seq) and fluorescence in situ hybridization (FISH), we identified chromosomal regions that colocalize frequently with the Oct4 locus in PSCs. These PSC-specific long-range interactions are established prior to transcriptional activation of endogenous Oct4 during reprogramming to induced PSCs and are facilitated by Klf4-mediated recruitment of cohesin. Depletion of Klf4 leads to unloading of cohesin at the Oct4 enhancer and disrupts long-range interactions prior to loss of Oct4 transcription and subsequent PSC differentiation, suggesting a causative role for Klf4 in facilitating long-range interactions independent of its transcriptional activity. Taken together, our results delineate the basic nuclear organization at the Oct4 locus in PSCs and suggest a functional role for Klf4-mediated higher-order chromatin structure in maintaining and inducing pluripotency. Show less

Background
Circular chromosome conformation capture, when coupled with next-generation sequencing (4C-Seq), can be used to identify genome-wide interaction of a given locus (a "bait" sequence) with all of its interacting partners. Conventional 4C approaches used restriction enzyme digestion to fragment chromatin, and recently sonication approach was also applied for this purpose. However, bioinformatics pipelines for analyzing sonication-based 4C-Seq data are not well developed. In addition,… Show more

Background
Circular chromosome conformation capture, when coupled with next-generation sequencing (4C-Seq), can be used to identify genome-wide interaction of a given locus (a "bait" sequence) with all of its interacting partners. Conventional 4C approaches used restriction enzyme digestion to fragment chromatin, and recently sonication approach was also applied for this purpose. However, bioinformatics pipelines for analyzing sonication-based 4C-Seq data are not well developed. In addition, data consistency as well as similarity between the two methods has not been explored previously. Here we present a comparative analysis of 4C-Seq data generated by both methods, using an enhancer element of Pou5f1 gene in mouse embryonic stem (ES) cells.

Results
From biological replicates, we found good correlation (r>0.6) for inter-chromosomal interactions identified in either enzyme or sonication method. Compared to enzyme approach, sonication method generated less distal intra-chromosomal interactions, possibly due to the difference in chromatin fragmentation. From all mapped interactions, we further applied statistical models to identify enriched interacting regions. Interestingly, data generated from the two methods showed 30% overlap of the reproducible interacting regions. The interacting sites in the reproducible regions from both methods are similarly enriched with active histone marks. In addition, the interacting sites identified from sonication-based data are enriched with ChIP-Seq signals of transcription factors Oct4, Klf4, Esrrb, Tcfcp2i1, and Zfx that are critical for reprogramming and pluripotency.

Conclusions
Both enzyme-based and sonication-based 4C-Seq methods are valuable tools to explore long-range chromosomal interactions. Due to the nature of sonication-based method, correlation analysis of the 4C interactions with transcription factor binding should be more straightforward.
Show less

The genes POU5F1 and SOX2 are critical for pluripotency and reprogramming, yet the chromosomal organization around these genes remains poorly understood. We assayed long-range chromosomal interactions on putative enhancers of POU5F1 and SOX2 genes in human embryonic stem cells (hESCs) using 4C-Seq technique. We discovered that their frequent interacting regions mainly overlap with early DNA replication domains. The interactomes are associated with active histone marks and enriched with… Show more

The genes POU5F1 and SOX2 are critical for pluripotency and reprogramming, yet the chromosomal organization around these genes remains poorly understood. We assayed long-range chromosomal interactions on putative enhancers of POU5F1 and SOX2 genes in human embryonic stem cells (hESCs) using 4C-Seq technique. We discovered that their frequent interacting regions mainly overlap with early DNA replication domains. The interactomes are associated with active histone marks and enriched with 5-hydroxymethylcytosine sites. In hESCs, genes within the interactomes have elevated expression. Additionally, some genes associated with the POU5F1 enhancer contribute to pluripotency. Binding sites for multiple DNA binding proteins, including ATF3, CTCF, GABPA, JUND, NANOG, RAD21 and YY1, are enriched in both interactomes. The RARG locus, frequently interacting with the POU5F1 locus, has abundant RAD21 binding sites co-localized with other protein binding sites. Thus the interactomes of these two pluripotency genes could be an important part of the regulatory network in hESCs. Show less

Meningiomas are central nervous system tumors that originate from the meningeal coverings of the brain and spinal cord. Most meningiomas are pathologically benign or atypical, but 3-5% display malignant features. Despite previous studies on benign and atypical meningiomas, the key molecular pathways involved in malignant transformation remain to be determined, as does the extent of epigenetic alteration in malignant meningiomas. In this study, we explored the landscape of DNA methylation in ten… Show more

Meningiomas are central nervous system tumors that originate from the meningeal coverings of the brain and spinal cord. Most meningiomas are pathologically benign or atypical, but 3-5% display malignant features. Despite previous studies on benign and atypical meningiomas, the key molecular pathways involved in malignant transformation remain to be determined, as does the extent of epigenetic alteration in malignant meningiomas. In this study, we explored the landscape of DNA methylation in ten benign, five atypical and four malignant meningiomas. Compared to the benign tumors, the atypical and malignant meningiomas demonstrate increased global DNA hypomethylation. Clustering analysis readily separates malignant from atypical and benign tumors, implicating that DNA methylation patterns may serve as diagnostic biomarkers for malignancy. Genes with hypermethylated CpG islands in malignant meningiomas (such as HOXA6 and HOXA9) tend to coincide with the binding sites of polycomb repressive complexes (PRC) in early developmental stages. Most genes with hypermethylated CpG islands at promoters are suppressed in malignant and benign meningiomas, suggesting the switching of gene silencing machinery from PRC binding to DNA methylation in malignant meningiomas. One exception is the MAL2 gene that is highly expressed in benign group and silenced in malignant group, representing de novo gene silencing induced by DNA methylation. In summary, our results suggest that malignant meningiomas have distinct DNA methylation patterns compared to their benign and atypical counterparts, and that the differentially methylated genes may serve as diagnostic biomarkers or candidate causal genes for malignant transformation. Show less