Papers by Shiaoching Gong
Nature communications, Feb 13, 2018
Enhancers regulate gene expression and have been linked with disease pathogenesis. Little is know... more Enhancers regulate gene expression and have been linked with disease pathogenesis. Little is known about enhancers that regulate human disease-associated genes in primary cells relevant for pathogenesis. Here we use BAC transgenics and genome editing to dissect, in vivo and in primary immune cells, enhancers that regulate human TNFAIP3, which encodes A20 and is linked with autoimmune diseases. A20 expression is dependent on a topologically associating subdomain (sub-TAD) that harbors four enhancers, while another >20 enhancers in the A20 locus are redundant. This sub-TAD contains cell- and activation-specific enhancers, including an enhancer (termed TT>A) harboring a proposed causal SLE-associated SNV. Deletion of the sub-TAD or the TT>A enhancer results in enhanced inflammatory responses, autoantibody production, and inflammatory arthritis, thus establishing functional importance in vivo and linking enhancers with a specific disease phenotype. These findings provide insigh...
Neuron, 2011
Currently there is no general approach for achieving specific optogenetic control of genetically ... more Currently there is no general approach for achieving specific optogenetic control of genetically defined cell types in rats, which provide a powerful experimental system for numerous established neurophysiological and behavioral paradigms. To overcome this challenge we have generated genetically restricted recombinase-driver rat lines suitable for driving gene expression in specific cell types, expressing Cre recombinase under the control of large genomic regulatory regions (200-300 kb). Multiple tyrosine hydroxylase (Th)::Cre and choline acetyltransferase (Chat)::Cre lines were produced that exhibited specific opsin expression in targeted cell types. We additionally developed methods for utilizing optogenetic tools in freely moving rats and leveraged these technologies to clarify the causal relationship between dopamine (DA) neuron firing and positive reinforcement, observing that optical stimulation of DA neurons in the ventral tegmental area (VTA) of Th::Cre rats is sufficient to support vigorous intracranial self-stimulation (ICSS). These studies complement existing targeting approaches by extending the generalizability of optogenetics to traditionally non-genetically-tractable but vital animal models.
ABSTRACTThe hemizygous R47H variant of TREM2, a microglia-specific gene in the brain, increases r... more ABSTRACTThe hemizygous R47H variant of TREM2, a microglia-specific gene in the brain, increases risk for late-onset Alzheimer’s disease (AD). In this study, we identified a subpopulation of microglia with disease-enhancing proinflammatory signatures associated with the R47H mutation in human AD brains and tauopathy mouse brains. Using transcriptomic analysis at the single-nuclei level from AD patients with the R47H or the common variant (CV)-TREM2 with matched sex, pathology and APOE status, we found that the R47H mutation was associated with cell type- and sex-specific transcriptional changes in human AD brains, with microglia exhibiting the most robust alterations. Further characterization revealed that R47H-associated microglial subpopulations had enhanced inflammatory signatures including hyperactivation of Akt, one of the signaling pathways downstream of TREM2. In a newly-generated tauopathy knock-in mouse model expressing one allele of human TREM2 (hTREM2) with either the R47H...
Molecular and Cellular Biology, 2001
Fas/CD95 is a key regulator of apoptotic signaling, which is crucial for the maintenance of homeo... more Fas/CD95 is a key regulator of apoptotic signaling, which is crucial for the maintenance of homeostasis in peripheral lymphoid organs. TDAG51 has been shown to play critical roles in the up-regulation of Fas gene expression and T-cell apoptosis in vitro. In order to identify the role of TDAG51 in vivo, we generated TDAG51-deficient (TDAG51 −/− ) mice. Northern blotting revealed no expression of TDAG51 in TDAG51 −/− mice, indicating that the TDAG51 gene was successfully targeted. TDAG51 −/− mice were healthy and showed no gross developmental abnormalities. While Fas-deficient mice display marked lymphadenopathy, splenomegaly, and lymphocytosis, TDAG51 −/− mice had no apparent defects in secondary lymphoid organs. Although TDAG51 is required for up-regulation of Fas expression in T-cell hybridomas, TDAG51 −/− mice expressed normal levels of Fas and had normal T-cell apoptosis. Therefore, we conclude that TDAG51 is not essential for Fas up-regulation and T-cell apoptosis in vivo. There...
The Journal of …, 1996
The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that ar... more The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins ...
The Journal of …, 1996
The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that ar... more The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins ...
PLoS Genetics, 2014
Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative dis... more Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis.
Genome research, 2002
Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable wa... more Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs....
Current Protocols in Neuroscience, 2001
BAC transgenesis is a powerful tool for the study of gene expression and gene function in the mou... more BAC transgenesis is a powerful tool for the study of gene expression and gene function in the mouse in vivo. In this unit, detailed protocols are provided for modification (i.e., marker gene insertion, deletion, or point mutation) of BACs by homologous recombination in E. coli. This method utilizes a shuttle vector that allows transient expression of the E. coli RecA gene to support homologous recombination in the BAC host bacteria. In addition, two protocols are provided for purification of BAC DNA for microinjection to generate transgenic mice. Since BAC DNA is prone to degradation, which may introduce positional effects in transgenic mice, two methods are given for purification of intact BAC DNA for subsequent microinjection.
Journal of Neuroscience, 2007
PLoS ONE, 2012
A major barrier to complex experimental design in mouse genetics is the allele problem: combining... more A major barrier to complex experimental design in mouse genetics is the allele problem: combining three or more alleles is time-consuming and inefficient. Here, we solve this problem for transgenic animals with a simple modification of existing BAC transgenesis protocols, and generate triple-colored 'prism' mice in which the major cell types of the brain: neurons, astrocytes, and oligodendrocytes, are each labeled with a distinct fluorophore. All three fluorophores are expressed from the same locus, yet each fluorophore is expressed in an independent temporal and spatial pattern. All three transgenes are generally co-inherited across multiple generations with stable genomic copy number and expression patterns. This generic solution should permit more sophisticated experimental manipulations to assess functional interactions amongst populations of cell types in vivo in a more rapid and efficient manner.
Neuron, 2005
neuordegeneration is mostly confined to striatal me-Saitama 351-0198 dium spiny neurons and, to a... more neuordegeneration is mostly confined to striatal me-Saitama 351-0198 dium spiny neurons and, to a lesser extent, to cortical Japan pyramidal neurons (Vonsattel and DiFiglia, 1998). These 7 PRESTO, Japan Science and Technology Agency two types of neurons constitute the vast majority (about Saitama 332-0012 80%-90%) of the total neurons in the respective brain Japan regions. With longer expansion of the polyQ repeats, cell death pathology in HD as well as other polyQ disorders becomes increasingly less distinct and more over-Summary lapping (Zoghbi and Orr, 2000). The cellular and molecular mechanisms underlying the selective neuronal Expanded polyglutamine (polyQ) proteins in Huntingtoxicity in HD as well as in other polyQ disorders remain ton's disease (HD) as well as other polyQ disorders largely unknown. are known to elicit a variety of intracellular toxicities, A number of pathogenic mechanisms have been imbut it remains unclear whether polyQ proteins can plicated in HD. First, mhtt has been shown to exert elicit pathological cell-cell interactions which are critidominant toxicity to cause disease-like phenotypes in cal to disease pathogenesis. To test this possibility, a variety of cellular and animal models. In transgenic we have created conditional HD mice expressing a mouse models of HD, overexpression of mhtt resulted neuropathogenic form of mutant huntingtin (mhttin late-onset behavioral and neuropathological pheno-exon1) in discrete neuronal populations. We show types mimicking the disease, but deletion of the htt that mhtt aggregation is a cell-autonomous process. gene in mice resulted in an embryonic lethal phenotype However, progressive motor deficits and cortical neuand not HD-like neurodegenerative phenotypes (Duyao ropathology are only observed when mhtt expression et al., 1995). A variety of toxic gain-of-function mechais in multiple neuronal types, including cortical internisms have been implicated in HD pathogenesis. A neurons, but not when mhtt expression is restricted common mechanism shared by HD as well as other to cortical pyramidal neurons. We further demon-polyQ disorders is the progressive accumulation of prostrate an early deficit in cortical inhibition, suggesting tein aggregates containing the mutant polyQ proteins that pathological interactions between interneurons in the affected neurons (Davies et al., 1997; DiFiglia et and pyramidal neurons may contribute to the cortical al., 1997). However, the pathogenic role of the polyQ manifestation of HD. Our study provides genetic eviprotein aggregates remains unresolved (Bates, 2003; dence that pathological cell-cell interactions elicited Klement et al., 1998; Arrasate et al., 2004). Other pathoby neuropathogenic forms of mhtt can critically congenic mechanisms implicated in HD include transcriptribute to cortical pathogenesis in a HD mouse model. tional dysregulation, mitochondrial deficits, proteosomal dysfunction, abnormal endocytosis, axonal transport deficits, and synaptic dysfunction (Zoghbi and Orr, Neuron 434 2000; Dunah et al., 2002; Li et al., 2000; Steffan et al., As summarized above, much has been learned about 2004; Panov et al., 2002; Bence et al., 2001; Gunawarcell-autonomous toxicities of mhtt. However, very little dena et al., 2003; Zeron et al., 2002). In addition to the is known about the possible role of mhtt in pathological gain-of-function pathogenic mechanisms, recent studies cell-cell interactions and their relative contribution to using cellular models of HD also suggested that loss of the overall pathogenesis of HD. A few recent studies normal htt function, including regulation of transcription in HD mice have suggested that pathological cell-cell and vesicular transport of BDNF, may also contribute interactions may be present in vivo. Such interactions to HD pathogenesis (Zuccato et al., 2001; Gauthier et include blocking transcription and/or vesicular transal., 2004). Together, current evidence suggests that a port of BDNF (Zuccato et al., 2001; Gauthier et al., combination of gain-of-function and loss-of-function 2004), generating abnormal corticostriatal neurotransmechanisms may be involved in pathogenesis of HD mission (Cepeda et al., 2003), and alterations in other (Ross, 2004). nonneuronal cell types, including astrocytes (Lievens et In HD, there is increasing evidence to support the al., 2001) and microglia (Sapp et al., 2001). Yet, there is idea that proteolysis of mhtt into toxic N-terminal fragno direct evidence to support that pathological cell-cell ments containing the polyQ repeat is a critical early interactions are critical to HD pathogenesis in vivo in pathogenic event. In the brains of HD patients, the fulla mouse model. In this study, we asked the following length mhtt was shown to be proteolytically cleaved questions: Can a neuropathogenic form of mhtt (mhttinto small N-terminal fragments (110-600 amino acids) exon1) elicit pathological cell-cell interactions in vivo? by a variety of proteases, including caspases (Martin-Are such interactions critical to cortical pathogenesis dale et al., 1998; Wellington et al., 2002), calpains (Kim in HD mice? et al., 2001; Gafni et al., 2004), and a yet unidentified aspartyl endopeptidase (Lunkes et al., 2002). In both Results HD patients and transgenic mice expressing the fulllength mhtt, accumulation of small aggregated mhtt Creating a Cre/LoxP Conditional Mouse N-terminal fragments in the nucleus and cytoplasm was Model of HD evident prior to the onset of motor deficits and neuro-Our strategy to test the cell-autonomy model versus degeneration (DiFiglia et al., 1997; Gutekunst et al., the cell-cell interaction model was to develop condi-1999; Hodgson et al., 1999; Wellington et al., 2002; tional HD mice that express a toxic mhtt fragment in Zhou et al., 2003). Furthermore, in cellular models of discrete neuronal populations: either in all neurons in HD, small mhtt N-terminal fragments are clearly more the brain or restricted to cortical pyramidal neurons, toxic to neurons than the full-length mhtt, suggesting one of the vulnerable neuronal types in HD. The cellthat small mhtt fragments are likely to be a critical neuautonomy model predicts that restricting mhtt expresropathogenic entity in HD (Hackam et al., 1998). Finally, sion to the cortical pyramidal neurons alone should be mhtt N-terminal fragments (including mhtt-exon1) can sufficient to cause significant pathology in these neuelicit a variety of HD-like phenotypes ranging from agrons. The cell-cell interaction model predicts that regregation and cellular toxicity in yeast (Muchowski et stricting mhtt expression to cortical pyramidal neurons al., 2000) and cultured cell models (Hackam et al., 1998; alone will be insufficient to cause significant pathology Arrasate et al., 2004) to progressive motor deficits and in these neurons. Only mhtt expression in multiple neuneuropathology in transgenic Drosophila (Jackson et ronal types should cause significant pathology in cortial., 1998) and transgenic mice (Mangiarini et al., 1996; cal pyramidal neurons secondary to pathological cell-Schilling et al., 1999; Yamamoto et al., 2000; Laforet et cell interactions with other types of neurons. To test al., 2001). Transgenic mice expressing mhtt N-terminal these models in vivo, we created conditional HD mice fragments exhibit a variety of HD-like phenotypes inin which expression of mhtt-exon1 with a 103 glutamine cluding progressive motor deficits, striatal and cortical repeat was completely dependent on Cre-mediated exatrophy, reactive gliosis, mhtt aggregation, and altercision of a transcription termination (STOP) sequence ation of gene expression (ibid; Menalled and Chesselet, flanked by two Cre binding sites (LoxP) (Nagy, 2000; 2002; Yu et al., 2003; Luthi-Carter et al., 2000). Together, Soriano, 1999; Srinivas et al., 2001) (Figure 1A). In this existing evidence supports the "toxic fragment hypothemouse model (RosaHD), mhtt-exon1 is targeted to the sis," which states that polyQ-containing mhtt N-terminal murine Rosa26 locus, hence its expression is driven by fragments generated by proteolysis of the full-length mhtt the endogenous murine Rosa26 promoter, which diare the critical pathogenic entity that mediates neuronal rects ubiquitous transgene expression (Zambrowicz et dysfunction and degeneration in HD (Leavitt et al., al., 1997). RosaHD mice were crossed with two Cre 1999; Ross, 2002). Therefore, cellular and transgenic transgenic mice: Nestin-Cre, which activates LoxP reanimal models expressing neuropathogenic mhtt N-tercombination in all neurons and glia in the brain (Tronche minal fragments are widely used to study HD pathoet al., 1999), and Emx1-Cre, which activates LoxP regenic mechanisms downstream of the proteolysis of combination in all cortical pyramidal neurons and glia, the full-length mhtt into the toxic N-terminal fragments. but not in cortical interneurons or striatal neurons (Iwa-Despite significant progress in HD research, many sato et al., 2000; Gorski et al., 2002). Therefore, a criticritical issues remain to be addressed (Tobin and cal difference between these two Cre mouse lines in Signer, 2000; Ross, 2002; Ross, 2004). One such issue the cortex is that Nestin-Cre is active in all pyramidal is whether neuronal dysfunction and degeneration in neurons (about 80% of the cortical neurons) and inhibi-HD are solely caused by cell-autonomous toxicities of tory interneurons (about 20% of the cortical neurons; mhtt (cell-autonomy model) or require pathological interactions between cells (cell-cell interaction model)? Somogyi et al., 1998); while Emx1-Cre is only active in
Neuron, 2011
Currently there is no general approach for achieving specific optogenetic control of geneticallyd... more Currently there is no general approach for achieving specific optogenetic control of geneticallydefined cell types in rats, which provide a powerful experimental system for numerous established neurophysiological and behavioral paradigms. To overcome this challenge we have generated genetically-restricted recombinase-driver rat lines suitable for driving gene expression in specific cell-types, expressing Cre recombinase under control of large genomic regulatory regions (200-300 Kb). Multiple tyrosine hydroxylase (Th)::Cre and choline acetyltransferase (Chat)::Cre lines were produced that exhibited specific opsin expression in targeted cell-types. We additionally developed methods for utilizing optogenetic tools in freely-moving rats, and leveraged these technologies to clarify the causal relationship between dopamine (DA) neuron firing and positive reinforcement, observing that optical stimulation of DA neurons in the ventral tegmental area (VTA) of Th::Cre rats is sufficient to support vigorous intracranial self-stimulation (ICSS). studies complement existing targeting approaches by extending generalizability of optogenetics to traditionally non-genetically-tractable but vital animal models.
Nature Neuroscience, 2008
DARPP-32 is a dual-function protein kinase/phosphatase inhibitor that is involved in striatal sig... more DARPP-32 is a dual-function protein kinase/phosphatase inhibitor that is involved in striatal signaling. The phosphorylation of DARPP-32 at threonine 34 is essential for mediating the effects of both psychostimulant and antipsychotic drugs; however, these drugs are known to have opposing behavioral and clinical effects. We hypothesized that these drugs exert differential effects on striatonigral and striatopallidal neurons, which comprise distinct output pathways of the basal ganglia. To directly test this idea, we developed bacterial artificial chromosome transgenic mice that allowed the analysis of DARPP-32 phosphorylation selectively in striatonigral and striatopallidal neurons. Using this new methodology, we found that cocaine, a psychostimulant, and haloperidol, a sedation-producing antipsychotic, exert differential effects on DARPP-32 phosphorylation in the two neuronal populations that can explain their opposing behavioral effects. Furthermore, we found that a variety of drugs that target the striatum have cell type-specific effects that previous methods were not able to discern.
Nature, 2010
Mutations in the X-linked MECP2 gene, which encodes the transcriptional regulator methyl-CpG-bind... more Mutations in the X-linked MECP2 gene, which encodes the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2), cause Rett syndrome and several neurodevelopmental disorders including cognitive disorders, autism, juvenile-onset schizophrenia and encephalopathy with early lethality. Rett syndrome is characterized by apparently normal early development followed by regression, motor abnormalities, seizures and features of autism, especially stereotyped behaviours. The mechanisms mediating these features are poorly understood. Here we show that mice lacking Mecp2 from GABA (c-aminobutyric acid)-releasing neurons recapitulate numerous Rett syndrome and autistic features, including repetitive behaviours. Loss of MeCP2 from a subset of forebrain GABAergic neurons also recapitulates many features of Rett syndrome. MeCP2-deficient GABAergic neurons show reduced inhibitory quantal size, consistent with a presynaptic reduction in glutamic acid decarboxylase 1 (Gad1) and glutamic acid decarboxylase 2 (Gad2) levels, and GABA immunoreactivity. These data demonstrate that MeCP2 is critical for normal function of GABA-releasing neurons and that subtle dysfunction of GABAergic neurons contributes to numerous neuropsychiatric phenotypes.
Nature, 2003
The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with... more The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.
Methods, 2006
Transgenesis using bacterial artificial chromosomes (BAC) offers greater fidelity in directing de... more Transgenesis using bacterial artificial chromosomes (BAC) offers greater fidelity in directing desirable expression of foreign genes. Application of this technology in the optically transparent zebrafish with fluorescent protein reporters enables unparalleled visual analysis of regulation of gene expression in a living organism. Here we describe a streamlined procedure of direct selecting multiple BAC clones based on public sequence databases followed by rapid modification with GFP or RFP for transgenic analysis in zebrafish. Experimental procedures for BAC DNA preparation, microinjection of zebrafish embryos and screening of transgenic zebrafish carrying GFP/RFP modified BAC clones are detailed.
Mammalian Genome, 2007
The LGI1 gene has been implicated in the development of epilepsy and the invasion phenotype of gl... more The LGI1 gene has been implicated in the development of epilepsy and the invasion phenotype of glial cells. Controversy over the specific tissue expression pattern of this gene has stemmed from conflicting reports generated using immunohistochemistry and the polymerase chain reaction. LGI1 is one of a four-member family of secreted proteins with high homology and here we demonstrate, using GFP-tagged constructs from the four LGI1family members, that commonly used antibodies against LGI1 cross-react with different family members. With the uncertainty surrounding the use of commercially available antibodies to truly establish the expression pattern of LGI1, we generated transgenic mice carrying the LGI1-containing BAC, RP23-127G7, which had been modified to express the GFP reporter gene under the control of the endogenous regulatory elements required for
Current Opinion in Immunology, 1997
The antigen receptor on B lymphocytes is the product of a series ~of gene rearrangements which en... more The antigen receptor on B lymphocytes is the product of a series ~of gene rearrangements which ends when a functional receptor gene is assembled. Recent work has shown that the receptor-associated molecules Iga and Igl3 provide the signals that lead to inhibition of further recombination. Furthermore, Igl~ has been implicated in initiating the last step of the recombination reaction.
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Papers by Shiaoching Gong