GPR108 Is a Highly Conserved AAV Entry Factor.

Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

GPR108, an NF-κB activator suppressed by TIRAP, negatively regulates TLR-triggered immune responses.

Higher vertebrates have evolved innate and adaptive immune systems to defend against foreign substances and pathogens. Sophisticated regulatory circuits are needed to avoid inappropriate immune responses and inflammation. GPR108 is a seven-transmembrane family protein that activates NF-κB strongly when overexpressed. Surprisingly, its action in a physiological context is that of an antagonist of Toll-like receptor (TLR)-mediated signaling. Cells from Gpr108-null mice exhibit enhanced cytokine secretion and NF-κB and IRF3 signaling, whereas Gpr108-null macrophages reconstituted with GPR108 exhibit blunted signaling. Co-expression of TLRs and GPR108 reduces NF-κB and IFNß promoter activation compared to expression of either TLRs or GPR108 alone. Upon TLR stimulation GPR108 abundance increases and the protein engages TLRs and their partners to reduce MyD88 expression and interfere with its binding to TLR4 through blocking MyD88 ubiquitination. In turn GPR108 is antagonized by TIRAP, an adaptor protein for TLR and MyD88. The interrelationships between GPR108 and innate immune signaling components are multifactorial and point to a membrane-associated signaling structure of significant complexity.

Gut microbiota profiles in treatment-naïve children with attention deficit hyperactivity disorder.

BACKGROUNDS: Although increasing evidence suggests a role for the gut microbiota in neurodevelopment, the actual structure and composition of microbiota in children with attention-deficit/hyperactivity disorder (ADHD) remain unclear. METHODS: Thus, the present study aimed to define the characteristics of gut microbiota in treatment-naive children with ADHD and to assess their relationship with the severity of ADHD symptoms. High-throughput pyrosequencing was used to investigate the microbiota composition in fecal matter from 51 children with ADHD and 32 healthy controls (HC). RESULTS: An operational taxonomical unit (OTU)-level analysis revealed a significant decrease in the fractional representation of Faecalibacterium in children with ADHD compared to HC. In individuals with ADHD, the abundance of Faecalibacterium was negatively associated with parental reports of ADHD symptoms. However, there was no significant difference in alpha diversity between the ADHD and control groups. CONCLUSIONS: This present findings support the involvement of microbiota alteration in psychiatric diseases and Faecalibacterium may represent a potential novel marker of gut microbiota in ADHD. Future studies are needed to validate these findings and to elucidate the temporal and causal relationships between these variables.

Pulmonary hypertension after prolonged hypoxic exposure in mice with a congenital deficiency of Cyp2j.

Cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids contribute to the regulation of pulmonary vascular tone and hypoxic pulmonary vasoconstriction. We investigated whether the attenuated acute vasoconstrictor response to hypoxic exposure of Cyp2j(-/-) mice would protect these mice against the pulmonary vascular remodeling and hypertension associated with prolonged exposure to hypoxia. Cyp2j(-/-) and Cyp2j(+/+) male and female mice continuously breathed an inspired oxygen fraction of 0.21 (normoxia) or 0.10 (hypoxia) in a normobaric chamber for 6 weeks. We assessed hemoglobin (Hb) concentrations, right ventricular (RV) systolic pressure (RVSP), and transthoracic echocardiographic parameters (pulmonary acceleration time [PAT] and RV wall thickness). Pulmonary Cyp2c29, Cyp2c38, and sEH mRNA levels were measured in Cyp2j(-/-) and Cyp2j(+/+) male mice. At baseline, Cyp2j(-/-) and Cyp2j(+/+) mice had similar Hb levels and RVSP while breathing air. After 6 weeks of hypoxia, circulating Hb concentrations increased but did not differ between Cyp2j(-/-) and Cyp2j(+/+) mice. Chronic hypoxia increased RVSP in Cyp2j(-/-) and Cyp2j(+/+) mice of either gender. Exposure to chronic hypoxia decreased PAT and increased RV wall thickness in both genotypes and genders to a similar extent. Prolonged exposure to hypoxia produced similar levels of RV hypertrophy in both genotypes of either gender. Pulmonary Cyp2c29, Cyp2c38, and sEH mRNA levels did not differ between Cyp2j(-/-) and Cyp2j(+/+) male mice after breathing at normoxia or hypoxia for 6 weeks. These results suggest that murine Cyp2j deficiency does not attenuate the development of murine pulmonary vascular remodeling and hypertension associated with prolonged exposure to hypoxia in mice of both genders.

Deficits in receptor-mediated endocytosis and recycling in cells from mice with Gpr107 locus disruption.

GPR107 is a type III integral membrane protein that was initially predicted to be a member of the family of G-protein-coupled receptors. This report shows that deletion of Gpr107 leads to an embryonic lethal phenotype that is characterized by a reduction in cubilin transcript abundance and a decrease in the representation of multiple genes implicated in the cubilin-megalin endocytic receptor complex (megalin is also known as LRP2). Gpr107-null fibroblast cells exhibit reduced transferrin internalization, decreased uptake of low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) cargo and resistance to toxins. Colocalization studies and proteomic analyses suggest that GPR107 associates with clathrin and the retromer protein VPS35 and that GPR107 might be responsible for the return of receptors to the plasma membrane from endocytic compartments. The highly selective deficits observed in Gpr107-null cells indicate that GPR107 interacts directly or indirectly with a limited subset of surface receptors.

Deletion of the murine cytochrome P450 Cyp2j locus by fused BAC-mediated recombination identifies a role for Cyp2j in the pulmonary vascular response to hypoxia.

Epoxyeicosatrienoic acids (EETs) confer vasoactive and cardioprotective functions. Genetic analysis of the contributions of these short-lived mediators to pathophysiology has been confounded to date by the allelic expansion in rodents of the portion of the genome syntenic to human CYP2J2, a gene encoding one of the principle cytochrome P450 epoxygenases responsible for the formation of EETs in humans. Mice have eight potentially functional genes that could direct the synthesis of epoxygenases with properties similar to those of CYP2J2. As an initial step towards understanding the role of the murine Cyp2j locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to CYP2J2, using a combination of homologous and site-directed recombination strategies. A mouse strain in which the locus deletion was complemented by transgenic delivery of BAC sequences encoding human CYP2J2 was also created. Systemic and pulmonary hemodynamic measurements did not differ in wild-type, null, and complemented mice at baseline. However, hypoxic pulmonary vasoconstriction (HPV) during left mainstem bronchus occlusion was impaired and associated with reduced systemic oxygenation in null mice, but not in null mice bearing the human transgene. Administration of an epoxygenase inhibitor to wild-type mice also impaired HPV. These findings demonstrate that Cyp2j gene products regulate the pulmonary vascular response to hypoxia.

Identification of long range regulatory elements of mouse alpha-globin gene cluster by quantitative associated chromatin trap (QACT).

Chromatin from different regions of the genome frequently forms steady associations that play important roles in regulating gene expression. The widely used chromatin conformation capture (3C) assay allows determination of the in vivo structural organization of an active endogenous locus. However, unpredicted chromatin associations within a given genomic locus can not be identified by 3C. Here, we describe a new strategy, quantitative associated chromatin trap (QACT), which incorporates a modified 3C method and a quantitative assay tool, to capture and quantitatively analyzes all possible associated chromatin partners (ACPs) of a given chromatin fragment. Using QACT, we have analyzed the chromatin conformation of the mouse alpha-globin gene cluster and proved the extensive interaction between HS26 and alpha-globin genes. In addition, we have identified a candidate alpha1-globin gene specific silencer 475A8 which shows the differentiation-stage specific DNase I hypersensitivity. Functional analysis suggests that 475A8 may regulate the alpha1-globin gene during terminal differentiation of committed erythroid progenitor cells. ChIP (chromatin immunoprecipitation) and cotransfection assays demonstrate that GATA-1, a hemopoietic specific transcriptional factor, may increase alpha1-globin gene expression by suppressing the function of 475A8 in terminally differentiated erythroid cells.

[Change of the chromosome conformation of human alpha-globin gene locus in transgenic mice].

OBJECTIVE: To establish chromosome conformation capture (3C) strategy and to use this method for exploring the effect of chromosome conformation on human alpha-globin gene expression in the human alpha-globin transgenic mouse. METHODS: Homozygous human alpha-globin transgenic male mouse was crossed with KM female mouse. The 14.5-day post-coitum (dpc) embryos were used for the isolation of fetal liver and fetal brain cells. Homogeneous single-cell suspension was treated with formaldehyde to crosslink the chromatin conformation in the nuclear. The cross-linked chromatin compound was digested with Nco I and then ligated with T4 DNA ligase. The ligated compound was reversely cross-linked and then the ligated genomic DNA was purified for PCR analysis. The primers were designed along the two sides of cut and ligated sites. Semi-quantitative PCR was used to analyze the chromosome conformation of the whole human alpha-globin gene locus in fetal liver and fetal brain cells. RESULTS: When HS40 fragment was used as the fixed fragment, in fetal brain cells, the ligation frequencies of HS40 fragment with other fragments were decreased as the linear distances to HS40 fragment were increasing; while in fetal liver cells, two active genes (alpha1 and alpha2) fragments showed higher ligation frequencies with HS40 fragment than other fragments. However, the fragment containing an inactive gene (xi) displayed the comparable low ligation frequency as that in fetal brain. When alpha2 fragment was used as the fixed fragment, similarly, in fetal brain cells the ligation frequencies of alpha2 fragment with other ones were decreased as the linear distances increasing; when in fetal liver cells, it showed higher ligation frequencies with two upstream regulatory elements (HS 40 and 33). However, it showed a little bit lower ligation frequency with another two upstream regulatory elements (HS10 and 8) than those in fetal brain. CONCLUSION: In fetal liver cells, the distant regulatory elements are in close proximity to the downstream of the expressed globin genes through looping out, the interval region; however, in fetal brain, they were not in vicinity to the expressed globin genes.

Exploring cellular memory molecules marking competent and active transcriptions.

BACKGROUND: Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation. RESULTS: We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription. CONCLUSION: Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

Active chromatin hub of the mouse alpha-globin locus forms in a transcription factory of clustered housekeeping genes.

RNA polymerases can be shared by a particular group of genes in a transcription "factory" in nuclei, where transcription may be coordinated in concert with the distribution of coexpressed genes in higher-eukaryote genomes. Moreover, gene expression can be modulated by regulatory elements working over a long distance. Here, we compared the conformation of a 130-kb chromatin region containing the mouse alpha-globin cluster and their flanking housekeeping genes in 14.5-day-postcoitum fetal liver and brain cells. The analysis of chromatin conformation showed that the active alpha1 and alpha2 globin genes and upstream regulatory elements are in close spatial proximity, indicating that looping may function in the transcriptional regulation of the mouse alpha-globin cluster. In fetal liver cells, the active alpha1 and alpha2 genes, but not the inactive zeta gene, colocalize with neighboring housekeeping genes C16orf33, C16orf8, MPG, and C16orf35. This is in sharp contrast with the mouse alpha-globin genes in nonexpressing cells, which are separated from the congregated housekeeping genes. A comparison of RNA polymerase II (Pol II) occupancies showed that active alpha1 and alpha2 gene promoters have a much higher RNA Pol II enrichment in liver than in brain. The RNA Pol II occupancy at the zeta gene promoter, which is specifically repressed during development, is much lower than that at the alpha1 and alpha2 promoters. Thus, the mouse alpha-globin gene cluster may be regulated through moving in or out active globin gene promoters and regulatory elements of a preexisting transcription factory in the nucleus, which is maintained by the flanking clustered housekeeping genes, to activate or inactivate alpha-globin gene expression.

Memory mechanisms of active transcription during cell division.

The developmental programs of eukaryotic organisms involve the programmed transcription of genes. A characteristic gene expression pattern is established and preserved in each different cell type. Therefore, gene activation at a particular time and its maintenance during cell division are significant for cellular differentiation and individual development. Although many studies have sought to explain the molecular mechanisms of gene expression regulation, the mechanism through which gene expression states are inherited during cell division has not been fully elucidated yet. This review illustrates the general principles and the complexities involved in the establishment and maintenance of active transcription through cell cycles. It focuses on the most-recent findings about the ways in which molecular memory marks for active transcription are coordinated with cell cycle events, such as replication, mitosis and nuclear organization, to mediate transcription memory across cell division events, which may establish a unifying memory process of active transcription.

Remembering the cell fate during cellular differentiation.

Higher eukaryote contains several hundreds of different cell types, each with a distinctive set of property defined by a unique gene expression pattern, even though every cell (with minor exception) shares the common genome. During cellular differentiation, the committed gene expression pattern is set up and propagated through numerous cell divisions. Therefore, cells must have evolved some elegant and inherent mechanisms to remember their expression states for the requirement of the stability of differentiation and development. Here we speculate a hypothetically cellular memory mechanism. In this hypothesis, the cell-cell variation during cellular differentiation may result from the inherent stochastic gene expression. The evolution of histone and distant regulatory sequences change the parameters of expression stochasticity. S-phase-dependent gene activation and epigenetic marks on chromatin provide means to discriminate transcriptionally active and repressive states. Eventually, mitotic memory mechanisms have been developed through which these expression states are transmitted through numerous cell divisions.