RESUMO
BACKGROUND & AIMS: Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic (TG) expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. METHODS: We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulfate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S ribosomal RNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expressing hREG3A were monitored for DSS-induced colitis after cohousing or feeding feces from control mice. Colitis was induced in another set of control and hREG3A-TG mice by administration of trinitrobenzene sulfonic acid; some mice were given intrarectal injections of the hREG3A protein. Colon tissues were collected from mice and analyzed by histology and immunohistochemistry to detect mucin 2, as well as by 16S ribosomal RNA fluorescence in situ hybridization, transcriptional analyses, and quantitative polymerase chain reaction. We measured levels of reactive oxygen species (ROS) in bacterial cultures and fecal microbiota using 2',7'-dichlorofluorescein diacetate and flow cytometry. RESULTS: The fecal microbiota of mice that express hREG3A had a significant shift in composition, compared with control mice, with enrichment of Clostridiales (Ruminococcaceae, Lachnospiraceae) and depletion of Bacteroidetes (Prevotellaceae); the TG mice developed less-severe colitis following administration of DSS than control mice, associated with preserved gut barrier integrity and reduced bacterial translocation, epithelial inflammation, and oxidative damage. A similar shift in the composition of the fecal microbiota occurred after a few months in TG mice heterozygous for REG3A that harbored a wild-type maternal microbiota at birth; these mice developed less-severe forms of colitis following DSS administration. Cohoused and germ-free mice fed feces from REG3A-TG mice and given DSS developed less-severe forms of colitis and had reduced lipopolysaccharide activation of the toll-like receptor 4 and increased survival times compared with mice not fed feces from REG3A-TG mice. REG3A TG mice developed only mild colonic inflammation after exposure to 2,4,6-trinitrobenzene sulfonic acid, compared with control mice. Control mice given intrarectal hREG3A and exposed to 2,4,6-trinitrobenzene sulfonic acid showed less colon damage and inflammation than mice not given intrarectal hREG3A. Fecal samples from REG3A-TG mice had lower levels of ROS than feces from control mice during DSS administration. Addition of hREG3A to bacterial cultures reduced levels of ROS and increased survival of oxygen-sensitive commensal bacteria (Faecalibacterium prausnitzii and Roseburia intestinalis). CONCLUSIONS: Mice with hepatocytes that express hREG3A, which travels to the intestinal lumen, are less sensitive to colitis than control mice. We found hREG3A to alter the colonic microbiota by decreasing levels of ROS. Fecal microbiota from REG3A-TG mice protect non-TG mice from induction of colitis. These findings indicate a role for reduction of oxidative stress in preserving the gut microbiota and its ability to prevent inflammation.
Assuntos
Bactérias/metabolismo , Colite/prevenção & controle , Colo/metabolismo , Microbioma Gastrointestinal , Hepatócitos/metabolismo , Proteínas Associadas a Pancreatite/metabolismo , Animais , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Colite/induzido quimicamente , Colite/metabolismo , Colite/microbiologia , Colo/microbiologia , Sulfato de Dextrana , Modelos Animais de Doenças , Transplante de Microbiota Fecal , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Viabilidade Microbiana , Estresse Oxidativo/efeitos dos fármacos , Proteínas Associadas a Pancreatite/genética , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Ácido TrinitrobenzenossulfônicoRESUMO
X-chromosome inactivation (XCI) ensures dosage compensation in mammals and is a paradigm for allele-specific gene expression on a chromosome-wide scale. Important insights have been made into the developmental dynamics of this process. Recent studies have identified several cis- and trans-acting factors that regulate the initiation of XCI via the X-inactivation centre. Such studies have shed light on the relationship between XCI and pluripotency. They have also revealed the existence of dosage-dependent activators that trigger XCI when more than one X chromosome is present, as well as possible mechanisms underlying the monoallelic regulation of this process. The recent discovery of the plasticity of the inactive state during early development, or during cloning, and induced pluripotency have also contributed to the X chromosome becoming a gold standard in reprogramming studies.
Assuntos
Cromossomos Humanos X , Inativação do Cromossomo X , Alelos , Animais , Diferenciação Celular , Cromossomos/ultraestrutura , Mecanismo Genético de Compensação de Dose , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica , Humanos , Camundongos , Modelos Genéticos , RNA Longo não Codificante , RNA não Traduzido/genéticaRESUMO
The initial differential treatment of the two X chromosomes during X-chromosome inactivation is controlled by the X-inactivation centre (Xic). This locus determines how many X chromosomes are present in a cell ('counting') and which X chromosome will be inactivated in female cells ('choice'). Critical control sequences in the Xic include the non-coding RNAs Xist and Tsix, and long-range chromatin elements. However, little is known about the process that ensures that X inactivation is triggered appropriately when more than one Xic is present in a cell. Using three-dimensional fluorescence in situ hybridization (FISH) analysis, we showed that the two Xics transiently colocalize, just before X inactivation, in differentiating female embryonic stem cells. Using Xic transgenes capable of imprinted but not random X inactivation, and Xic deletions that disrupt random X inactivation, we demonstrated that Xic colocalization is linked to Xic function in random X inactivation. Both long-range sequences and the Tsix element, which generates the antisense transcript to Xist, are required for the transient interaction of Xics. We propose that transient colocalization of Xics may be necessary for a cell to determine Xic number and to ensure the correct initiation of X inactivation.
Assuntos
Impressão Genômica , RNA não Traduzido/fisiologia , Células-Tronco/fisiologia , Inativação do Cromossomo X , Cromossomo X/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Feminino , Hibridização in Situ Fluorescente , Masculino , Camundongos , RNA Longo não Codificante , RNA não Traduzido/genéticaRESUMO
Epigenetic mechanisms lead to the stable regulation of gene expression without alteration of DNA and trigger initiation and/or maintenance of cell-type-specific transcriptional profiles. Indeed, modulation of chromatin structure and the global 3D organization of the genome and nuclear architecture participate in the precise control of transcription. Thus, dissection of these epigenetic mechanisms is essential for our understanding of gene regulation. In this chapter, we describe challenging combinations of immunofluorescence, and RNA and DNA fluorescent in situ hybridization and their application to our studies of a remarkable example of epigenetic control of gene expression in female mammals, the process of X chromosome inactivation.
Assuntos
Cromatina/genética , Epigênese Genética , Hibridização in Situ Fluorescente/métodos , Microscopia de Fluorescência/métodos , RNA/metabolismo , Transcrição Gênica , Inativação do Cromossomo X , Animais , Núcleo Celular/genética , DNA/metabolismo , Células-Tronco Embrionárias , Feminino , Regulação da Expressão Gênica , Camundongos , Cromossomo X/metabolismoRESUMO
A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the early female (XX) embryo and in differentiating female ES cells but not in their male (XY) counterparts. Counting depends on the X inactivation center (Xic), which contains the Xist gene encoding a nuclear RNA, which coats the inactive X chromosome and induces gene silencing. A 37-kb sequence lying 3' to the Xist gene is known to prevent initiation of XCI in male differentiating ES cells. This region contains the major and minor promoters of the Tsix gene, which runs antisense to Xist, and the DXPas34 tandem repeat lying close to the Tsix major promoter. We have addressed the role of these elements in counting by using male ES cells. Targeted deletion of DXPas34 impaired recruitment of RNA-polymerase II and TFIIB at the Tsix major promoter, resulting in low levels of Tsix expression in ES cells and moderate ectopic initiation of XCI upon differentiation. A deletion extending 3' to Xist and including the Tsix major promoter resulted in almost complete impairment of Tsix transcription and in efficient ectopic XCI upon differentiation of male ES cells. Internal deletions within the Tsix gene did not affect significantly the level of antisense transcription within Xist and had only minor effects upon differentiation. Our results identify a function for DXPas34 in murine XCI and demonstrate the critical role of Tsix transcription in preventing XCI in differentiating male ES cells and in normal functioning of the counting pathway.