Smarcad1 mediates microbiota-induced inflammation in mouse and coordinates gene expression in the intestinal epithelium.

BACKGROUND: How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue. RESULTS: Specific deletion of Smarcad1 in the mouse intestinal epithelium leads to colitis resistance and substantial changes in gene expression, including a striking increase of expression of several genes linked to innate immunity. Absence of Smarcad1 leads to changes in chromatin accessibility and significant changes in histone H3K9me3 over many sites, including genes that are differentially regulated upon Smarcad1 deletion. We identify candidate members of the gut microbiome that elicit a Smarcad1-dependent colitis response, including members of the poorly understood TM7 phylum. CONCLUSIONS: Our study sheds light onto the role of the chromatin remodeling machinery in intestinal epithelial cells in the colitis response and shows how a highly conserved chromatin remodeling factor has a distinct role in anti-microbial defense. This work highlights the importance of the intestinal epithelium in the colitis response and the potential of microbial species as pharmacological and probiotic targets in the context of inflammatory diseases.

SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae.

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.

The SNF2-family member Fun30 promotes gene silencing in heterochromatic loci.

Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.

Rescuing yeast mutants with human genes.

The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae have, in addition to being extensively studied themselves, both been utilized for the last quarter century as experimental systems for the isolation of genes from other organisms. Mutations conferring growth defects in either of the two yeast strains have frequently been complemented by expression of cDNA libraries from heterologous species, often human. Many successful experiments have utilized available yeast mutations to allow successful complementation by a human gene, which can thus be deduced to have the same, or an overlapping function as the mutated yeast gene. However complementation in yeast has also been used with success to study two fields, apoptosis and steroid receptor signalling, which, at first glance, seem to be foreign to the yeast life cycle.

A natural hypomorphic variant of the apoptosis regulator Gimap4/IAN1.

The Gimap/IAN family of GTPases has been implicated in the regulation of cell survival, particularly in lymphomyeloid cells. Prosurvival and prodeath properties have been described for different family members. We generated novel serological reagents to study the expression in rats of the prodeath family member Gimap4 (IAN1), which is sharply up-regulated at or soon after the stage of T cell-positive selection in the thymus. During these investigations we were surprised to discover a severe deficiency of Gimap4 expression in the inbred Brown Norway (BN) rat. Genetic analysis linked this trait to the Gimap gene cluster on rat chromosome 4, the probable cause being an AT dinucleotide insertion in the BN Gimap4 allele (AT(+)). This allele encodes a truncated form of Gimap4 that is missing 21 carboxyl-terminal residues relative to wild type. The low protein expression associated with this allele appears to have a posttranscriptional cause, because mRNA expression was apparently normal. Spontaneous and induced apoptosis of BN and wild-type T cells was analyzed in vitro and compared with the recently described mouse Gimap4 knockout. This revealed a "delayed" apoptosis phenotype similar to but less marked than that of the knockout. The Gimap4 AT(+) allele found in BN was shown to be rare in inbred rat strains. Nevertheless, when wild rat DNA samples were studied the AT(+) allele was found at a high overall frequency ( approximately 30%). This suggests an adaptive significance for this hypomorphic allele.

Eosinophilic bowel disease controlled by the BB rat-derived lymphopenia/Gimap5 gene.

BACKGROUND & AIMS: Many models of autoimmunity are associated with lymphopenia. Most involve a T-helper cell (Th)1-type disease, including the diabetic BioBreeding (BB) rat. To investigate the roles of identified susceptibility loci in disease pathogenesis, we bred PVG-RT1(u), lymphopenia (lyp)/lyp rats, congenic for the iddm1 (RT1(u)) and iddm2 (lyp, Gimap5(-/-)) diabetes susceptibility loci on the PVG background. Surprisingly, these rats developed a spontaneous, progressive, inflammatory bowel disease. To understand the disease pathogenesis, we undertook investigations at the genetic, histologic, and cellular levels. METHODS: Genetically lymphopenic rats and congenic wild-type partners were compared for gross pathologic, histologic, and immunologic parameters, the latter including cytokines and autoantibodies. RESULTS: Genetic analysis demonstrated that homozygosity at the lyp locus was required for disease. All rats developed disease, and the median age at humane killing was approximately 36 weeks. This panintestinal disease showed a conspicuous eosinophilic infiltrate in the submucosa and muscle layers, but the villi were unaffected. Diseased rats showed splenomegaly and massive enlargement of the mesenteric lymph nodes. This pathology resembles human eosinophilic gastroenteritis, and several further features indicate a Th2 basis. The rats developed high serum IgE and made IgG autoantibodies that detected a nonleukocytic cell present in the intestinal wall of all rats (including germ free). CONCLUSIONS: The T-lymphopenic state associated with GIMAP5 deficiency renders rats generally susceptible to T-cell-mediated autoimmunity, but the immunoregulatory bias (Th1/Th2) of any disease depends on other genetic (or environmental) factors. In the present model, we suggest that defective peripheral tolerance to an intestine-specific autoantigen leads to uncontrolled inflammation of the intestinal wall.

Expression of the Ian family of putative GTPases during T cell development and description of an Ian with three sets of GTP/GDP-binding motifs.

Reports suggest that two members of the novel immune-associated nucleotide (Ian) GTPase family, Ian1 and Ian5, play roles in T cell development. We performed real-time PCR analysis of the expression of Ian genes of the rat during T cell maturation, in macrophages and in cell lines. We found that all of the genes were expressed at relatively low levels at the early double-negative thymocyte stage but were expressed more strongly at later cell stages. Our study also revealed the fact that the previously reported Ian9, Ian10 and Ian11 genes are, instead, parts of a single gene for which we retain the name Ian9, potentially encoding a GTPase with a highly unusual triplicated structure. Antisera were developed against both Ian1 and Ian9. We established that Ian9 is produced as an approximately 75-kDa protein in both T cells and thymocytes. We observed that levels of both Ian1 and Ian9 proteins are profoundly reduced in T cells from lymphopenic rats as compared with wild-type rats. It was demonstrated that thymocytes and B cells from lymphopenic rats (Ian5 null) did not show enhanced sensitivity to gamma-irradiation-induced apoptosis.

Using yeast to place human genes in functional categories.

The availability of the draft sequence of the human genome has created a pressing need to assign functions to each of the 35,000 or so genes that it defines. One useful approach for this purpose is to use model organisms for both bioinformatic and functional comparisons. We have developed a complementation system, based on the model eukaryote Saccharomyces cerevisiae, to clone human cDNAs that can functionally complement yeast essential genes. The system employs two regulatable promoters. One promoter, tetO (determining doxycycline-repressible expression), is used to control essential S. cerevisiae genes. The other, pMET3 (which is switched off in the presence of methionine), is employed to regulate the expression of mammalian cDNAs in yeast. We have demonstrated that this system is effective for both individual cDNA clones and for cDNA libraries, permitting the direct selection of functionally complementing clones. Three human cDNA libraries have been constructed and screened for clones that can complement specific essential yeast genes whose expression is switched off by the addition of doxycycline to the culture medium. The validity of each complementation was checked by showing that the yeast cells stop their growth in the presence of doxycycline and methionine, which represses the expression of the yeast and mammalian coding sequence, respectively. Using this system, we have screened 25 tetO replacement strains and succeeded in isolating human cDNAs complementing six essential yeast genes. In this way, we have uncovered a novel human ubiquitin-conjugating enzyme, have isolated a human cDNA clone that may function as a signal peptidase and have demonstrated that the functional segment of the human Psmd12 proteosome sub-unit contains a PINT domain.

Molecular characterization of the novel rat NK receptor 1C7.

A novel receptor, named 1C7 or NKp30 and involved in natural cytotoxicity, was recently identified. This receptor is encoded by the 1C7 gene, which is located within the class III region of the human MHC, HLA. It is a member of the immunoglobulin gene superfamily (IgSF) and, remarkably, is expressed at the mRNA level as six different splice variants in human. Recent investigations have indicated that the 1c7 gene of the mouse is silenced by in-frame stop codons. In this study, the molecular characterization of the rat 1c7 gene is described. cDNA derived from this gene encode a protein of 192 amino acid residues predicted to contain a single IgV-set domain in the extracellular region and a positively charged residue in the transmembrane region. Expression of the gene was detected in freshly isolated rat Natural Killer (NK) and T splenocytes. Transfection of rat 1C7 into the NK cell line RNK-16 induced cytolytic activity against glioma as well as lymphoma tumor cells. In addition, binding of a r1C7-Fc fusion protein by a panel of target cells correlated with susceptibility to killing by RNK-16-1C7 effector cells. These results indicate that the r1C7 molecule could function as an NK activating receptor as previously reported for the human NKp30 receptor molecule.