Association Of Transcription Factors Research Articles

BcTaf14 regulates growth and development, virulence, and stress responses in the phytopathogenic fungus Botrytis cinerea.

TATA box-binding protein (TBP)-associated factor 14 (Taf14), a transcription-associated factor containing a conserved YEATS domain and an extra-terminal (ET) domain, is a multifunctional protein in Saccharomyces cerevisiae. However, the role of Taf14 in filamentous phytopathogenic fungi is not well understood. In this study, the homologue of ScTaf14 in Botrytis cinerea (named BcTaf14), a destructive phytopathogen causing grey mould, was investigated. The BcTaf14 deletion strain (ΔBcTaf14) showed pleiotropic defects, including slow growth, abnormal colony morphology, reduced conidiation, abnormal conidial morphology, reduced virulence, and altered responses to various stresses. The ΔBcTaf14 strain also exhibited differential expression of numerous genes compared to the wild-type strain. BcTaf14 could interact with the crotonylated H3K9 peptide, and mutation of two key sites (G80 and W81) in the YEATS domain disrupted this interaction. The mutation of G80 and W81 affected the regulatory effect of BcTaf14 on mycelial growth and virulence but did not affect the production and morphology of conidia. The absence of the ET domain at the C-terminus rendered BcTaf14 unable to localize to the nucleus, and the defects of ΔBcTaf14 were not recovered to wild-type levels when BcTaf14 without the ET domain was expressed. Our results provide insight into the regulatory roles of BcTaf14 and its two conserved domains in B. cinerea and will be helpful for understanding the function of the Taf14 protein in plant-pathogenic fungi.

Immunoprecipitation-Mass Spectrometry (IP-MS) of Protein-Protein Interactions of Nuclear-Localized Plant Proteins.

Transcription factors that act within a gene regulatory network (GRN) often interact with other proteins such as chromatin remodeling factors, histone modifiers, and other co-regulators. Characterizing these interactions is crucial for understanding the function and mechanism of action of atranscription factor. Here, a method for the identification of protein-protein interactions of nuclear-localized, transcription-associated factors is described. The method is based on the immunoprecipitation (IP) of a fluorophore-tagged target, followed by mass spectrometry (MS), peptide identification, and quantification of interacting proteins. By applying label-free quantification to IPs and their input protein extracts, statistically controlled protein enrichment ratios uncover high-confidence interaction partners of the target. A complete step-by-step procedure, including sample preparation, MS settings, data analysis, and visualization is provided.

Deciphering the role of unique bacterial transcription-associated factor HelD

Deciphering the role of unique bacterial transcription-associated factor HelD

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

Gene Regulatory Networks Governing the Generation and Regeneration of Blood

Blood is an example of a highly regenerative tissue and its regeneration depends on the presence of stem cells residing in the bone marrow in humans. A better understanding of how these stem cells are programmed would benefit their use in clinical practice and shed light on the mechanisms by which the unique properties of stem cells are established. Our approach is to delineate the gene regulatory networks (GRNs) that specify these cells during their development in the embryo, and we use the amphibian experimental model because a wealth of evidence shows that the mechanisms used are conserved in mammals including humans. Blood stem cells are made during the intraembryonic wave of hematopoiesis during embryonic development where they emerge from endothelial precursors in the floor of the dorsal aorta (DA). These cells are derived from lateral plate mesoderm and so we have focused on the subset of cells in the lateral plate mesoderm fated to become blood and endothelium known as definitive hemangioblasts. We have found that their programming results from the activities of vascular endothelial growth factor A (VEGFA) and bone morphogenetic protein (BMP) signaling and the inhibition by miRNA of transforming growth factor beta signaling. VEGFA is first generated in the somites adjacent to the lateral plate mesoderm, and one of the responses of the lateral plate mesoderm is to activate endogenous VEGFA expression. BMP has multiple inputs into the programming of these cells via the activation of the transcription factor (TF), Gata2, and of the VEGFA receptor. These actions culminate in the expression of the leukemia-associated TF, Scl/Tal1, which is essential for blood fate specification. The activity of VEGFA in driving endothelial development resides in the small isoform, but the medium and large isoforms are required to initiate the blood stem cell program in the floor of the DA. The expression of the small isoform is dependent on the blood TF with leukemia connections, Tel1/Etv6, whereas the larger isoforms depend on another transcription-associated factor with leukemia connections, Eto2, raising the possibility that the regulation of VEGFA expression may be the mode of action of these leukemic factors. The action of Tel1/Etv6 in directly activating VEGFA expression in the somites was unexpected because this TF had only been reported to repress transcription. Using chromatin immunoprecipitation technology, we were able to show that Tel1/Etv6 does indeed work by repressing the expression of a VEGFA repressor, FoxC3, but it also acts directly to activate VEGFA expression, working together with Klf4. Finally, we have also looked at the mesodermal population that gives rise to the earlier waves of hematopoiesis, which do not generate a stem cell. We find significant differences including differential use of TFs of the E-Twenty-Six (ETS) family. In conclusion, we have elucidated the GRN responsible for preparing the lateral mesoderm for blood stem cell production.

The C-Terminal Domain of RNA Polymerase II Is a Multivalent Targeting Sequence that Supports Drosophila Development with Only Consensus Heptads

The C-terminal domain (CTD) of RNA polymerase II (Pol II) is composed of repeats of the consensus YSPTSPS and is an essential binding scaffold for transcription-associated factors. Metazoan CTDs have well-conserved lengths and sequence compositions arising from the evolution of divergent motifs, features thought to be essential for development. Onthe contrary, we show that a truncated CTD composed solely of YSPTSPS repeats supports Drosophila viability but that a CTD with enough YSPTSPS repeats to match the length of the wild-type Drosophila CTD is defective. Furthermore, a fluorescently tagged CTD lacking the rest of Pol II dynamically enters transcription compartments, indicating that the CTD functions as a signal sequence. However, CTDs with too many YSPTSPS repeats are more prone to localize to static nuclear foci separate from the chromosomes. We propose that the sequence complexity of the CTD offsets aberrant behavior caused by excessive repetitive sequences without compromising its targeting function.

MRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription

SummarymRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription.

Dynamic Changes in the Protein Localization in the Nuclear Environment in Pancreatic β-Cell after Brief Glucose Stimulation.

Characterization of molecular mechanisms underlying pancreatic β-cell function in relation to glucose-stimulated insulin secretion is incomplete, especially with respect to global response in the nuclear environment. We focus on the characterization of proteins in the nuclear environment of β-cells after brief, high glucose stimulation. We compared purified nuclei derived from β-cells stimulated with 17 mM glucose for 0, 2, and 5 min using quantitative proteomics, a time frame that most likely does not result in translation of new protein in the cell. Among the differentially regulated proteins, we identified 20 components of the nuclear organization processes, including nuclear pore organization, ribonucleoprotein complex, and pre-mRNA transcription. We found alteration of the nuclear pore complex, together with calcium/calmodulin-binding chaperones that facilitate protein and RNA import or export to/from the nucleus to the cytoplasm. Putative insulin mRNA transcription-associated factors were identified among the regulated proteins, and they were cross-validated by Western blotting and confocal immunofluorescence imaging. Collectively, our data suggest that protein translocation between the nucleus and the cytoplasm is an important process, highly involved in the initial molecular mechanism underlying glucose-stimulated insulin secretion in pancreatic β-cells.

DreamBase: DNA modification, RNA regulation and protein binding of expressed pseudogenes in human health and disease.

Although thousands of pseudogenes have been annotated in the human genome, their transcriptional regulation, expression profiles and functional mechanisms are largely unknown. In this study, we developed dreamBase (http://rna.sysu.edu.cn/dreamBase) to facilitate the investigation of DNA modification, RNA regulation and protein binding of potential expressed pseudogenes from multidimensional high-throughput sequencing data. Based on ∼5500 ChIP-seq and DNase-seq datasets, we identified genome-wide binding profiles of various transcription-associated factors around pseudogene loci. By integrating ∼18 000 RNA-seq data, we analysed the expression profiles of pseudogenes and explored their co-expression patterns with their parent genes in 32 cancers and 31 normal tissues. By combining microRNA binding sites, we demonstrated complex post-transcriptional regulation networks involving 275 microRNAs and 1201 pseudogenes. We generated ceRNA networks to illustrate the crosstalk between pseudogenes and their parent genes through competitive binding of microRNAs. In addition, we studied transcriptome-wide interactions between RNA binding proteins (RBPs) and pseudogenes based on 458 CLIP-seq datasets. In conjunction with epitranscriptome sequencing data, we also mapped 1039 RNA modification sites onto 635 pseudogenes. This database will provide insights into the transcriptional regulation, expression, functions and mechanisms of pseudogenes as well as their roles in biological processes and diseases.

Kaposi\u2019s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV establishes a life-long infection in its host and alternates between a latent and lytic infection state. During lytic infection, lytic-related genes are expressed in a temporal manner and categorized as immediate early, early, and late gene transcripts. ORF34 is an early-late gene that interacts with several viral transcription-associated factors, however its physiological importance remains poorly understood. Here, we investigated the role of ORF34 during KSHV infection by generating ORF34-deficient KSHV, using a bacterial artificial chromosome system. Our results reveal that ORF34-deficient KSHV exhibited significantly attenuated late gene expression and viral production but did not affect viral DNA replication. ORF34 interacted with transcription factors ORF18, ORF24, ORF31, and ORF66, and a novel ORF34-interaction partner, ORF23. The C-terminal region of ORF34 was important for interaction with ORF24 and viral production. Our data support a model, in which ORF34 serves as a hub for recruiting a viral transcription complex to ORF24 to promote late viral gene expression.

Functional transcription factor target discovery via compendia of binding and expression profiles.

Genome-wide experiments to map the DNA-binding locations of transcription-associated factors (TFs) have shown that the number of genes bound by a TF far exceeds the number of possible direct target genes. Distinguishing functional from non-functional binding is therefore a major challenge in the study of transcriptional regulation. We hypothesized that functional targets can be discovered by correlating binding and expression profiles across multiple experimental conditions. To test this hypothesis, we obtained ChIP-seq and RNA-seq data from matching cell types from the human ENCODE resource, considered promoter-proximal and distal cumulative regulatory models to map binding sites to genes, and used a combination of linear and non-linear measures to correlate binding and expression data. We found that a high degree of correlation between a gene’s TF-binding and expression profiles was significantly more predictive of the gene being differentially expressed upon knockdown of that TF, compared to using binding sites in the cell type of interest only. Remarkably, TF targets predicted from correlation across a compendium of cell types were also predictive of functional targets in other cell types. Finally, correlation across a time course of ChIP-seq and RNA-seq experiments was also predictive of functional TF targets in that tissue.

Functional transcription factor target discovery via compendia of binding and expression profiles

Genome-wide experiments to map the DNA-binding locations of transcription-associated factors (TFs) have shown that the number of genes bound by a TF far exceeds the number of possible direct target genes. Distinguishing functional from non-functional binding is therefore a major challenge in the study of transcriptional regulation. We hypothesized that functional targets can be discovered by correlating binding and expression profiles across multiple experimental conditions. To test this hypothesis, we obtained ChIP-seq and RNA-seq data from matching cell types from the human ENCODE resource, considered promoter-proximal and distal cumulative regulatory models to map binding sites to genes, and used a combination of linear and non-linear measures to correlate binding and expression data. We found that a high degree of correlation between a gene’s TF-binding and expression profiles was significantly more predictive of the gene being differentially expressed upon knockdown of that TF, compared to using binding sites in the cell type of interest only. Remarkably, TF targets predicted from correlation across a compendium of cell types were also predictive of functional targets in other cell types. Finally, correlation across a time course of ChIP-seq and RNA-seq experiments was also predictive of functional TF targets in that tissue.

Nonsecreted cytoplasmic alpha-fetoprotein: a newly discovered role in intracellular signaling and regulation. An update and commentary.

The concept of a non-secreted cytoplasmic-bound form of alpha-fetoprotein is not a new notion in AFP biological activities. Cytoplasmic AFP (CyAFP) is a long known but forgotten protein in search of a function other than a histochemical biomarker. In this report, CyAFP is presented as an "old" protein with a newly described intracellular function. In 1976, CyAFP was shown to be a product of hepatoma cells utilizing 14Cleucine incorporation and demonstrated by autoradiographic procedures. The synthesis of CyAFP without secretion was demonstrated to occur in both malignant and non-malignant cells encompassing hepatomas, ascite fluid cells, immature rodent uterus, MCF-7 breast cancers, and cytosols from human breast cancer patients. Using computer protein matching and alignments in AFP versus members of the nuclear receptor superfamily, a consecutive series of leucine zipper (heptad) repeats in AFP was previously reported, suggesting the possibility for protein-to-protein interactions. The potential for heptad heterodimerization between protein-binding partners provided the rationale for proposing that CyAFP might have the capability to form molecular hetero-complexes with cytoplasmic based transcription factors. More recent investigations have now provided experimental evidence that CyAFP is capable of colocalizing and interacting with transcription-associated factors. Such proteins can modulate intracellular signaling leading to regulation of transcription factors and initiation of growth in human cancer cells. Although circulating serum AFP is known as a growth-enhancing factor during development, cytoplasmic AFP has a lethal role in the oncogenesis, growth, and metastasis of adult liver cancer.

On the identification of potential regulatory variants within genome wide association candidate SNP sets.

BackgroundGenome wide association studies (GWAS) are a population-scale approach to the identification of segments of the genome in which genetic variations may contribute to disease risk. Current methods focus on the discovery of single nucleotide polymorphisms (SNPs) associated with disease traits. As there are many SNPs within identified risk loci, and the majority of these are situated within non-coding regions, a key challenge is to identify and prioritize variants affecting regulatory sequences that are likely to contribute to the phenotype assessed.MethodsWe focused investigation on SNPs within lung and breast cancer GWAS loci that reached genome-wide significance for potential roles in gene regulation with a specific focus on SNPs likely to disrupt transcription factor binding sites. Within risk loci, the regulatory potential of sub-regions was classified using relevant open chromatin and epigenetic high throughput sequencing data sets from the ENCODE project in available cancer and normal cell lines. Furthermore, transcription factor affinity altering variants were predicted by comparison of position weight matrix scores between disease and reference alleles. Lastly, ChIP-seq data of transcription associated factors and topological domains were included as binding evidence and potential gene target inference.ResultsThe sets of SNPs, including both the disease-associated markers and those in high linkage disequilibrium with them, were significantly over-represented in regulatory sequences of cancer and/or normal cells; however, over-representation was generally not restricted to disease-relevant tissue specific regions. The calculated regulatory potential, allelic binding affinity scores and ChIP-seq binding evidence were the three criteria used to prioritize candidates. Fitting all three criteria, we highlighted breast cancer susceptibility SNPs and a borderline lung cancer relevant SNP located in cancer-specific enhancers overlapping multiple distinct transcription associated factor ChIP-seq binding sites.ConclusionIncorporating high throughput sequencing epigenetic and transcription factor data sets from both cancer and normal cells into cancer genetic studies reveals potential functional SNPs and informs subsequent characterization efforts.

Presenting ENCODE

The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription-factor association, chromatin structure and histone modification. In this overview, the Consortium guides the readers through the project itself, the data and their integrated analyses. Eighty per cent of the human genome now has at least one biochemical function assigned to it. In addition to expanding our understanding of how gene expression is regulated on a genome-wide scale, the newly identified functional elements should help researchers to interpret the results of genome-wide associated studies because many correspond to sites associated with human disease. This paper describes the first extensive map of human DNaseI hypersensitive sites — markers of regulatory DNA — in 125 diverse cell and tissue types. Integration of this information with other data sets generated by ENCODE (Encyclopedia of DNA Elements) identified new relationships between chromatin accessibility, transcription, DNA methylation and regulatory-factor occupancy patterns. Evolutionary-conservation analysis revealed signatures of recent functional constraint within DNaseI hypersensitive sites. DNaseI footprinting detects DNA sequences that are protected from cleavage by DNaseI because they are bound by regulatory factors. Studying these footprints in 41 diverse cell and tissue types, the authors describe millions of short sequence elements that are conserved recognition sequences for DNA-binding proteins. The effort nearly doubles the size of the human cis-regulatory lexicon and provides insight into chromatin states and levels of evolutionary conservation. A large collection of novel regulatory-factor recognition motifs that closely parallel major regulators of development, differentiation and pluripotency is also described. This manuscript describes the effort of the ENCODE (Encyclopedia of DNA Elements) Consortium to examine the principles of human transcriptional regulatory networks, using a subset of 119 transcription factors. The results are integrated with other genomic information to form a multi-level meta-network in which different levels have distinct properties. The findings will aid future interpretations of human genomics and help us to understand the basic principles of human biology and disease. These authors describe the ENCODE (Encyclopedia of DNA Elements) effort to provide a complete catalogue of primary and processed RNAs found either in specific sub-cellular compartments or throughout the cell. They show that three-quarters of the human genome can be transcribed, and provide a wealth of information about the range and levels of expression, localization, processing fates and modifications of both known and previously unannotated RNAs. Collectively, these observations suggest that the current concept of a gene should be revisited. In this ENCODE (Encyclopedia of DNA Elements) manuscript, the authors use chromosome conformation capture carbon copy (5C2) to look at the relationships between functional elements and distal target genes in 1% of the human genome in three dimensions. They describe numerous long-range interactions between promoters and distal sites that include elements resembling enhancers, promoters and CTCF-bound sites, their genomic distribution and complex interactions. Because only ∼7% of looping interactions are with the nearest gene, genomic proximity is not a simple predictor for long-range interactions.

GREAT improves functional interpretation of cis-regulatory regions

We developed the Genomic Regions Enrichment of Annotations Tool (GREAT) to analyze the functional significance of cis-regulatory regions identified by localized measurements of DNA binding events across an entire genome. Whereas previous methods took into account only binding proximal to genes, GREAT is able to properly incorporate distal binding sites and control for false positives using a binomial test over the input genomic regions. GREAT incorporates annotations from 20 ontologies and is available as a web application. Applying GREAT to data sets from chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) of multiple transcription-associated factors, including SRF, NRSF, GABP, Stat3 and p300 in different developmental contexts, we recover many functions of these factors that are missed by existing gene-based tools, and we generate testable hypotheses. The utility of GREAT is not limited to ChIP-seq, as it could also be applied to open chromatin, localized epigenomic markers and similar functional data sets, as well as comparative genomics sets.

Over-expression of transcription associated factor genes co-expressed with genes of the mevalonate pathway, upstream of isoprenoid biosynthesis, in Arabidopsis cultured cells

From a list of 3087 Arabidopsis putative transcription associated factor (TAF) genes including those possibly involved in transcription processes via their protein interaction, five TAF genes were related to genes upstream of isopentenyl diphosphate biosynthesis, i.e., the mevalonate pathway, by analyzing their network relationships with gene-to- gene Pearson's correlation coefficients calculated from 1388 DNA microarray results. We up-regulated the five candidate genes under the control of a cauliflower mosaic virus 35S promoter in Arabidopsis suspension-cultured T87 cells, and evaluated gene expression in the transgenic cells by DNA microarray analysis. All five of the genes were substantially up- regulated in each transgenic cell line, whereas none of the enzyme genes for the mevalonate pathway exhibited any considerable up-regulation. The inconsistency between the co-expression relationship and lack of consequent up-regulation suggests that it is difficult to find genes controlling isoprenoid upstream metabolites through co-expression analysis. Nevertheless, the results of this study are a basis for further study of isoprenoid synthesis.

Splicing and transcription-associated proteins PSF and p54nrb/nonO bind to the RNA polymerase II CTD.

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II (pol II) plays an important role in promoting steps of pre-mRNA processing. To identify proteins in human cells that bind to the CTD and that could mediate its functions in pre-mRNA processing, we used the mouse CTD expressed in bacterial cells in affinity chromatography experiments. Two proteins present in HeLa cell extract, the splicing and transcription-associated factors, PSF and p54nrb/NonO, bound specifically and could be purified to virtual homogeneity by chromatography on immobilized CTD matrices. Both hypo- and hyperphosphorylated CTD matrices bound these proteins with similar selectivity. PSF and p54nrb/NonO also copurified with a holoenzyme form of pol II containing hypophosphorylated CTD and could be coimmunoprecipitated with antibodies specific for this and the hyperphosphorylated form of pol II. That PSF and p54nrb/NonO promoted the binding of RNA to immobilized CTD matrices suggested these proteins can interact with the CTD and RNA simultaneously. PSF and p54nrb/NonO may therefore provide a direct physical link between the pol II CTD and pre-mRNA processing components, at both the initiation and elongation phases of transcription.

The 90- and 110-kDa Human NFAR Proteins Are Translated from Two Differentially Spliced mRNAs Encoded on Chromosome 19p13

The NFAR gene (nuclear factor associated with dsRNA) encodes a putative transcription-associated factor that we have shown is a substrate for the interferon-inducible, dsRNA-dependent protein kinase, PKR. However, our protein expression analysis has revealed that NFAR exists as two major protein species of 90 kDa (NFAR-1) and 110 kDa (NFAR-2) in the cell. To resolve the genetic identity of NFAR-1 and -2, we carried out sequence analysis of genomic and cDNA NFAR clones and determined that the coding region of this gene spans 16.2 kb and comprises 21 exons. Our data indicate that NFAR-1 and -2 arise from a single gene on chromosome 19p13 and are generated through alternative splicing events. NFAR-1 (HGMW-approved symbol ILF3) was found to comprise 1 extra exon, 18, that contains several stop codons to ensure termination of the protein at amino acid 702. In contrast, NFAR-2 lacks this exon, though it comprises an additional 3 coding exons (exons 19, 20, and 21) located at the carboxyl region to generate an extended product of 894 amino acids. Our studies, the first to elucidate the gene structure and chromosomal assignment of NFAR, establish the genetic basis for future NFAR research in humans.

The MouseAireGene: Comparative Genomic Sequencing, Gene Organization, and Expression

Mutations in the human AIRE gene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; OMIM 240300). Previously, we have cloned hAIRE and shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization of Aire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of the AIRE gene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT-PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product.