Intronic Promoters Research Articles

Trans-splicing in the cestode Hymenolepis microstoma is constitutive across the life cycle and depends on gene structure and composition

Spliced leader (SL) trans-splicing is a key process during mRNA maturation of many eukaryotes, in which a short sequence (SL) is transferred from a precursor SL-RNA into the 5′ region of an immature mRNA. This mechanism is present in flatworms, in which it is known to participate in the resolution of polycistronic transcripts. However, most trans-spliced transcripts are not part of operons, and it is not clear if this process may participate in additional regulatory mechanisms in this group. In this work, we present a comprehensive analysis of SL trans-splicing in the model cestode Hymenolepis microstoma. We identified four different SL-RNAs which are indiscriminately trans-spliced to 622 gene models. SL trans-splicing is enriched in constitutively expressed genes and does not appear to be regulated throughout the life cycle. Operons represented at least 20% of all detected trans-spliced gene models, showed conservation to those of the cestode Echinococcus multilocularis, and included complex loci such as an alternative operon (processed as either a single gene through cis-splicing or as two genes of a polycistron). Most insertion sites were identified in the 5′ untranslated region (UTR) of monocistronic genes. These genes frequently contained introns in the 5′ UTR, in which trans-splicing used the same acceptor sites as cis-splicing. These results suggest that, unlike other eukaryotes, trans-splicing is associated with internal intronic promoters in the 5′ UTR, resulting in transcripts with strong splicing acceptor sites without competing cis-donor sites, pointing towards a simple mechanism driving the evolution of novel SL insertion sites.

Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells.

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) – the promoter that regulates immediate early (IE) gene expression – is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in in vitro generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

Alternative promoters drive human cytomegalovirus reactivation from latency

Reactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP has poor activity in HPCs, it is unclear how viral transactivators are expressed during reactivation. It has been presumed that viral gene expression is reinitiated via de-repression of the MIEP. We demonstrate that immediate early transcripts arising from reactivation originate predominantly from alternative promoters within the canonical major immediate early locus. Disruption of these intronic promoters results in striking defects in re-expression of viral genes and viral genome replication in the THP-1 latency model. Furthermore, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings shift the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.

Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-l-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters

Background2-Keto-l-gulonic acid (2-KGA), the precursor of vitamin C, is currently produced by two-step fermentation. In the second step, l-sorbose is transformed into 2-KGA by the symbiosis system composed of Ketogulonicigenium vulgare and Bacillus megaterium. Due to the different nutrient requirements and the uncertain ratio of the two strains, the symbiosis system significantly limits strain improvement and fermentation optimization.ResultsIn this study, Ketogulonicigenium robustum SPU_B003 was reported for its capability to grow well independently and to produce more 2-KGA than that of K. vulgare in a mono-culture system. The complete genome of K. robustum SPU_B003 was sequenced, and the metabolic characteristics were analyzed. Compared to the four reported K. vulgare genomes, K. robustum SPU_B003 contained more tRNAs, rRNAs, NAD and NADP biosynthetic genes, as well as regulation- and cell signaling-related genes. Moreover, the amino acid biosynthesis pathways were more complete. Two species-specific internal promoters, P1 (orf_01408 promoter) and P2 (orf_02221 promoter), were predicted and validated by detecting their initiation activity. To efficiently produce 2-KGA with decreased CO2 release, an innovative acetyl-CoA biosynthetic pathway (XFP-PTA pathway) was introduced into K. robustum SPU_B003 by expressing heterologous phosphoketolase (xfp) and phosphotransacetylase (pta) initiated by internal promoters. After gene optimization, the recombinant strain K. robustum/pBBR-P1_xfp2502-P2_pta2145 enhanced acetyl-CoA approximately 2.4-fold and increased 2-KGA production by 22.27% compared to the control strain K. robustum/pBBR1MCS-2. Accordingly, the transcriptional level of the 6-phosphogluconate dehydrogenase (pgd) and pyruvate dehydrogenase genes (pdh) decreased by 24.33 ± 6.67 and 8.67 ± 5.51%, respectively. The key genes responsible for 2-KGA biosynthesis, sorbose dehydrogenase gene (sdh) and sorbosone dehydrogenase gene (sndh), were up-regulated to different degrees in the recombinant strain.ConclusionsThe genome-based functional analysis of K. robustum SPU_B003 provided a new understanding of the specific metabolic characteristics. The new XFP-PTA pathway was an efficient route to enhance acetyl-CoA levels and to therefore promote 2-KGA production.

Alternative intronic promoters in development and disease.

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.

Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species.

BackgroundRed yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts.ResultsThe upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in Rhodotorula toruloides were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene (LDP1) displayed much stronger activity (4–11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene (ACC1) and fatty acid synthase β subunit gene (FAS1) exhibited intermediate strength, displaying 50–160 and 20–90% levels of GPD1 promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of LDP1 (LDP1in promoter) enhanced its promoter activity by 1.6–3.0 folds. Similarly, the strength of ACC1 promoter was enhanced by 1.5–3.2 folds if containing intron 1. The intron 1 sequences of ACL1 and FAS1 also played significant regulatory roles. When driven by the intronic promoters of ACC1 and LDP1 (ACC1in and LDP1in promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene (DGA1) by LDP1in promoter was significantly more efficient than GPD1 promoter in enhancing lipid accumulation.ConclusionIntronic sequences play an important role in regulating gene expression in R. toruloides. Three intronic promoters, LDP1in, ACC1in and FAS1in, are excellent promoters for metabolic engineering in the oleaginous and carotenogenic yeast, R. toruloides. Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0600-x) contains supplementary material, which is available to authorized users.

Principles of microRNA Regulation Revealed Through Modeling microRNA Expression Quantitative Trait Loci.

Extensive work has been dedicated to study mechanisms of microRNA-mediated gene regulation. However, the transcriptional regulation of microRNAs themselves is far less well understood, due to difficulties determining the transcription start sites of transient primary transcripts. This challenge can be addressed using expression quantitative trait loci (eQTLs) whose regulatory effects represent a natural source of perturbation of cis-regulatory elements. Here we used previously published cis-microRNA-eQTL data for the human GM12878 cell line, promoter predictions, and other functional annotations to determine the relationship between functional elements and microRNA regulation. We built a logistic regression model that classifies microRNA/SNP pairs into eQTLs or non-eQTLs with 85% accuracy; shows microRNA-eQTL enrichment for microRNA precursors, promoters, enhancers, and transcription factor binding sites; and depletion for repressed chromatin. Interestingly, although there is a large overlap between microRNA eQTLs and messenger RNA eQTLs of host genes, 74% of these shared eQTLs affect microRNA and host expression independently. Considering microRNA-only eQTLs we find a significant enrichment for intronic promoters, validating the existence of alternative promoters for intragenic microRNAs. Finally, in line with the GM12878 cell line derived from B cells, we find genome-wide association (GWA) variants associated to blood-related traits more likely to be microRNA eQTLs than random GWA and non-GWA variants, aiding the interpretation of GWA results.

The effect of 2, 3, 7, 8-Tetrachlorodibenzo -p-Dioxin in the human 3’IgH regulatory region and immunoglobulin expression. (IRC11P.440)

Abstract The transcription of Ig heavy chain (Igh) is regulated by numerous regulatory elements including 3’Igh regulatory region (3’IghRR).Several transcription factors are involved in modulating the 3’IghRR and aryl hydrocarbon receptor (AhR). TCDD is an environmental contaminant and a ligand for AhR known to interfere with Immunoglobulins (Ig) expression in animal models. Our lab have identified in previous study the mouse 3’Ig heavy chain regulatory region (3’IgH RR) as a sensitive transcriptional target of TCDD, which inhibits mouse 3’IgH RR activation and induces AhR binding to dioxin response element (DRE) within 3’IgH RR. In human the hs1, 2 enhancer is polymorphic which has been correlated with several autoimmune diseases. We have identified TCDD-induced activation of the hu-hs1, 2 enhancer and a decrease in enhancer activity with deletion of the polymorphic region. Pervious study in our lab suggested that the hu-hs1, 2 enhancer may be a negative regulator of 3’IgH RR activity and Ig expression. The objective of this study is to study the cooperative effect of the Human 3’IgHRR Enhancers in intronic promoters and the possibility of TCDD interfering with this activity in the presence and absence of the stimulation and to study the polymorphic alleles effect in the human 3’IgHRR basal activity. We used human CL-01 B cell for this study and multiple luciferase reporter constructs driven by human Igγ intronic promoter and regulated by the human 3’IgHRR enhancers .

Dual Promoter Usage as Regulatory Mechanism of let-7c Expression in Leukemic and Solid Tumors

Let-7c, an intronic microRNA (miRNA) embedded in the long non-coding gene LINC00478, can act as a tumor suppressor by targeting oncogenes. Previous studies indicated that in acute promyelocytic leukemia (APL), a subtype of acute myelogenous leukemia (AML) bearing the leukemia promoting PML/RARα fusion protein, let-7c expression seems to be controlled by the host gene promoter, in which canonical Retinoic Acid Responsive Elements (RAREs) are bound by PML/RARα in an all transretinoic acid (ATRA)-sensitive manner. Here, let-7c transcriptional regulation was further investigated and a novel intronic promoter upstream of the pre-miRNA was identified. This new promoter has transcriptional activity strongly indicating that at least two promoters need to be considered for let-7c transcription: the distal host gene and the proximal intronic promoter. Therefore, epigenetic modifying enzymes and histone acetylation and methylation status were analyzed on both let-7c promoters. It was demonstrated that ATRA treatment leads to let-7c upregulation inducing a more open chromatin conformation of the host gene promoter, with an enrichment of epigenetic marks that correlate with a more active transcriptional state. Conversely, the epigenetic marks on the intronic promoter are not significantly affected by ATRA treatment. Interestingly, in solid tumors such as prostate and lung adenocarcinoma it was found that both host and intronic promoters are functional. These data suggest that while the host gene promoter may control let-7c expression in AML, in a nonleukemic tumor context instead the intronic promoter contributes or preferentially regulates let-7c transcription. Alternative promoter usage represents a regulatory mechanism of let-7c expression in different tissues. Mol Cancer Res; 12(6); 878-89. ©2014 AACR.

Control of the STAT6–BCL6 Antagonism by SWAP-70 Determines IgE Production

Asthma and allergies are major health concerns in which Ig isotype E plays a pivotal role. Ag-bound IgE drives mast cells and basophils into exocytosis, thereby promoting allergic and potentially anaphylactic reactions. The importance of tightly regulated IgE production is underscored by severe immunological conditions in humans with elevated IgE levels. Cytokines direct IgH class-switching to a particular isotype by initiation of germline transcription (GLT) from isotype-specific intronic (I) promoters. The switch to IgE depends on IL-4, which stimulates GLT of the Iε promoter, but is specifically and strongly impaired in Swap-70(-/-) mice. Although early events in IL-4 signal transduction (i.e., activation of the JAK/STAT6 pathway) do not require SWAP-70, SWAP-70 deficiency results in impaired Iε GLT. The affinity of STAT6 to chromatin is reduced in absence of SWAP-70. Chromatin immunoprecipitation revealed that SWAP-70 binds to Iε and is required for association of STAT6 with Iε. BCL6, known to antagonize STAT6 particularly at Iε, is increased on Iε in absence of SWAP-70. Other promoters bound by BCL6 and STAT6 were found unaffected. We conclude that SWAP-70 controls IgE production through regulation of the antagonistic STAT6 and BCL6 occupancy of Iε. The identification of this mechanism opens new avenues to inhibit allergic reactions triggered by IgE.

PROmiRNA: a new miRNA promoter recognition method uncovers the complex regulation of intronic miRNAs

The regulation of intragenic miRNAs by their own intronic promoters is one of the open problems of miRNA biogenesis. Here, we describe PROmiRNA, a new approach for miRNA promoter annotation based on a semi-supervised statistical model trained on deepCAGE data and sequence features. We validate our results with existing annotation, PolII occupancy data and read coverage from RNA-seq data. Compared to previous methods PROmiRNA increases the detection rate of intronic promoters by 30%, allowing us to perform a large-scale analysis of their genomic features, as well as elucidate their contribution to tissue-specific regulation. PROmiRNA can be downloaded from http://promirna.molgen.mpg.de.

Intronic promoters and their noncoding transcripts: A new source of cancer‐associated genes

Recent studies of mammalian genomes suggest that alternative promoters are associated with various disorders, including cancer. Here we present an intronic promoter of the murine proteinase 3 gene, which drives the expression of an alternative mRNA in intron 2 of the prtn3 gene. The proximal promoter sequences were identified and a series of promoter deletion constructs were used to identify the sequence elements that are required for basal promoter activity. Expression of the homeobox transcription factor CUX1 p75 isoform was found to suppress the activity of the alternative PR3 promoter. Data base analyses, multiple alignments and expression data showed that the intronic PR3 promoter is active in leukemia and other tumor cells as well as in mouse embryo, male mammary gland and bone marrow. In the spleen, the transcript is exclusively expressed by Gr-1(int) /CD11b(+) cells, which are also known as myeloid-derived suppressor cells (MDSCs). In humans, an alternative transcript of the PR3-gene could be detected in the bone marrow and in various cancer cell lines but not in primary leukemia cells, suggesting a species-overarching function of this kind of promoter. Therefore, the alternative PR3 promoter and its mRNA may be useful tools to investigate the fate of hematopoietic stem cells.

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

Identification of C-terminally and N-terminally truncated estrogen receptor α variants in the mouse

We re-examined mouse ERα mRNA variants using rapid amplification of cDNA ends (RACE) and RT-PCR. Our analysis showed the presence of several mRNA variants containing unique 5′- or 3′-nucleotide sequences. We mapped the cDNA sequences on the mouse genome, and identified four novel 3′-terminal and 5′-leader exons in the intronic region between exons 4 and 5. RT-PCR analysis revealed that the expression patterns of the C-terminally truncated ERα products (CTERPs) were similar to that of Wild-type ERα and that the N-terminally truncated ERα products (NTERPs) appeared to have different expression profiles. Moreover, we constructed expression vectors and analyzed the subcellular localization and the transcriptional activation abilities of the variant proteins in transfected HEK293 cells using immunocytochemistry and luciferase reporter assay. The CTERP variants localized in the nuclei and constitutively activated estrogen response element (ERE)-driven promoters, while the NTERP variant was located in the extra-nuclear regions and had no ability to activate the ERE promoters in the presence or absence of 10 nM estradiol. Our results indicate that the mouse ERα gene is more complex than previously thought in terms of genomic organization and that alternative splicing and alternative usage of intronic promoters contribute to the remarkable diversity of ERα mRNAs and proteins.

Analysis of nucleosome positioning in promoters of miRNA genes and protein-coding genes

Nucleosome positioning in promoters is important for gene transcription regulation. In this paper, with a nucleosome prediction model, curvature profile, the characteristics of nucleosome positioning in promoters are analyzed for miRNA genes and protein-coding genes. In the vicinity of transcription start site (TSS), there is a nucleosome-free region (NFR) followed by a positioned nucleosome at ∼200 bp downstream of TSS. A similar characteristic is observed in independent intronic promoters and intergenic promoters, namely, both types of promoters have a longer NFR in 0–−400 bp upstream of TSS. Moreover, transcription factor binding sites (TFBSs) locate in the NFR with a high concentration. However, nucleosome pattern in dependent intronic promoters are like that in protein-coding promoters, with two nucleosomes positioned at −200–−400 bp and −400–−600 bp upstream of TSS. The results indicate nucleosome positioning is probably different in independent miRNA promoters and protein-coding promoters; and positioning seems to be an important factor not only in regulation of protein-coding gene, but also in that of miRNA gene.

Structure and activity of putative intronic miRNA promoters

MicroRNAs (miRNAs) are RNA sequences of approximately 22 nucleotides that mediate post-transcriptional regulation of specific mRNAs. miRNA sequences are dispersed throughout the genome and are classified as intergenic (between genes) or intronic (embedded into a gene). Intergenic miRNAs are expressed by their own promoter, and until recently, it was supposed that intronic miRNAs are transcribed from their host gene. Here, we performed a genomic analysis of currently known intronic miRNA regions and observed that approximately 35% of intronic miRNAs have upstream regulatory elements consistent with promoter function. Among all intronic miRNAs, 30% have associated Pol II regulatory elements, including transcription start sites, CpG islands, expression sequence tags, and conserved transcription factor binding sites, while 5% contain RNA Pol III regulatory elements (A/B box sequences). We cloned intronic regions encompassing miRNAs and their upstream Pol II (miR-107, miR-126, miR-208b, miR-548f-2, miR-569, and miR-590) or Pol III (miR-566 and miR-128-2) sequences into a promoterless plasmid, and confirmed that miRNA expression occurs independent of host gene transcription. For miR-128-2, a miRNA overexpressed in acute lymphoblastic leukemia, ChIP analysis suggests dual regulation by both intronic (Pol III) and host gene (Pol II) promoters. These data support complex regulation of intronic miRNA expression, and have relevance to disregulation in disease settings.

Expression of a Structurally Unique Osteoclastic Protein-tyrosine Phosphatase Is Driven by an Alternative Intronic, Cell Type-specific Promoter

An osteoclastic protein-tyrosine phosphatase (PTP-oc), essential for osteoclast activity, shows sequence identity with the intracellular domain of GLEPP1, a renal receptor-like transmembrane PTP. PTP-oc has been assumed to be a truncated variant of GLEPP1, resulting from alternative splicing. However, the 5'-untranslated region sequence of PTP-oc mRNA contains 217 bp from an intron of GLEPP1. There are no splicing acceptor sites at the PTP-oc transcription site. The intronic sequence flanking the 5' end of the PTP-oc transcription start site contains potential promoter elements essential for transcriptional initiation. To test the hypothesis that the PTP-oc gene has an alternative, tissue-specific, intronic promoter, the promoter activity of a 1.3-kb PCR fragment covering the 5'-flanking region of the PTP-oc gene was measured. The putative PTP-oc promoter fragment showed strong promoter activity in U937 cells. Mutation of the putative TATA box within the PTP-oc promoter abolished 60-90% of its promoter activity. The PTP-oc promoter fragment showed strong promoter activity in cells that express PTP-oc (U937 cells and RAW264.7 cells) but not in cells that do not express the enzyme (skin fibroblasts, TE85 cells, and HEK293 cells). These findings strongly support the conclusion that the 1.3-kb intronic fragment contains the tissue-specific, PTP-oc proximal promoter. Deletion and functional analyses indicate that the proximal 5' sequence flanking the TATA box of the PTP-oc contains potential repressor elements. The removal of the putative repressor elements led to the apparent loss of tissue specificity. In summary, we conclude that an intronic promoter within the GLEPP1 gene drives the expression of the PTP-oc in a cell type-specific manner. This GLEPP1/PTP-oc gene system is one of the very few systems in which two important tissue-specific enzymes are derived from the same gene by the use of alternative intronic promoters.

Role for RFX Transcription Factors in Non-neuronal Cell-specific Inactivation of the Microtubule-associated Protein MAP1A Promoter

Microtubule-associated protein MAP1A is expressed abundantly in mature neurons and is necessary for maintenance of neuronal morphology and localization of some molecules in association with the microtubule-based cytoskeleton. Previous studies indicated that its complementary expression together with MAP1B during nervous system development is regulated at the transcriptional level and that the mouse Map1A gene is transcribed under the control of 5' and intronic promoters. In this study, we investigated the regulatory mechanisms that govern the neuronal cell-specific activation of the MAP1A 5' promoter. We found that two regulatory factor for X box (RFX) binding sites in exon1 of both the mouse and human genes are important for effective transcriptional repression observed only in non-neuronal cells by reporter assays. Among RFX transcription factor family members, RFX1 and 3 mainly interact with repressive elements in vitro. Cotransfection studies indicated that RFX1, which is expressed ubiquitously, down-regulated the MAP1A 5' promoter activity in non-neuronal cells. Unexpectedly, RFX3, which is abundantly expressed in neuronal cells, down-regulated the transactivity as well, when it was expressed in non-neuronal cells. Both RFX1 and 3 did not down-regulate the transactivity in neuronal cells. These results suggest that RFX1 and 3 are pivotal factors in down-regulation of the MAP1A 5' promoter in non-neuronal cells. The cell type-specific down-regulation, however, does not depend simply on which RFX interacts with the elements, but seems to depend on underlying profound mechanisms.

Novel insights into class switch recombination.

The discovery of activation-induced cytidine deaminase, a putative RNA editing enzyme essential for both class switch recombination and somatic hypermutation, marked the field of isotype switching studies in the year 2000. More recent work from the same group now highlights some essential mechanistic aspects of the switch recombination process. In particular, much has been learnt about the relationship between transcription and recombination and the transcriptional competence of intronic promoters on looped-out circular DNA uncoupled from the proximal and distal enhancers. These findings have far-reaching implications, particularly for studies of class switch recombination in tissues. Although these important advances do not directly relate to interleukin-4-dependent immunoglobulin E switching, the conceptual and experimental tools developed through these studies are certain to foster progress in the immunoglobulin E field.

Characterization of two promoters that regulate alternative transcripts in the microtubule-associated protein (MAP) 1A gene

We cloned and characterized the mouse gene for microtubule-associated protein (MAP) 1A, an important protein for neuronal morphology and mitotic spindle formation. We also investigated the 5′ untranslated region of the gene to characterize the promoter units. Two alternative transcripts different in the 5′ region were identified by 5′ RACE. Both transcripts were principally observed in the brain. Genomic cloning revealed that exons 1, 2, and 4 generate the 5′ part of a long transcript, whereas exons 3 and 4 generate a short transcript. Putative 5′ and intronic promoters flanking exons 1 and 3, respectively, are GC-rich and lack a canonical TATA box. DNase I footprinting from mouse cells revealed that several potential cis-elements were occupied by nuclear proteins. A reporter assay system in conjunction with a number of deletion and mutation constructs was used to test the two putative promoters. Both putative promoters showed transactivity and their function was dependent upon Sp1 sites. In addition, an NF-1 site, an HNF3B site, and an AP-1/ATF site were necessary for basal promoter activity of the intronic promoter. Our data provide insight into the regulatory mechanisms that govern the expression of the MAP1A gene.