Enhancer Motifs Research Articles

Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts.

The multiplicity of proteins compared with genes in mammals owes much to alternative splicing. Splicing signals are so subtle and complex that small perturbations may allow the production of new mRNA variants. However, the flexibility of splicing can also be a liability, and several genetic diseases result from single-base changes that cause exons to be skipped during splicing. Conventional oligonucleotide strategies can block reactions but cannot restore splicing. We describe here a method by which the use of a defective exon was restored. Spinal muscular atrophy (SMA) results from mutations of the Survival Motor Neuron (SMN) gene. Mutations of SMN1 cause SMA, whereas SMN2 acts as a modifying gene. The two genes undergo alternative splicing with SMN1, producing an abundance of full-length mRNA transcripts, whereas SMN2 predominantly produces exon 7-deleted transcripts. This discrepancy is because of a single nucleotide difference in SMN2 exon 7, which disrupts an exonic splicing enhancer containing an SF2ASF binding site. We have designed oligoribonucleotides that are complementary to exon 7 and contain exonic splicing enhancer motifs to provide trans-acting enhancers. These tailed oligoribonucleotides increased SMN2 exon 7 splicing in vitro and rescued the incorporation of SMN2 exon 7 in SMA patient fibroblasts. This treatment also resulted in the partial restoration of gems, intranuclear structures containing SMN protein that are severely reduced in patients with SMA. The use of tailed antisense oligonucleotides to recruit positively acting factors to stimulate a splicing reaction may have therapeutic applications for genetic disorders, such as SMA, in which splicing patterns are altered.

Finding of TRE (TPA responsive element) in the sequence of human taurine transporter promoter.

Activity of the taurine transporter (TAUT) is regulated by signal transduction in response to diverse stimuli including tumor promoters such as phobol ester. Regulation of the transcription rate of TAUT appears to play an important role in exerting biological roles of taurine in mammalian tissues in adverse environments. Although cDNA of human TAUT has been cloned and sequenced in placenta, thyroid cells, and retinal pigment epithelial cells, the promoter region of TAUT has never been reported. In order to clone the upstream region of the human TAUT promoter, we have compared TAUT cDNA sequences with the entire human genome sequence. Polymerase chain reaction (PCR) was performed from genomic DNA prepared from a SK-Hep-1 cell line for the amplification of the TAUT promoter region including the partial exon (150 bp) and the 5' untranslated region (UTR, 380 bp). The PCR product of the promoter region, which was 1800 bp long, was ligated into the pGEM-T vector, and sequenced. The 5' flanking region of the TAUT promoter was analysed for the identification of enhancer and regulation motifs. Surprisingly we found the consensus TPA responsive element (TGAGTCAG) which is responsible for gene regulation by the protein kinase C (PKC)-mediated signal transduction pathway. The well known fact that proto-oncogene AP1 (cFos/cJun heterodimer or cJun/cJun homodimer) binds to TRE implies that TAUT expression might be closely linked to tumor promotion. Since AP1 activity is also tightly regulated in nerve cells, AP1-regulated TAUT transcription might be an important step in nerve cell function. Furthermore, the TFIID binding site, cap signal for transcription initiation, PEA3 motif, heat shock factor binding motif, and many other motifs were found in the TAUT promoter region, and require characterization.

Identification of genetic determinants that regulate tumorigenicity of Friend murine leukemia virus in rats.

A neuropathogenic variant of Friend murine leukemia virus (FrMLV), clone A8, has been shown to cause thymoma and infiltration of leukemic cells to organs at 7-8 weeks post-infection in rats with a more rapid progression than clone 57. We have previously reported that the determinant for induction of aggressive leukemia in rats is located in the ClaI-AatII fragment containing the long terminal repeat (LTR) and the 5' half of the 5' leader sequence of A8 virus. Further studies of chimeric viruses restricted the determinant for the induction of thymoma to only the 0.6-kb ClaI-KpnI fragment of A8. This fragment contains a 0.1 kb region of the 3' terminus of the env gene, the intergenic region, the U3, and the 5' half of the R region in the LTR. Major differences in the fragment between A8 and 57 viruses were found in the U3 region, especially in the enhancer motifs. These results indicate that the enhancer region of A8-LTR contributes to the manifestation of thymoma with rapid progression in rats.

HnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure.

The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point.

Characterization of a carbohydrate response element regulating the gene for human galactose-1-phosphate uridyltransferase

Human galactose-1-phosphate uridyltransferase (hGALT) is a central enzyme in the conserved pathway by which galactose is converted to energy, UDP-galactose and UDP-glucose. A natural mutation that deleted −119 to −116 bp (delGTCA) of the promoter decreased hGALT mRNA and enzyme activity and prompted analysis of hGALT gene regulation. Regulatory domains were identified by inspection and confirmed in a reporter system. Previous studies by others were confirmed that HepG2 cells grown in d-glucose increased hGALT enzyme activity and mRNA by 30%. We extended these observations by sequencing the promoter region and identifying a potential carbohydrate response element (ChoRE). The response to glucose rose to 190% when a plasmid construct containing a luciferase reporter and only the −165 bp region as a promoter was transfected into HepG2 and NIH:OVCAR-3. By contrast, fibroblasts transfected with the identical construct failed to respond to glucose. Within the −165 bp region there were two enhancer (E-box) motifs that encompassed the delGTCA mutation. The deletion diminished the positive regulatory response, but an additional GTCA repeat unexpectedly increased the response. Using this postulated ChoRE as a probe in electrophoretic mobility shift assays, multiple nuclear proteins bound and one was identified as a member of the basic/helix–loop–helix/leucine zipper enhancer-binding (b/HLH/LZ) family. Increased binding of proteins correlated with increased hGALT expression when the spacing between E-box motifs was enlarged but the carbohydrate response was dampened. When the 3 ′ E-box was mutated, b/HLH/LZ binding and gene expression were abolished. We conclude that the hGALT promoter region contains a ChoRE in which the spacing between and the sequence of its E-box motifs are critical. One nuclear protein of the b/HLH/LZ family is necessary, but not sufficient for the carbohydrate response.

Embryonic expression and regulation of the large zinc finger protein KRC.

KRC fusion proteins bind to the kappaB enhancer motif and to the signal sequences of V(D)J recombination. Here we have characterized endogenous KRC in mouse embryos and lymphoma cell lines. Starting from midgestation, neuronal- and lymphoid-restricted expression of KRC was observed from the dorsal root ganglia, trigeminal ganglion, thymus, and cerebral cortex. Several B-cell lines produced an alternatively spliced KRC transcript of 4.5 kb and a 115-kDa DNA-binding protein isoform. Additionally, that KRC transcript was induced by lipopolysaccharide, a potent activator of cells in immunity and inflammation. In genetic-engineered B cells stably transfected with inducible expression vectors for the recombination activating genes RAG1, RAG2, or both, the avidity of KRC to DNA was markedly decreased when RAG1 and RAG2 were overexpressed. We hypothesize that KRC may function in developing thymocytes and neurons, where its role might be transcription regulation or DNA recombination.

An exon splice enhancer mutation causes autosomal dominant GH deficiency.

Familial isolated GH deficiency type II (IGHD II) is caused, in some cases, by heterogeneous IVS3 mutations that affect GH mRNA splicing. We report here our finding an A-->G transition of the fifth base of exon 3 (E3+ 5 A-->G) in affected individuals from an IGHD II family. This mutation disrupts a (GAA)(n) exon splice enhancer (ESE) motif immediately following the weak IVS2 3' splice site. The mutation also destroys an MboII site used to demonstrate heterozygosity in all affected family members. To determine the effect of ESE mutations on GH mRNA processing, GH(3) cells were transfected with expression constructs containing the normal ESE, +5 A-->G, or other ESE mutations, and cDNAs derived from the resulting GH mRNAs were sequenced. All ESE mutations studied reduced activation of the IVS2 3' splice site and caused either partial E3 skipping, due to activation of an E3+ 45 cryptic 3' splice site, or complete E3 skipping. Partial or complete E3 skipping led to loss of the codons for amino acids 32-46 or 32-71, respectively, of the mature GH protein. Our data indicate that the E3+ 5 A-->G mutation causes IGHD II because it perturbs an ESE required for GH splicing.

TNFalpha decreases alphaMHC expression by a NO mediated pathway: role of E-box transcription factors for cardiomyocyte specific gene regulation.

Tumor necrosis factor alpha(TNFalpha) is thought to play a key role in the pathogenesis of cardiac failure. In the myocardium, TNFalpha enhances the expression of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) has been shown to affect beta-agonist-dependent cardiac contractility and relaxation. It is not clear, however, whether TNFalpha mediated NO release has sustained cardiac effects, by altering expression of cardiomyocyte specific genes such as alpha-myosin heavy chain (alphaMHC). Neonatal rat ventricular cardiomyocytes (CM) were stimulated with TNFalpha and/or the NOS inhibitor nitro-L-arginine (L-NNA). Protein binding to the E-box enhancer element in the alphaMHC promoter was evaluated by electrophoretic mobility shift assay (EMSA) and transcriptional activity of the E-box consensus motif was determined by luciferase assay. mRNA levels of the endogenous alphaMHC gene were assessed by RT-PCR. In vivo studies were performed in transgenic mice with cardiac specific over-expression of TNFalpha. CM treated with TNFalpha exhibited decreased levels of alphaMHC transcripts (69 +/- 8% of control), the effect of TNFalpha was reversed by L-NNA (94 +/- 14% of control). As shown by EMSA, TNFalpha reduced protein binding to the alphaMHC E-box enhancer motif via NO dependent pathways. Addition of the NO-donor sodium nitroprusside (SNP) to CM nuclear extracts dose dependently disrupted protein binding to the alphaMHC E-box. Furthermore, exposure of CM to TNFalpha or SNP decreased transcription from an E-box luciferase-reporter construct (TNFalpha: 74 +/- 12%; SNP 250 microM: 72 +/- 10%; SNP 500 microM: 66 +/- 11% of control). In myocardial tissue of TNFalpha transgenic mice, increased nitrotyrosine staining, decreased protein binding to the alphaMHC E-box motif and reduced expression of alphaMHC (62 +/- 26%) were observed. The present study shows that TNFalpha reduces alphaMHC transcript levels in cardiomyocytes. Our data obtained in cultured CM and in TNFalpha transgenic mice support the notion that TNFalpha exerts these effects by NO and E-box dependent mechanisms in vitro and possibly in vivo.

The κB transcriptional enhancer motif and signal sequences of V(D)J recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study

BackgroundThe ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements. The amino-DNA binding domain (ZAS-N) and the carboxyl-DNA binding domain (ZAS-C) of a representative family member, named κB DNA binding and recognition component (KRC), were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing. The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains.ResultsBoth fusion proteins selected sequences that were similar to the κB motif or the canonical elements of the V(D)J recombination signal sequences (RSS) from a pool of degenerate oligonucleotides. Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer. In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS.ConclusionsThe RSS are cis-acting DNA motifs which are essential for V(D)J recombination of antigen receptor genes. Due to its specific binding affinity for RSS and κB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(D)J recombination.

Identification of cis-elements regulating expression of Fgf10 during limb development.

Fibroblast growth factor 10 (FGF10) is known to be expressed in limb mesenchymal cells and to function as a mesenchymal signaling factor involved in epithelial-mesenchymal interactions during limb development. To elucidate regulation of Fgf10 expression, we isolated the promoter region of Fgf10 containing its 2.0 kb upstream 5'-fragment from the initiation codon and its 0.9 kb downstream fragment. Transcriptional activity of the fragment was examined with transgenic mice, using a lacZ-reporter system. Although no significant expression of the reporter gene was observed for the 0.2 kb 5'-fragment, expression was detected in the apical ectodermal ridge of the limb bud and developing cartilage of the limb for the 2.0 kb and 0.7 kb 5'-fragments, respectively. From comparison of the mouse sequences of the 2.0 kb fragment with corresponding sequences of human and chicken Fgf10, we identified 17 conserved putative enhancer motifs for AER expression and other unidentified expressions. For limb cartilage expression, we found putative enhancer sequences conserved among the three species in the 0.7 kb 5'-fragment. In the fragment, three DNA binding motifs were identified in the mouse and human sequence, although they are not conserved in the corresponding chicken sequence.

The drosophila aeroplane mutant is caused by an I-element insertion into a tissue-specific teashirt enhancer motif.

In Drosophila melanogaster, aeroplane (ae) is a regulatory allele of teashirt (tsh), and the mutant wing posture phenotype of homozygous ae flies is caused by a defect in the hinge region of the wing, whereby the base of the wing at the proximal ventral radius is fused to the thorax in the region of the pleural wing process. The apparent paralysis of the wings and the drooping halteres are caused by an I-element insertion into a 3' noncoding sequence of tsh. The cis-acting regulatory element interrupted by the I element is required, to drive tsh expression in the regions of the developing adult that give rise to proximal wing and haltere tissues. Loss of this expression results in the fusion of the proximal structures of the wing and halteres to the thoracic cuticle. Further characterization of this tsh regulatory motif has now identified an additional enhancer activity directing tsh expression in tissues forming portions of the midgut. Subdivision of this midgut enhancer activity has identified putative negatively acting motifs.

Strong RNA Splicing Enhancers Identified by a Modified Method of Cycled Selection Interact with SR Protein

A modified method of cycled selection was used to characterize splicing enhancers for exon inclusion from a pool of beta-globin-based three exon/two intron pre-mRNAs with a variable number of random nucleotides incorporated in the internal exon. The pre-mRNAs generated by this method contained random sequences ranging from 0 to 18 nucleotides in length. This method was used to isolate particular splicing enhancer motifs from a previously enriched pool of extremely diverse enhancers. After four cycles of selection for mRNA containing the internal exon, a distinct enhancer motif (GACGAC...CAGCAG) was highly enriched. This motif served as strong splicing enhancers in a heterogeneous exon. We have shown here that the selected enhancer motif promotes exon inclusion through specific interaction with SRp30. We have also shown that although present in many of our selected splicing enhancers conforming to this motif, a typical purine-rich enhancer sequence is dispensable for either enhancer activity or binding with SRp30.

The DNA-binding ability of HIVEP3/KRC decreases upon activation of V(D)J recombination.

Somatic V(D)J recombination of the immune receptor genes is mediated by the recombination signal sequence (RSS) and the recombination-activating genes RAG1 and RAG2. Previously, proteins binding specifically to the RSS have been characterized in nuclear extracts of T and B lymphocytes. Further elucidation of the role of those RSS-binding proteins in V(D)J recombination, however, has been hampered by the fact that their identities have not been established. Here, we show that the major RSS-binding protein present in the nuclear extracts of B lymphocytes is an Mr 135,000 species. Notably, its affinity for the RSS decreased when RAG1 and RAG2 were induced. In immunoblot analyses and gel supershift assays, we showed that KRC antisera react with the Mr 135,000 RSS-binding protein. We previously cloned KRC from a thymocyte expression library using 32P-RSS as a ligand and showed that KRC fusion proteins bind specifically to the RSS and to the kappaB enhancer motif. The lymphoid expression and DNA-binding characteristics suggest that KRC may be involved in lymphocyte development.

Mammalian Scratch: a neural-specific Snail family transcriptional repressor.

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix--loop--helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter

We cloned the Slc14a2 gene and determined the genomic organization of the rat urea transporter UT-A. Slc14a2, the gene encoding the rat UT-A transporter, extends for more that 300 kb. The four known rat mRNA isoforms: UT-A1, UT-A2, UT-A3, and UT-A4 are transcribed from 24 exons. The Slc14a2 genomic map also accounts for 3′-untranslated sequences expressed alternatively in UT-A1, UT-A2, and UT-A3. We previously identified a TATA-less, tonicity-responsive promoter controlling the transcription of UT-A1, UT-A3, and UT-A4 from a single initiation site in the 5′-flanking region of the gene. Here, we describe a second, internal promoter in intron 12, which controls the transcription of UT-A2 starting from exon 13. This region contains a TATA motif upstream from the UT-A2 transcription start site, and shows consensus sequences for the cAMP response element (CRE) and for the tonicity enhancer (TonE) motif. Stimulation by cAMP induces UT-A2 mRNA expression in mIMCD3 cells, and luciferase activity in mIMCD3 cells transfected with those pGL3 constructs including the CRE sequences. Although long-term exposure to hypertonicity induces UT-A2 expression in mIMCD3 cells, hypertonicity does not induce significantly the activity of the promoter in intron 12. In summary, we describe the genomic structure of the rat UT-A urea transporter, encoded by the Slc14a2 gene. Our findings suggest that two promoters regulate transcription of the four UT-A isoforms, and that stimulation of transcription by vasopressin, mediated by cAMP and CRE sequences, and controlled by an intronic promoter, may contribute to the increase in UT-A2 expression during water deprivation.

The MuDR transposon terminal inverted repeat contains a complex plant promoter directing distinct somatic and germinal programs

SummaryThe Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near‐identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20‐fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell‐cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.

The <i>Drosophila</i> <i>aeroplane</i> mutant is caused by an I-element insertion into a tissue-specific <i>teashirt</i> enhancer motif

The <i>Drosophila</i> <i>aeroplane</i> mutant is caused by an I-element insertion into a tissue-specific <i>teashirt</i> enhancer motif

Nuclear Receptor Response Elements Mediate Induction of Intestinal MDR1 by Rifampin

Intestinal P-glycoprotein, which is encoded by the MDR1 gene, plays an important role in the absorption and presystemic elimination of many xenobiotics. Hence, an understanding of the factors regulating its expression and function is of substantial interest. In addition to genetic factors, exposure to drugs such as rifampin can profoundly affect its expression. So far, the mechanisms by which rifampin induces MDR1 expression are poorly understood. Recent studies demonstrate that the nuclear receptor PXR (pregnane X receptor) is involved in xenobiotic induction of CYP3A4. Because CYP3A4 and MDR1 are often co-induced, we investigated whether a similar mechanism is also involved in MDR1 induction. The human colon carcinoma cell line LS174T was used as an intestinal model to study induction because in these cells the endogenous MDR1 gene is highly inducible by rifampin. The 5'-upstream region of human MDR1 was examined for the presence of potential PXR response elements. Several binding sites were identified that form a complex regulatory cluster at about -8 kilobase pairs. Only one DR4 motif within this cluster is necessary for induction by rifampin. We conclude that induction of MDR1 is mediated by a DR4 motif in the upstream enhancer at about -8 kilobase pairs, to which PXR binds.

Structure of the Human Zinc Finger Protein HIVEP3: Molecular Cloning, Expression, Exon–Intron Structure, and Comparison with Paralogous Genes HIVEP1 and HIVEP2

Here we report the cloning and characterization of HIVEP3, the newest member in the human immunodeficiency virus type 1 enhancer-binding protein family that encodes large zinc finger proteins and regulates transcription via the κB enhancer motif. The largest open reading frame of HIVEP3 contains 2406 aa. and is ∼80% identical to the mouse counterpart. The HIVEP3 gene is located in the chromosomal region 1p34 and is at least 300 kb with 10 exons. RNA studies show that multiple HIVEP3 transcripts are differentially expressed and regulated. Additionally, transcription termination occurs in the ultimate exon, exon 10, or in exon 6. Therefore, HIVEP3 may produce protein isoforms that contain or exclude the carboxyl DNA binding domain and the leucine zipper by alternative RNA splicing and differential polyadenylation. Sequence homologous to HIVEP3 exon 6 is not found in mouse nor are the paralogous genes HIVEP1 and HIVEP2. Zoo-blot analysis suggests that sequences homologous to the human exon 6 are present only in primates and cow. Therefore, a foreign DNA harboring a termination exon likely was inserted into the HIVEP3 locus relatively recently in evolution, resulting in the acquisition of novel gene regulatory mechanisms as well as the generation of structural and functional diversity.

The MuDR transposon terminal inverted repeat contains a complex plant promoter directing distinct somatic and germinal programs.

The Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near-identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20-fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell-cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.