Cis-acting Genetic Elements Research Articles

A common structural core in the internal ribosome entry sites of picornavirus, hepatitis C virus, and pestivirus.

Cap-independent translations of viral RNAs of enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, hepatitis A and C viruses (HAV and HCV), and pestivirus are initiated by the direct binding of 40S ribosomal subunits to a cis-acting genetic element termed the internal ribosome entry site (IRES) or ribosome landing pad (RLP) in the 5' noncoding region (5'NCR). RNA higher ordered structure models for these IRES elements were derived by a combined approach using thermodynamic RNA folding, Monte Carlo simulation, and phylogenetic comparative analysis. The structural differences among the three groups of picornaviruses arise not only from point mutations, but also from the addition or deletion of structural domains. However, a common core can be identified in the proposed structural models of these IRES elements from enteroviruses and rhinoviruses, cardioviruses and aphthoviruses, and HAV. The common structural core identified within the picornavirus IRES is also conserved in the 5'NCR of the divergent viruses, HCV, and pestiviruses. Furthermore, the proposed structural motif shares a structural feature similar to that observed in the catalytic core of the group 1 intron. The conserved structural motif from these divergent sequences that looks like the common core region of group 1 introns is probably a crucial element involved in the IRES-dependent translation.

Level of human TCRBV3S1 (V beta 3) expression correlates with allelic polymorphism in the spacer region of the recombination signal sequence.

One of the causes of variations in the expressed human T cell receptor (TCR) BV (V beta) repertoire is genetic variation in the germline DNA. Herein evidence is provided that allelic polymorphism may affect recombination frequency for a specific V gene. Two alleles of the TCR BV3 differ only at a single nucleotide position (C/T) within the 23-bp spacer region of the recombination signal sequence. These alleles are associated with variable percentages of BV3 cells in the peripheral blood, as shown in families and in unrelated normal donors. Individuals homozygous for allele 2 have a mean of 8.1% BV3 cells, heterozygous individuals have a mean of 4.7% BV3 cells, and homozygotes for allele 1 have a mean of 1.2% BV3 cells in CD3+ CD4+ peripheral blood T cells. Since the correlation is tight in unrelated individuals and other genetic differences were not found in the vicinity of BV3, we suggest that the spacer region sequence itself modifies recombination efficiency. This allelic system provides an example of a novel mechanism by which cis-acting genetic elements may affect recombination in a natural in vivo system.

A 5' Portion of the ICAM-1 Gene Confers Tissue-Specific Differential Expression Levels and Cytokine Responsiveness

Intercellular adhesion molecule-1 (ICAM-1), a cell-adhesion molecule critically involved in leukocyte trafficking and adherence, displays tissue-specific and cytokine-specific expression profiles. Although human dermal microvascular endothelial cells (HDMEC) constitutively express ICAM-1, keratinocytes (HK) do not. Interleukin-1 (IL-1) upregulates ICAM-1 expression in HDMEC, but fails to do so in either HK or A431, a human squamous carcinoma cell line, even though both have IL-1 receptors and express ICAM-1 on exposure to other cytokines. We have previously characterized a human ICAM-1 genomic clone that contains the 5' flanking transcriptional regulatory region. To test the hypothesis that tissue- and cytokine-specific ICAM-1 gene expression results from the interaction of constitutive and inducible tissue-specific trans-acting factors with distinct cis-elements of the ICAM-1 gene, various ICAM-1-based reporter gene (CAT) plasmids were constructed. Transcriptional activity of these various constructs was assessed after transient transfection into HDMEC and A431. A critical ICAM-1 region was identified that conferred enhanced expression of CAT in HDMEC and suppressed expression of CAT in A431. This same region further enhanced CAT expression in transfected HDMEC treated with IL-1 alpha, yet no such enhancement was seen with IL-1 treatment of identically transfected A431. However, treatment of A431 transfectants with IFN gamma did result in enhanced CAT expression, demonstrating reversal of A431 cell context suppression of the ICAM-1-based reporter gene construct. These data implicate the existence of both tissue- and cytokine-specific responsive elements in the 5' flanking region of the ICAM-1 gene and demonstrate that regulatory effects directed by such elements are dependent upon their cellular context. Moreover, they provide the basis for identification of specific cis-acting genetic elements, the trans-acting factors with which they interact, and the molecular mechanisms by which they regulate transcription of the ICAM-1 gene.

Cis-acting elements that confer lung epithelial cell expression of the CC10 gene.

To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.

Evolution of messenger RNA structure and regulation in the genus Mus: the androgen-inducible RP2 mRNAs.

The RP2 gene is one of several genes that are regulated by androgens in the mouse kidney. Previous studies have demonstrated that androgen inducibility of RP2 transcription varies among species within the genus Mus, indicating extensive evolutionary modification of the participating regulatory elements. Thus, while a five-fold induction of transcription occurs in M. domesticus, none is detectable in M. hortulanus or M. caroli. In the present paper, we have sequenced cDNAs representing the RP2 mRNAs of M. caroli and M. saxicola and have compared them with each other and with M. domesticus. Several findings from the sequence comparisons indicate that the encoded 41-kD polypeptide is physiologically functional. First, divergence within noncoding regions of the mRNAs exceeds that within coding regions. Second, the 357-codon open reading frame has been maintained among the species, with approximately 90% of the amino acid replacements being conservative. Finally, substitution rates at synonymous sites within the coding regions are from twofold to threefold greater than those at nonsynonymous sites. The genetic elements responsible for variations in RP2 inducibility among species were studied by cis/trans analyses of mice heterozygous for RP2 alleles, using a primer extension assay to measure expression of species-specific mRNAs. The results show that the absence of transcriptional induction in M. hortulanus is due to a cis-acting genetic element, while that in M. caroli is due to a trans-acting element. Thus, the androgen-resistant RP2 phenotypes of these two species derive from distinct genetic events.

Two spatially distinct genetic elements constitute a bipartite DNA replication origin in the minute virus of mice genome

Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.

Cis-Acting Determinants of Basal and Lipid-regulated Apolipoprotein A-IV Expression in Mice

The levels of apolipoprotein A-IV (apoA-IV) mRNA are regulated by dietary lipid in the liver of both the mouse and rat. Thirteen different inbred mouse strains were fed a high lipid diet, and the effect on apoA-IV liver mRNA levels was examined. It was found that each strain responded in one of two ways. Mice of four strains had higher liver apoA-IV mRNA levels as compared with syngeneic mice fed a normal chow diet. Mice of the other nine strains had decreased liver apoA-IV mRNA levels as compared with syngeneic mice fed a normal chow diet. Using F1 hybrids between mice from BALB/c, C3H, and C57BL/6 and between 129 and C57BL/6, as well as recombinant inbred strains derived from a cross between BALB/c and C57BL/6, we have shown that both the normal level of liver apoA-IV mRNA in the chow-fed mice and the lipid-dependent regulation of apoA-IV mRNA levels are controlled by cis-acting genetic elements. The apoA-IV mRNA levels in mice fed a normal diet varied dramatically among strains, with the largest difference (90-fold) being between the 129/J inbred strain and the C57BL/6J strain. In addition, we have examined the expression of apoA-IV during mouse development. ApoA-IV mRNA is expressed early in mouse liver (16 days postcoitum), whereas others have shown previously that rat liver apoA-IV mRNA is undetectable until 14 days after birth. ApoA-IV mRNA levels in the intestine and apoA-I mRNA levels in the liver and intestine, by contrast, mirror the pattern seen in the rat.

Repression of polyoma virus DNA replication by 5′-flanking region of mouse DNA polymerase β gene containing transcriptional silencer elements

Dual cis-acting silencer elements are located upstream of the mouse DNA polymerase beta gene (Yamaguchi, M., Hayashi, Y., and Matsukage, A. (1989) J. Biochem. (Tokyo) 105, 79-83). In order to examine possible involvement of transcriptional silencer elements in the regulation of DNA replication, we have utilized a transient replication system of the plasmid DNA carrying replication origin of polyoma virus DNA in mouse MOP8 cells, which is constitutively producing polyoma virus large T-antigen. The polyoma virus origin of DNA replication is composed of three cis-acting genetic elements called alpha, beta, and core, in which alpha and beta elements correspond to enhancer domains. When the 5'-flanking regions of the DNA polymerase beta gene containing silencer elements were placed at the late gene border of alpha element, they effectively repressed the DNA replication. However, when placed at the early gene border of core element, it only marginally repressed the DNA replication. These results suggest that the silencer elements at cis position repress polyoma virus DNA replication by impeding the enhancer function that activates the DNA replication.

Promoter inactivation or inhibition by sequence-specific methylation and mechanisms of reactivation

In studies on adenovirus promoters, predominantly on the late E2A promoter of adenovirus type 2 (Ad2), we have demonstrated by a number of experimental approaches that the sequence-specific methylation of three 5'-CCGG-3' sequences inactivates this promoter. Recently, we have developed a cell-free transcription system in which the methylation-inactivation of eukaryotic promoters can be studied in detail. It has also been shown that methylation-caused promoter inactivation can be reversed by the 289 amino acid E1A protein of Ad2 or of adenovirus type 5. In the presence of this protein with a transactivating effect, transcription is initiated at the authentic cap site of the methylated late E2A promoter. A similar reactivation of the methylated late E2A promoter can also be effected by a cis-acting genetic element, i.e., the strong enhancer of human cytomegalovirus. Further studies will be directed toward the biochemical mechanisms of promoter silencing by sequence-specific methylations.

Diversity and regulation of expression of human leukocyte antigen class II molecules

The initiation of an immune response requires that a foreign antigen be degraded, and that one of the degradative fragments be presented in the context of a human leukocyte antigen (HLA) class II molecule to an antigen-specific helper T cell. The success of this process is maximized by the diversity of the class II molecules possessed by a given person. The expression of the HLA class II molecules is highly selective. Aberrant expression has been postulated to be responsible for autoimmune disease. The interaction of inducible tissue-specific transacting factors with cis-acting genetic elements adjacent to the coding portion of the class II genes is responsible for both normal and aberrant expression of class II molecules. The cDNA encoding one such transacting factor that binds to the cis-acting element known as the Y box has been cloned.

Lack of evidence for association of meiotic nondisjunction with particular DNA haplotypes on chromosome 21.

The hypothesis of a predisposition to meiotic nondisjunction for chromosome 21 carrying a specific molecular haplotype has been tested. The haplotype in question is defined by the restriction fragment length polymorphisms for the D21S1/D21S11 loci. Our results obtained on a sample of Northern Italian families with the occurrence of trisomy 21 (Down syndrome) failed to support this hypothesis, contradicting a previous study [Antonarakis, S. E., Kittur, S. D., Metaxotou, C., Watkins, P. C. & Patel, A. S. (1985) Proc. Natl. Acad. Sci. USA 82, 3360-3364]. These findings rule out an association between any specific D21S1/D21S11 haplotype (as well as other haplotypes for the D21S13, ETS2, and D21S23 loci) and a putative cis-acting genetic element favoring the meiotic missegregation of chromosome 21. For this reason, no preventive screening for couples at risk for trisomy 21 may be based on any of the haplotypes tested.

Upstream promoter element of the human metallothionein-IIA gene can act like an enhancer element.

Initiation of transcription by RNA polymerase II in eukaryotes is strongly increased by cis-acting genetic elements, known as activators or enhancers. Enhancers, first detected in simian virus 40 (SV40), were subsequently also found to control the expression of several cellular genes. The human metallothionein-IIA (hMT-IIA) gene, although inducible by heavy metals and glucocorticoids, is widely expressed in most cell types in the absence of inducers. Here we show that the high basal level of transcription of the hMT-IIA gene is due to the presence of an enhancer element within the hMT-IIA promoter region. The structural and functional organization of this cellular enhancer element in two direct repeats is strikingly similar to that of the enhancer element of SV40. This suggests a possible functional and evolutionary relationship between enhancers and upstream promoter elements.

Genetic and physical studies of a portion of the white locus participating in transcriptional regulation and in synapsis-dependent interactions in Drosophila adult tissues.

We have identified and sequenced the portion of the white locus affected by an idiosyncratic set of white mutant alleles (the wsp alleles). The affected white locus portion (wsp region) extends from ca. 590 base pairs (bp) to ca. 1270 bp 5' to the apparent start site for the major white transcription unit. Based on the properties of these mutant alleles, we infer the existence of two distinct cis-acting regulatory elements in the wsp region and a third element mapping 3' to this region (3' to position ca. -670). Our analysis allows us to define the apparent position of one of the two wsp region elements with substantial precision. Examination of the DNA sequences in this region suggests that it is functionally similar to the enhancers identified in vertebrates. This same element participates in synapsis-dependent genetic interactions, suggesting a largely unexpected relationship between enhancer-like, cis-acting genetic elements and the genetic elements responsible for the synapsis-dependent genetic interactions in trans revealed by the existence of transvection effects. Our results further suggest that a presumptive regulatory locus (suppressor-of-white-spotted) regulates white transcription in adult tissues and is not involved in regulating white expression in larvae. We discuss the regulation of white expression in light of our studies. We also demonstrate unusual structures for an X-ray-induced deletion and a spontaneous deletion.

Variation in the major urinary protein multigene family in wild-derived mice.

The levels of expression and genomic organization of genes coding for the major urinary proteins (MUPs) were examined in several stocks of wild-derived mice. Levels of MUP mRNA in the liver varied considerably with M. musculus Brno and M. castaneus males having several-fold more MUP RNA than inbred C57BL/6 males, whereas M. hortulanus, M. caroli and M. cervicolor displayed levels much lower than C57BL/6. Analysis of RNA with MUP cDNAs specific to two different subfamilies of MUP genes revealed that M. caroli and M. cervicolor primarily expressed a MUP mRNA that was less abundant in C57BL/6, suggesting differential expression of subfamilies of genes within the MUP multigene complex. Although inbred males usually have five-fold more MUP mRNA than inbred females, male to female ratios for wild-derived stocks ranged from one to several hundred. Southern blots of genomic DNA hybridized to MUP subfamily probes revealed differences in restriction fragment sizes as well as possible variation in the number of MUP genes in some species. Analysis of urinary proteins from hybrids between C57BL/6 and M. spretus suggested that low MUP expression in M. spretus females was due to cis-acting genetic elements.

An evolutionary switch in tissue-specific gene expression. Abundant expression of alpha 1-antitrypsin in the kidney of a wild mouse species.

alpha 1-Antitrypsin (AT), one of the major proteinase inhibitors in mammalian serum, is generally considered to be synthesized exclusively in the liver. We have found that a wild-derived Mus species, Mus caroli, expresses AT mRNA in kidney at levels approaching that in liver; no other mouse, inbred or wild-derived, exhibits this striking property. Liver and kidney mRNAs from M. caroli encode very similar AT polypeptides that are distinct from that encoded by Mus musculus liver mRNA. In vivo, liver AT is secreted into the bloodstream, while kidney AT, which is processed differently from the liver protein, is excreted into the urine. Analysis of RNA from a hybrid between M. musculus and M. caroli indicates that a cis-acting genetic element may be responsible for the difference in AT expression. Restriction enzyme digestion patterns of AT genomic sequences in M. caroli DNA are considerably different from those in M. musculus; in addition, these sequences are undermethylated in liver DNA from M. musculus and in liver and kidney DNA from M. caroli, reflecting the respective patterns of expression. Further studies of the altered tissue specificity of AT expression that is apparent in these two related species should lead to new insights into the nature and evolution of genetic determinants of tissue-specific phenotypes.

Integration-specific retrovirus expression in embryonal carcinoma cells.

The undifferentiated embryonal carcinoma (EC) cell line PCC4 aza 1 was infected with a selectable amphotropic retrovirus. Although EC cells are generally refractory to retroviral gene expression, we found that approximately 1 of 5,000-10,000 recombinant proviruses was transcribed in these cells. Cells containing an active recombinant provirus were cloned and expanded. Nucleic acid analysis revealed approximately one intact provirus per cell. Viral RNA levels were different for each cell clone examined [between 0.05% and 0.5% of the poly(A)+ RNA], and transcription was initiated and terminated in the long terminal repeats as in permissive differentiated cells. The cells retained an undifferentiated phenotype and remained positive for stage-specific embryonic antigen 1 and negative for H-2 surface antigens. The cells retained the block to the expression of other proviruses integrated at other chromosomal locations. The data suggest that a cis-acting genetic element, at or near the chromosomal site of integration, or mutations in the viral control elements themselves may be responsible for provirus expression in these cells.

Genetic control of levels of murine kidney glucuronidase mRNA in response to androgen.

A cis-acting genetic element, designated Gus-r, regulates the androgen-induced rates of murine glucuronidase (EC 3.2.1.31) synthesis in kidney tubule cells and is tightly linked to the glucuronidase structural gene, Gus-s. To investigate the molecular mechanism underlying this regulation, we have cloned a glucuronidase-specific cDNA sequence in plasmid pBR322. This cloned DNA has been utilized as a probe in blot hybridization analyses to determine whether the control of androgen responsiveness of kidney glucuronidase synthesis by Gus-r is exerted over the level or the translatability of glucuronidase mRNA. Three important observations emerged from these studies: (i) glucuronidase mRNA exists as a single size class of approximately 2,800 nucleotides; (ii) androgen stimulation of glucuronidase synthesis is directly related to the level of glucuronidase mRNA; and (iii) strain differences in levels of kidney glucuronidase mRNA accumulated in response to androgen are controlled by alleles of Gus-r. Thus, Gus-r regulates the androgen responsiveness of glucuronidase synthesis by controlling the amount of glucuronidase mRNA available for translation and is a cis-acting genetic element that regulates the hormonal responsiveness of a specific mRNA.

Regulation of the tissue specificity of an enzyme by a cis-acting genetic element: evidence from interspecific Drosophila hybrids.

Homologous genes for alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) are expressed in qualitatively different patterns during the development of two closely related species of Hawaiian Drosophila. In interspecific hybrids, each parental structural allele is expressed according to the developmental program characteristic of the species from which it is derived. This provides strong evidence for a cis acting control element.