Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

Isolation and characterization of Histone1 gene and its promoter from tea plant (Camellia sinensis)

One 1.2 kbp long sequence was cloned by using PCR with primers that were designed from cDNA sequence of CsH1 gene (Genbank: EU716314) from tea plant (Camellia sinensis). According to the 1.2 kbp sequence, a 0.6 kbp sequence was isolated from tea plant genomic DNA using DNA Walking Method. Sequence analysis revealed that the 1.2 kbp sequence is a CsH1 gene consisting of 1 exon and 2 introns, the border of exton and intron sequences conforming to the GT–AG rule, and the 0.6 kbp sequence was found to be the promoter of CsH1 gene which contains basic promoter elements, TATA-box and CAAT-box. Abscisic acid responsiveness cis-acting element, elictor-responsive element, GA response element, light response cis-acting element and TC-rich repeats were also represented. To further study the activity of this promoter, the sequence was used to drive a GUS fusion gene in Agrobacterium-mediated transformation of tea plant somatic embryos, leaf discs and calli of tobacco (Nicotiana tabacum L.) where a high level of GUS expression was both observed in the tobacco calli and tea plant somatic embryos. These results suggest that the CsH1 gene promoter isolated is capable of conferring nuclear gene expression.     

Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, which is a structural homolog of the RAD2 and RAD27 DNA repair genes

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.?cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression.     

Evidence that the plastid signal and light operate via the samecis-acting elements in the promoters of nuclear genes for plastid proteins

Nuclear-encoded genes for proteins of the photosynthetic maschinery represent a particular subset of genes. Their expression is cooperatively stimulated by discrete factors including the developmental stage of plastids and light. We have analyzed in transgenic tobacco the plastid- and light-dependent expression of a series of 5′ promoter deletions of various nuclear genes from spinach, of fusions of defined promoter segments with the 90-bp 35S RNA CaMV minimal promoter, as well as with mutations in sequences with homologies to characterizedcis-elements, to address the question of whether the plastid signal and light operate via the same or differentcis-acting elements. In none of the 160 different transgenic lines (representing 32 promoter constructs from seven genes) analyzed, could significant differences be identified in the responses to the two regulatory pathways. The data are compatible with the idea that both signals control the expression of nuclear genes for plastid proteins via the samecis-acting elements.     

The Yeast Checkpoint Kinase Dun1 Downregulates DIN7 in the Absence of DNA Damage

Yeast DIN7 is a DNA damage-inducible gene. Its expression is increased in the absence of Dun1, a DNA damage checkpoint kinase. We identified a DIN7 promoter region responsible for Dun1-mediated downregulation and found that DIN7 expression was not further increased in response to hydroxyurea in Δdun1 cells. Thus DIN7 repression by Dun1 can be released upon DNA damage.     

Nonautonomous inverted repeat Alien transposable elements are associated with genes of both monocotyledonous and dicotyledonous plants

Alien are highly repeated plant transposable elements characterized by their small size (approx. 400 bp), high A + T content, target site specificity, potential to form stable secondary structures and possession of a conserved 28-bp terminal inverted repeat (TIR). Besides the TIR, they contain subterminal inverted repeat motifs (SIRM), as well as the 5′-CATGCAT domain which has been reported to be a cis-acting regulatory element of gene expression in some plant species. Although they were first identified in the intron of the bell pepper (Capsicum annuum) Sn-2 gene and in the promoter region of the potato starch phosphorylase-encoding gene, Alien arranged in tandem are present in the promoter of patatin class-II genes. PCR on the bell pepper genomic DNA using the Alien TIR consensus sequence as primer yielded DNA fragments of nearly 400 bp. These fragments have characteristics of transposable elements and contain numerous motifs reminiscent of Alien elements. Importantly, PCR on genomic DNA extracts from various monocotyledonous and dicotyledonous plants using the TIR consensus sequence as primer and subsequent hybridization with different Alien probes revealed that these elements are ubiquitously present and highly repeated in the genomes of higher plants.     

Role of micronucleus-limited DNA in programmed deletion of mse2.9 during macronuclear development of Tetrahymena thermophila

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.     

Analysis of the promoter region of the gene LIP1 encoding triglyceride lipase from Fusarium graminearum

Triglyceride lipases catalyze the reversible degradation of glycerol esters with long-chain fatty acids into fatty acids and glycerol. In silico analysis of 5′-end flanking sequence of the gene LIP1 encoding a triglyceride lipase from the wheat head blight pathogen Fusarium graminearum revealed the presence of several cis-regulatory elements. To delineate the function of these regulatory elements, we constructed a series of deletion mutants in the LIP1 promoter region fused to the open reading frame of a green fluorescent protein (GFP) and assayed the promoter activity. Analysis of GFP expression levels in mutants indicated that a 563-bp promoter sequence was sufficient to drive the expression of LIP1 and regulatory elements responsible for the gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements spanned within the 563-372 bp region were mutated using a targeted mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be required for the basal expression of LIP1, glucose suppression and fatty acid induction, respectively.     

Cloning of a Nicotiana plumbaginifolia protoplast-specific enhancer-like sequence

We have isolated a 1.5-kb plant DNA fragment (called insert 7) from Nicotiana plumbaginifolia DNA that contains a protoplast-specific enhancer-like sequence. The presence of this sequence on a plasmid carrying a chimeric nos-npt-II gene conferring kanamycin resistance to plant cells, produces an overexpression of the npt-II gene during at least eight days after protoplast transformation. This effect on the expression of the nos promoter was independent of the orientation and was observed both on circular and linearized plasmids. On the contrary, insert 7 had no influence when present on another plasmid (in trans) in cotransformation experiments. The overexpression of the nos-npt-II gene due to the presence of insert 7 on the transforming plasmid is correlated with a higher level of synthesis of the corresponding RNA. Insert 7 did not affect the level of expression of the nos-npt-II gene in stably transformed calli, or in regenerated plants. However, the overexpression was again detected in protoplasts prepared from leaves of stably transformed plants. This 1.5-kb plant DNA fragment contains highly repetitive DNA sequences, specific to N. plumbaginifolia. However, the enhancer-like activity is localized on a 600-bp unique sequence of insert 7. Insert 7 had no detectable effect on the transient expression of another gene, the nopaline synthase gene present at a longer distance on the same plasmid.