CG-rich Regions Research Articles

Extracting Gene Markers by the Identification of Functional CG Rich Genomic Regions

Extracting Gene Markers by the Identification of Functional CG Rich Genomic Regions

Effect of Methylation on the Nanomechanical Properties of Double-Stranded DNA

In mammalian cells 60-90% of cytosines in the genome are methylated. The methylation sites are unevenly distributed and are often found in clusters called “CpG islands”. Approximately 70% of promoters in the human genome contain or are preceeded by CG-rich regions, suggesting that methylation may be important in gene regulation. Cyclization-kinetic and nucleosome-binding assays suggest that methylation may significantly affect DNA flexibility. However, a direct effect of methylation on the mechanics of DNA is yet unknown. To investigate the impact of methylation on DNA mechanics, here we manipulated single molecules of methylated dsDNA and compared their nanomechanical properties with those of unmethylated DNA.A 3500-base-pair sequence of lambda-phage DNA composed almost entirely of CpG islands was cloned by using PCR containing dm5CTP to produce the fully methylated product. Individual DNA molecules were mechanically manipulated in stretch and relaxation cycles by using custom-built dual-beam counter-propagating optical tweezers. Force versus extension data were fitted with the extensible wormlike-chain model to obtain the contour length, the persistence length (entropic component of rigidity) and the stretch modulus (enthalpic component of rigidity) of dsDNA. Methylation reduced the contour length and stretch modulus of dsDNA from 1036±22 nm to 966±8 nm and from 1225±115 pN to 373±30 pN, respectively. Persistence length was 34±2 nm for the non-methylated and 35±2 nm in case of the hypermethylated DNA. The observed changes may be caused by a complex shift in tertiary structure, accounting for both the reduction of the contour length and the increase of the intrinsic compliance of the dsDNA chain. The methylation-induced effects on the nanomechanical properties of dsDNA may play an important role in the regulation of steric access to its sequence-specific sites.

Epigenetic Regulation of Cancer Stem Cell Genes in Triple-Negative Breast Cancer

Expression of specific breast cancer stem cells (BCSCs) is seen in aggressive tumors, but their regulation is unclear. Epigenetic changes influence gene expression and are implicated in breast cancer progression. We hypothesized that promoter methylation regulates specific BCSC-related genes [CD44, CD133, CD24, MSH1 (alias, Musashi-1), and ALDH1] and that this epigenetic profile can identify aggressive subtypes, such as triple-negative breast cancer (TNBC). Methylation analysis was performed using MassARRAY EpiTYPER sequencing; CpG-rich sites were identified in the promoter regions of BCSC genes, except ALDH1. These sites were screened by treatment with 5-aza-2'-deoxycytidine in four TN and five non-TNBC cell lines. The specific regulatory CpG site demonstrating the most significant inverse correlation between CpG site methylation and mRNA expression was identified for CD44, CD133, and Musashi-1, but not for CD24. Methylation of CD44, CD133, and Musashi-1 was evaluated in 91 American Joint Committee on Cancer stage I to III primary breast cancer tumors, and these sites were significantly hypomethylated in TNBC versus non-TNBC. The IHC staining of primary tumors with the highest and lowest methylation levels revealed the strongest staining in hypomethylated specimens, suggesting that hypomethylation leads to gene activation. We demonstrate that methylation is a significant mechanism regulating CD44, CD133, and Musashi-1, and that gene hypomethylation correlates with TNBC. Assessment of epigenetic changes in BCSC genes may provide a more accurate classification of TNBC and could be developed as potential therapeutic targets.

Molecular analysis of a sunflower gene encoding an homologous of the B subunit of a CAAT binding factor

A genomic DNA fragment containing the complete LEAFY COTYLEDON1-LIKE (HaL1L) gene was retrieved by chromosome walking. Its sequence was confirmed and elongated by screening a sunflower genomic DNA BAC Library. HaL1L, whose cDNA had already been sequenced and characterized, encodes a NF-YB subunit of a CCAAT box-binding factor (NF-Y) involved in the early stages of zygotic and somatic embryogenesis in the Helianthus genus. In the HaL1L 5'-flanking region, elements specific to a putative TATA-box promoter and two "CG isles" were identified. An investigation of the methylation status of these CG rich DNA regions showed that differentially methylated cytosines were recognizable in the DNA of embryos on the fifth day after pollination in comparison to leaf DNA suggesting that during plant development epigenetic regulation of HaL1L transcription was achieved by methylating cytosine residues. We also searched the HaL1L nucleotide sequence for cis-regulatory elements able to interact with other transcription factors (TFs) involved in the HaL1L regulation. Of the elements identified, one of the most intriguing is WUSATA, the target sequence for the WUSCHEL (WUS) TF, which may be part of a complex regulation network controlling embryo development. In this article, we show that the WUSATA target site, located in the intron of HaL1L, is able to bind the TF WUS. Interestingly, we found auxin and abscisic acid responsive motifs in the HaL1L promoter region suggesting that this gene may additionally by under hormonal control. Finally, the presence of a cytoplasmic polyadenylation signal downstream to the coding region indicates that this gene may also be controlled at the translation level by a temporarily making the pre-synthesized HaL1L mRNA unavailable for protein synthesis.

Evolution of genomic sequence inhomogeneity at mid-range scales

BackgroundMid-range inhomogeneity or MRI is the significant enrichment of particular nucleotides in genomic sequences extending from 30 up to several thousands of nucleotides. The best-known manifestation of MRI is CpG islands representing CG-rich regions. Recently it was demonstrated that MRI could be observed not only for G+C content but also for all other nucleotide pairings (e.g. A+G and G+T) as well as for individual bases. Various types of MRI regions are 4-20 times enriched in mammalian genomes compared to their occurrences in random models.ResultsThis paper explores how different types of mutations change MRI regions. Human, chimpanzee and Macaca mulatta genomes were aligned to study the projected effects of substitutions and indels on human sequence evolution within both MRI regions and control regions of average nucleotide composition. Over 18.8 million fixed point substitutions, 3.9 million SNPs, and indels spanning 6.9 Mb were procured and evaluated in human. They include 1.8 Mb substitutions and 1.9 Mb indels within MRI regions. Ancestral and mutant (derived) alleles for substitutions have been determined. Substitutions were grouped according to their fixation within human populations: fixed substitutions (from the human-chimp-macaca alignment), major SNPs (> 80% mutant allele frequency within humans), medium SNPs (20% - 80% mutant allele frequency), minor SNPs (3% - 20%), and rare SNPs (<3%). Data on short (< 3 bp) and medium-length (3 - 50 bp) insertions and deletions within MRI regions and appropriate control regions were analyzed for the effect of indels on the expansion or diminution of such regions as well as on changing nucleotide composition.ConclusionMRI regions have comparable levels of de novo mutations to the control genomic sequences with average base composition. De novo substitutions rapidly erode MRI regions, bringing their nucleotide composition toward genome-average levels. However, those substitutions that favor the maintenance of MRI properties have a higher chance to spread through the entire population. Indels have a clear tendency to maintain MRI features yet they have a smaller impact than substitutions. All in all, the observed fixation bias for mutations helps to preserve MRI regions during evolution.

Enriched transcription factor binding sites in hypermethylated gene promoters in drug resistant cancer cells

In the human genome, 'CpG islands', CG-rich regions located in or near gene promoters, are normally unmethylated. However, in cancer cells, CpG islands frequently gain methylation, resulting in silencing of growth-limiting tumor suppressor genes. To our knowledge, the potential relationship between CpG island hypermethylation, transcription factor (TF) binding in local promoter regions and transcriptional control has not been previously explored in a genome-wide context. In this study, we utilized bioinformatics tools and TF binding site(TFBs) databases to globally analyze sequences methylated in a laboratory model for the development of drug-resistant cancer. Our results demonstrated that four TFBS were enriched in hypermethylated sequences. More interestingly, overrepresentation of these TFBS was observed in hyper-/hypo-methylated sequences where signi.cant changes in methylation levels were observed in drug-resistant cancer cells. In summary, we believe that these.ndings offer a means to further explore the relationship between DNA methylation and gene expression in drug resistance and tumorigenesis.

Fragile X repeats are potent inducers of complex, multiple site rearrangements in flanking sequences in Escherichia coli

(CGG·CCG) n repeats induce the formation of complex, multiple site rearrangements and/or gross deletions in flanking DNA sequences in Escherichia coli plasmids. DNA sequence analyses of mutant clones revealed the influence of (a) the length (24, 44 or 73 repeats), (b) the orientation of the CGG·CCG region relative to the unidirectional origin, and (c) its transcription status. Complex rearrangements had occurred in the mutant clones since some products contained deletions, inversions and insertions and some products had only gross deletions. Furthermore, the CGG·CCG repeats repeatedly induced, up to 22 times, the formation of identical (to the bp) mutagenic products indicating the powerful nature of the complex processes involved. Also, the mutations were bidirectional from the CGG·CCG tract. The healed junctions had CG-rich microhomologies of 1–6 bp, CG-rich regions and putative cruciforms and slipped structures. Hence, the fragile X syndrome mutagenic spectrum has been found, at least in part, in our model system.

Characterizations of Hirudo medicinalis DNA promoters for targeted gene expression

The expression of exogenous genes in neurons and other cells has become a powerful means for studying the function of encoded proteins. We report here on the isolation and functional analysis of three Hirudo medicinalis actin gene promoters and the 5′ UTR of a leech elongation factor-1α ( HmEF-1α) gene. In situ hybridization labeling revealed that the EF-1α gene and one of the actins had pan-neuronal expression, whereas, the other two actin genes were expressed by the embryo's body wall musculature. Comparative analysis shows that they all display many features typical of actin and EF-1α promoters from other species, including canonical TATA box sequences and predicted general transcription factor binding sites (such as CCATT, CarB boxes and CG-rich regions). The ability of these 5′ UTR sequences to drive expression of the enhanced green fluorescent protein (EGFP), leech cytoplasmic actin and leech synaptobrevin was examined. Direct intracellular nuclear, but not cytoplasmic, microinjection of each of the promoter sequences was found to produce reliably cellular expression of the reporter construct in both neuronal and muscle cells. These results introduce reliable and effective methods to selectively express genes in individual cells of the leech in vivo during embryonic development.

Chrosomal banding inParodon hilarii(Pisces Parodontidae), with emphassis on constitutive heterochromatin

Abstract Several banding methods were employed to analyze the chromosomes of the fish Parodon hilarii, i.e., C-banding, digestion with the restriction endonuclease Hae III, G-banding (trypsin), incorporation of 5-BrdU, and base-specific fluorochrome staining, with emphasis on the constitutive heterochromatin. The pattern obtained after the chromosomal cleavage with the restriction enzyme Hae III was similar to that of the C-banding. The incorporation of 5-bromodeoxiuridin (5-BrdU) in the chromosomes was able to detect several heterochromatin segments of late replication, with exception of some C-banded sites which were not evidenced after the 5-BrdU treatment. The terminal segment of the long arm of the 15th chromosome pair, which corresponds to a NOR site, seems to be composed by AT-rich heterochromatic regions interspersed with CG-rich regions. Besides, the long arm of the W chromosome shows a structural differentiation concerning the distribution of the heterochromatin.

DNA hydrolysis by monoclonal autoantibody BV 04-01.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

DNA hydrolysis by monoclonal anti-ssDNA autoantibody BV 04-01: Origins of catalytic activity

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3′-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.

Differential effects of distamycin analogues on amplification of human gene sequences by polymerase-chain reaction.

In this report we analyse the effects of distamycin and five distamycin analogues on amplification by polymerase-chain reaction (PCR) of two gene sequences displaying a different A+T/G+C content. The first was a 5' region of the human oestrogen receptor (ER) gene, containing a (TA)26 stretch; the second was a CG-rich sequence of the human Ha-ras oncogene. The results obtained unequivocally demonstrate that the addition of one pyrrole ring significantly improves the ability of distamycin derivatives to interfere with PCR-mediated amplification of the human ER genomic region carrying a (TA)26 stretch. The distamycin analogues analysed differ in the number of pyrrole rings and in the presence of an N-formyl, an N-formimidoyl or a retroamide group at position X1. Among compounds carrying the same number of pyrrole rings, those carrying an N-formyl or an N-formimidoyl group retain a similar inhibitory activity. The retroamide analogues, on the contrary, are much less efficient in inhibiting PCR-mediated amplification of the 5'ER region. With respect to sequence selectivity both distamycin and distamycin analogues exhibit a sequence preference, since they do not inhibit PCR amplification of Ha-ras CG-rich gene regions, with the exception of a distamycin analogue carrying four pyrrole rings.

CG dinucleotide clusters in MHC genes and in 5' demethylated genes.

In the DNA of higher vertebrates the dinucleotide CG is unique in two respects: it occurs far less frequently than would be expected on the basis of the content of cytosine and guanine in a given DNA segment ("CG suppression") and it contains predominantly 5-methyl-cytosine, the only modified nucleotide common in vertebrate DNA. Here we point out the existence of CG clusters, i.e. localized lapses in the usual CG suppression, in two categories of DNA segments from vertebrates: around the polymorphic exons of major histocompatibility complex (MHC) genes and in the 5' regions of certain other genes. These observations contradict the recent suggestion that CG frequency is uniform over long contiguous segments of DNA containing several genes. A model for the origin of these CG clusters as a consequence of regional demethylation of germline DNA is supported by analysis of other sequence features of these regions as well as by previously published data on the methylation status in sperm DNA of two of these CG-rich regions.

The thermal inactivation of transforming activity at low ionic strength

A sample of triply marked pneumococcal DNA has been thermally denatured at pH 7 and ionic strengths of 0·1 M and 3 × 10 −4 M . The inactivation temperatures for the markers have been compared to the hyperchromicity midpoint and the marker densities reported by Rolfe & Ephrussi-Taylor (1961). We find that: (1) at low ionic strength, the transition breadth for inactivation is much greater than at high μ ; (2) the inactivation midpoint temperatures at low μ are much greater than the ambient hyperchromicity T m , while at high μ , this difference is small; ( c ) the differences between inactivation midpoints for different markers are increased at low μ . We suggest that the inactivation of each marker involves the denaturation of CG-rich critical regions. The critical regions for low ionic strength denaturation are shorter than the critical regions for high ionic strength denaturation. They are richer in GC content and more heterogeneous in base composition. We interpret the increased transition breadth at low ionic strength as evidence for a series of partially denatured states for each marker molecule.