Comparative study and prediction of DNA fragments associated with various elements of the nuclear matrix

Scaffold/matrix-associated region (S/MAR) sequences are DNA regions that are attached to the nuclear matrix, and participate in many cellular processes. The nuclear matrix is a complex structure consisting of various elements. In this paper we compared frequencies of simple nucleotide motifs in S/MAR sequences and in sequences extracted directly from various nuclear matrix elements, such as nuclear lamina, cores of rosette-like structures, synaptonemal complex. Multivariate linear discriminant analysis revealed significant differences between these sequences. Based on this result we have developed a program, ChrClass (Win/NT version, ftp.bionet.nsc.ru/pub/biology/chrclass/chrclass.zip), for the prediction of the regions associated with various elements of the nuclear matrix in a query sequence. Subsequently, several test samples were analyzed by using two S/MAR prediction programs (a ChrClass and MAR-Finder) and a simple MRS criterion (S/MAR recognition signature) indicating the presence of S/MARs. Some overlap between the predictions of all MAR prediction tools has been found. Simultaneous use of the ChrClass, MRS criterion and MAR-Finder programs may help to obtain a more clearcut picture of S/MAR distribution in a query sequence. In general, our results suggest that the proportion of missed S/MARs is lower for ChrClass, whereas the proportion of wrong S/MARs is lower for MAR-Finder and MRS.     

A comparative study of S/MAR prediction tools

Background  

S/MARs are regions of the DNA that are attached to the nuclear matrix. These regions are known to affect substantially the expression of genes. The computer prediction of S/MARs is a highly significant task which could contribute to our understanding of chromatin organisation in eukaryotic cells, the number and distribution of boundary elements, and the understanding of gene regulation in eukaryotic cells. However, while a number of S/MAR predictors have been proposed, their accuracy has so far not come under scrutiny.     

Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7.

Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/non-imprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARs in imprinting.     

Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy

Scaffold or matrix-attachment regions (S/MARs) are thought to be involved in the organization of eukaryotic chromosomes and in the regulation of several DNA functions. Their characteristics are conserved between plants and humans, and a variety of biological activities have been associated with them. The identification of S/MARs within genomic sequences has proved to be unexpectedly difficult, as they do not appear to have consensus sequences or sequence motifs associated with them. We have shown that S/MARs do share a characteristic structural property, they have a markedly high predicted propensity to undergo strand separation when placed under negative superhelical tension. This result agrees with experimental observations, that S/MARs contain base-unpairing regions (BURs). Here, we perform a quantitative evaluation of the association between the ease of stress-induced DNA duplex destabilization (SIDD) and S/MAR binding activity. We first use synthetic oligomers to investigate how the arrangement of localized unpairing elements within a base-unpairing region affects S/MAR binding. The organizational properties found in this way are applied to the investigation of correlations between specific measures of stress-induced duplex destabilization and the binding properties of naturally occurring S/MARs. For this purpose, we analyze S/MAR and non-S/MAR elements that have been derived from the human genome or from the tobacco genome. We find that S/MARs exhibit long regions of extensive destabilization. Moreover, quantitative measures of the SIDD attributes of these fragments calculated under uniform conditions are found to correlate very highly (r2>0.8) with their experimentally measured S/MAR-binding strengths. These results suggest that duplex destabilization may be involved in the mechanisms by which S/MARs function. They suggest also that SIDD properties may be incorporated into an improved computational strategy to search genomic DNA sequences for sites having the necessary attributes to function as S/MARs, and even to estimate their relative binding strengths.     

A map of nuclear matrix attachment regions within the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1

There is abundant evidence that the DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. To explore the DNA domain organization of the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1, we have identified a significant portion of the scaffold/matrix attachment regions (S/MARs) within this region. Forty independent putative S/MAR elements were assigned within the 16q22.1 locus. More than 90% of these S/MARs are AT rich, with GC contents as low as 27% in 2 cases. Thirty-nine (98%) of the S/MARs are located within genes and 36 (90%) in gene introns, of which 15 are in first introns of different genes. The clear tendency of S/MARs from this region to be located within the introns suggests their regulatory role. The S/MAR resource constructed may contribute to an understanding of how the genes in the region are regulated and of how the structural architecture and functional organization of the DNA are related.     

Scaffold/matrix attachment regions and intrinsic DNA curvature

Recent approaches have failed to detect nucleotide sequence motifs in Scaffold/Matrix Attachment Regions (S/MARs). The lack of any known motifs, together with the confirmation that some S/MARs are not associated to any peculiar sequence, indicates that some structural elements, such as DNA curvature, have a role in chromatin organization and on their efficiency in protein binding. Similar to DNA curvature, S/MARs are located close to promoters, replication origins, and multiple nuclear processes like recombination and breakpoint sites. The chromatin structure in these regulatory regions is important to chromosome organization for accurate regulation of nuclear processes. In this article we review the biological importance of the co-localization between bent DNA sites and S/MARs. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 598–606.     

Accuracy and power of bayes prediction of amino acid sites under positive selection

Bayes prediction quantifies uncertainty by assigning posterior probabilities. It was used to identify amino acids in a protein under recurrent diversifying selection indicated by higher nonsynonymous (d(N)) than synonymous (d(S)) substitution rates or by omega = d(N)/d(S) > 1. Parameters were estimated by maximum likelihood under a codon substitution model that assumed several classes of sites with different omega ratios. The Bayes theorem was used to calculate the posterior probabilities of each site falling into these site classes. Here, we evaluate the performance of Bayes prediction of amino acids under positive selection by computer simulation. We measured the accuracy by the proportion of predicted sites that were truly under selection and the power by the proportion of true positively selected sites that were predicted by the method. The accuracy was slightly better for longer sequences, whereas the power was largely unaffected by the increase in sequence length. Both accuracy and power were higher for medium or highly diverged sequences than for similar sequences. We found that accuracy and power were unacceptably low when data contained only a few highly similar sequences. However, sampling a large number of lineages improved the performance substantially. Even for very similar sequences, accuracy and power can be high if over 100 taxa are used in the analysis. We make the following recommendations: (1) prediction of positive selection sites is not feasible for a few closely related sequences; (2) using a large number of lineages is the best way to improve the accuracy and power of the prediction; and (3) multiple models of heterogeneous selective pressures among sites should be applied in real data analysis.     

Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.     

Regulatory potential of S/MAR elements in transient expression

S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from the previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer-blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells.     

IRSS: a web-based tool for automatic layout and analysis of IRES secondary structure prediction and searching system in silico

Background  

Internal ribosomal entry sites (IRESs) provide alternative, cap-independent translation initiation sites in eukaryotic cells. IRES elements are important factors in viral genomes and are also useful tools for bi-cistronic expression vectors. Most existing RNA structure prediction programs are unable to deal with IRES elements.     

Viral IRES Prediction System - a Web Server for Prediction of the IRES Secondary Structure In Silico

The internal ribosomal entry site (IRES) functions as cap-independent translation initiation sites in eukaryotic cells. IRES elements have been applied as useful tools for bi-cistronic expression vectors. Current RNA structure prediction programs are unable to predict precisely the potential IRES element. We have designed a viral IRES prediction system (VIPS) to perform the IRES secondary structure prediction. In order to obtain better results for the IRES prediction, the VIPS can evaluate and predict for all four different groups of IRESs with a higher accuracy. RNA secondary structure prediction, comparison, and pseudoknot prediction programs were implemented to form the three-stage procedure for the VIPS. The backbone of VIPS includes: the RNAL fold program, aimed to predict local RNA secondary structures by minimum free energy method; the RNA Align program, intended to compare predicted structures; and pknotsRG program, used to calculate the pseudoknot structure. VIPS was evaluated by using UTR database, IRES database and Virus database, and the accuracy rate of VIPS was assessed as 98.53%, 90.80%, 82.36% and 80.41% for IRES groups 1, 2, 3, and 4, respectively. This advance useful search approach for IRES structures will facilitate IRES related studies. The VIPS on-line website service is available at http://140.135.61.250/vips/.     

Identification of consensus sequence from matrix attachment regions and functional analysis of its activity in stably transfected Chinese hamster ovary cells

Matrix attachment regions (MARs) can enhance transgene expression levels and maintain stability. However, the consensus sequence from MARs and its functional analysis remains to be examined. Here, we assessed a possible consensus sequence from MARs and assessed its activity in stably transfected Chinese hamster ovary (CHO) cells. First, we analyzed the effects of 10 MARs on transfected CHO cells and then analyzed the consensus motifs from these MARs using a bioinformatics method. The consensus sequence was synthesized and cloned upstream or downstream of the eukaryotic vector. The constructs were transfected into CHO cells and the expression levels and stability of enhanced green fluorescent protein were detected by flow cytometry. The results indicated that eight of the ten MARs increased transgene expression in transfected CHO cells. Three consensus motifs were found after bioinformatics analyses. The consensus sequence tandemly enhanced transgene expression when it was inserted into the eukaryotic expression vector; the effect of the addition upstream was stronger than that downstream. Thus, we found a MAR consensus sequence that may regulate the MAR-mediated increase in transgene expression.     

Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.     

S/MAR与基因表达

在真核生物的细胞核内,基因组是通过ＤＮＡ的核骨架附着（ＳＡＲ）或称核基质附着区（ＭＡＲ）（简记为Ｓ／ＭＡＲ）锚定在核骨架网状系统上的．Ｓ／ＭＡＲ既有一定的特征,又有多样性,研究认为它参与了ＤＮＡ复制调控和转录调控等多种核内生化过程,通过重组,在目的基因一侧或两侧带上Ｓ／ＭＡＲ后作基因转染或基因动植物,发现整合后的基因表达有时可增强几倍,甚至上万倍和／或显示位置独立效应,有些研究还报道,Ｓ／ＭＡＲ能