Objective The recent publication of the first draft human genome sequence opened up the possibility for an extensive analysis of genome functional elements. Undoubtedly, these analyses will first be performed for coding sequences. The complete annotation of eukaryotic genomes, however, can not be limited to genes, but has to include the position of the associated non-coding regulatory elements. This information is indispensable for understanding the complex functional interplay between genes in the framework of higher genomic structures. Unfortunately, the data on genome positions of the multitude of regulatory sequences, like enhancers, promoters, transcription terminators, replication origins, are very limited, especially at the whole genome level. Therefore, the development of whole genome experimental approaches to the identification of genome elements that can not be recognized solely by their nucleotide sequences seems highly desirable.One important subclass of this type of sequences is nuclear scaffold/matrix attachment regions or S/MARs, that is those regions of chromatin loops or domains that are widely believed to anchor to the nuclear matrix and/or chromosome scaffold. Recent experimental evidence indicates that S/MARs can serve as bordering elements or insulators. These functional elements are capable to shield regulatory elements (e.g. enhancers) of genes within one functional chromatin domain from interference of the regulatory elements situated in adjacent domains, therefore permitting independent regulation of large gene clusters. In this connection the combination of S/MARs biochemical studies with functional assays developed to study insulator activity would be very fruitful. In case insulation capacity is an intrinsic property of S/MAR elements, the genome can be readily dissected on functionally insulated domains by means of isolation of S/MARs, their sequencing and identification of corresponding sites in the coming whole genome sequence.To address this problem two principal experimental approaches will be applied in the proposed project. In one of them already isolated by participating groups S/MAR elements will be probed for insulator properties, which will allow to link together S/MAR and insulator functional activities. One of the methods which permit to elucidate the insulator activity of S/MARs in vivo is the Drosophila enhancer blocking assay utilizing the ability of insulators to protect white mini-gene from the activity of 'upstream' eye enhancer in transgenic flies. These transgenic lines will be obtained by germ line transformation of white- Drosophila embryos with P-element based vectors containing a white expression cassette where S/MARs, suspected for insulator activity, will be inserted between enhancer and promoter of white gene.An alternative approach will be based on selection of the insulators from long stretches of human DNA following by their characterization as S/MAR elements.The direct selection of insulator elements from long (1-2 Mb) sequenced stretch of genomic DNA can be based on the re-combines mediated cassette exchange (RMCE) method developed in the participating group from National Research Centre for Biotechnology. Short fragments library prepared from cosmid contig spanning 1-2 Mb sequenced region of human genome can be cloned in the exchange vector between promoter and enhancer elements of negatively selectable marker. After transfection of the established cell line, containing the same cassette, with this library together with recombinase expression vector some cells, where initial cassette will be exchanged for cassette containing insulator sequence, will express reduced amount of the selection marker and therefore became resistant to negative selection. The insulator sequences could be rescued from the survived cell population, cloned in plasmid vector and analysed. The analysis will include checking the sequences for nuclear matrix binding properties, sequencing and direct placement into genomic context of the sequenced genome locus. This will allow reconstructing functionally independent domains and revealing genes contained within the same functional domains.The following main results are expected:1. Establishment of interrelations between S/MAR and insulator sequences;2. Isolation of novel human insulator elements, comparing their structure, and construction of map of independently regulated domains;3. Juxtaposition of loop domains specified by S/MARs and transcriptional domains specified by insulators;4. Isolation of novel human silencer sequences and location of genes, which can be regulated by these sequences;5. Establishment of universality of some insulators capable of functioning both in mammals and Drosophila;6. Testing the hypothesis about involvment of PcG and trxG proteins in the insulating activity of some insulators;7. Comparison of fuctional properties of placed in the BX-C background salient human insulators with gypsy, scs and Fab-7 insulators locating in the same genomic context. Programme(s) IC-INTAS - International Association for the promotion of cooperation with scientists from the independent states of the former Soviet Union (INTAS), 1993- Topic(s) 4 - Life Sciences OPEN - OPEN Call Call for proposal Data not available Funding Scheme Data not available Coordinator GBF, Gesellschaft für Biotechnologische Forschung, mbH EU contribution No data Address Mascheroder Weg 1 38124 Braunschweig Germany See on map Total cost No data Participants (3) Sort alphabetically Sort by EU Contribution Expand all Collapse all Department of Zoology and animal biology Switzerland EU contribution No data Address Quai E. Ansermet 30 1211 Geneva See on map Total cost No data Russian Academy of Sciences Russia EU contribution No data Address Kurchatov Sq., 2 123182 Moscow See on map Total cost No data Russian Academy of Sciences Russia EU contribution No data Address 16/10 Miklukho-Maklaya 117871 Moscow See on map Total cost No data
