DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

112. Search and Identification of Proteins that Bind Specifically to the Satellite DNAs

Ivan B. Lobov and Olga I. Podgornaya

Institute of Cytology RAS, 4 Tikhoretskii Ave., 194064 St. Petersburg, Russia

ivan_lobov@hotmail.com

In the nucleus, chromosomes and individual chromosome domains are arranged by a non-random fashion. This organization is cell type-specific and undergoes rearrangements under the conditions that alter gene expression. Tandemly organized transcriptionally silent non-coding sequences, satellite DNAs (satDNAs), are localized in gene-poor heterochromatic chromosome regions. In interphase nucleus, these regions have a tendency to fuse, forming large chromocenters in a cell type-specific manner. Euchromatic regions can also associate with chromocenters that causes gene silencing as a result of transcription repression effect of heterochromatin.

Heterochromatin properties are mediated by proteins specifically associated with satDNAs that, however, are poorly characterized. To furthering our understanding of the role that satDNAs play in genome, we undertook search for proteins that bind specifically satDNAs of mouse and human. Chromosomal DNA anchored to the nuclear matrix (NM) or scaffold at the specific sites called M/SARs (for Matrix or Scaffold Attachment Regions) and via large blocks of satDNAs. We used electrophoretic mobility shift essay to reveal NM DNA-binding protein specific for the mouse major satDNA. We have developed a reliable approach for mild non-denaturing extraction of NM proteins that are generally insoluble under physiological conditions. The main DNA-binding protein revealed in these experiments was identified as a mouse homologue of SAF-A, an M/SAR-binding protein. We have also found that in interphase nuclei SAF-A predominantly decorates and covers heterochromatic areas.

Using Southwestern assay we have also identified four abundant DNA-binding proteins (p150, p120, p83 and p66) in nuclei and NM preparations. These proteins bound specifically to mouse major satDNA and fragment of alphoid satDNA from locus alpha21-II of human chromosome 21. p120 and p66 were identified as SAF-A and lamin B correspondingly. p150 and p83 are apparently identical with SAF-B and ARBP, well-characterized M/SAR-binding proteins. Using an electrophoretic assay and computer modeling of DNA structure we have found that proteins prefer intrinsically bent DNA fragments over the strait ones. Thus, despite the lack of sequence homology, different satDNAs share structural features that might serve as a recognition signal for DNA-binding proteins of the NM.

Our data raise the possibility that different M/SAR-binding proteins can bind specifically to certain subsets of satDNAs of different species. The ability of NM proteins to recognize both M/SARs and satDNAs might serve as a general mechanism of gene association with heterochromatin.