Question

SAR/MAR sequences were postulated to form the bases of these radial loops.  SAR/MAR sequences were defined experimentally by one approach for SARs and by a different experimental approach for MARs. This question is about our experimental foundation for thinking about models for chromosome folding and nuclear organization as discussed in class which lead to the idea of a radial loop model of chromosome origination. SAR/MAR sequences were postulated to form the bases of these radial loops.  SAR/MAR sequences were defined experimentally by one approach for SARs and by a different experimental approach for MARs.

SAR/MAR sequences later turned out to be enriched in DNA sequences that show high binding affinity for topoisomerase 2.  Attention then turned to identifying proteins that bind to SAR/MAR sequences in mitotic chromosomes.  One protein in fact was found to be topoisomerase 2.

i.  What experimental approach was taken to discover proteins that bound to SAR/MAR sequences in mitotic chromosomes?

ii.  What is the current name for the second protein found that bound to SAR/MAR sequences?

Answer 1

Ans (I): The scaffold or matrix attached regions (SAR/MAR) are specific nucleotide sequences providing sites to DNA loop structures to bind with scaffold or matrix proteins. In an experimental approach, the nuclear matrix was prepared with the mild treatment of lithium diiodosalycilate referred to as LIS resulting in the extraction of histones and related nuclear proteins. This was followed by treatment with various restriction enzymes followed by centrifugation. The DNA was purified and run on an agarose gel and further confirmed with southern hybridization. In another experimental approach, the nuclear matrix was prepared devoid of histones and DNA by DNase I treatment followed by 2M NaCl treatment. It was treated with radiolabelled 32P gene fragments followed by centrifugation. Agarose gel electrophoresis experiment was performed to analyze bound gene fragments and further confirmed with autoradiography.

Ans (II): The second protein which is well-known for binding SAR/MAR sequences is popularly known as DNA-binding protein SATB2 or special AT-rich sequence-binding protein 2 encoded by SATB2 gene. It is a transcription factor involved in the regulation of gene expression. It has a significant role in local chromatin remodeling.