Specific interaction of mouse major satellite with MAR-binding protein SAF-A

A DNA-binding activity specific to the major mouse satellite (satMa) has been detected in a nuclear matrix protein extract by electrophoretic mobility shift assays (EMSA) after fractionation by ion exchange chromatography. An antibody raised against the satMa-protein complexes recovered from preparative EMSA recognizes on Western blots one major polypeptide with an apparent molecular mass of 120 kDa. The protein also has a similar affinity for a matrix-associated region (MAR) fragment. We demonstrate that the protein is a murine homologue of SAF-A which has been shown to bind selectively to MARs and is responsible for the satMa-binding activity in the chromatographic fractions. SatMa has significant homology to the mouse minor satellite fragments, but its binding of SAF-A shows much less affinity. No protected regions of significant length were found by footprinting, but multiple T residues scattered within the satMa sequence are protected, indicating that the whole fragment is involved in the binding to SAF-A. Combined immunofluorescence (SAF-A) and FISH (satMa) with in situ nuclear matrix procedures reveal that SAF-A and satMa colocalize. SAF-A appears as bright dots in interphase nuclei, presumably associated with MARs, predominantly surrounding and covering heterochromatic areas. A scheme based on morphological observations and biochemical data of SAF-A double satMa/MAR specificity is discussed.