The regulatory element 3' to the A gamma-globin gene binds to the nuclear matrix and interacts with special A-T-rich binding protein 1 (SATB1), an SAR/MAR-associating region DNA binding protein.
A cis-acting DNA regulatory element 3' to the A gamma-globin gene contains eight distinct regions of DNA-protein interaction distributed over 750 bp of DNA. The sequences of two foot-printed regions (sites I and IV) are A-T rich and generate a highly retarded complex on gel shift analysis with nuclear extract from human erythroleukemia (K562) cells. We have purified a 98-kD protein that reproduces this gel shift. Tryptic cleavage and peptide sequence analysis demonstrated that the 98-kD protein is identical to a recently cloned protein, special A-T-rich binding protein 1 (SATB1), that binds selectively to nuclear matrix/scaffold-associated regions of DNA (MARs/SARs). We have shown by functional analysis that the 3' A gamma regulatory element associates with the nuclear matrix. SATB1 mRNA was identified in K562 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated its transcript in several other hematopoietic lines. Antisera to SATB1 caused ablation of the gel shift complex generated by both the crude nuclear extract and the purified 98-kD protein with the site I oligonucleotide. Furthermore, oligonucleotides that bind SATB1 inhibited formation of the site I gel shift complex when added as excess unlabeled competitor. An immunoblot analysis of the site I gel shift complex documented the presence of SATB1. Binding of SATB1 to two sites within the 3' A gamma regulatory element and its MAR/SAR activity suggests that this element may influence gene expression through interaction with the nuclear matrix.
Cunningham
J M
JM
Clinical Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.
Purucker
M E
ME
Jane
S M
SM
Safer
B
B
Vanin
E F
EF
Ney
P A
PA
Lowrey
C H
CH
Nienhuis
A W
AW
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D036961:Matrix Attachment Region Binding Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015530:Nuclear Matrix / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D036961:Matrix Attachment Region Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; C076189:SATB1 protein, human; D005914:Globins