A nuclear targeting determinant for SATB1, a genome organizer in the T cell lineage

SATB1 is a nuclear protein, which acts as a cell-type specific genome organizer and gene regulator essential for T cell differentiation and activation. Several functional domains of SATB1 have been identified. However, the region required for nuclear localization remains unknown. To delineate this region, we employed sequence analysis to identify phylogenetically diverse members of the SATB1 protein family, and used hidden Markov model (HMM)-based analysis to define conserved regions and motifs in this family. One of the regions conserved in SATB1- and SATB2-like proteins in mammals, fish, frog and bird, is located near the N-terminus of family members. We found that the N-terminus of human SATB1 was essential for the nuclear localization of the protein. Furthermore, fusing residues 20-40 to a cytoplasmic green fluorescence protein (GFP) fused to pyruvate kinase (PK) was sufficient to quantitatively translocate the pyruvate kinase into the nucleus. The nuclear targeting sequence of human SATB1 (residues 20-40) is novel and does not contain clusters of basic residues, typically found in 'classical' nuclear localization signals (NLSs). We investigated the importance of four well-conserved residues (Lys29, Arg32, Glu34, and Asn36) in this nuclear targeting sequences. Remarkably, full-length SATB1 harboring a single point mutation at either Lys29 or Arg32, but not Glu34 or Asn36, did not enter the nucleus. Our results indicate that SATB1 N-terminal residues 20-40 represent a novel determinant of nuclear targeting.