PDZ domain-mediated dimerization and homeodomain-directed specificity are required for high-affinity DNA binding by SATB1

Abstract

To better understand DNA recognition and transcription activity by SATB1, the T-lineage-enriched chromatin organizer and transcription factor, we have determined its optimal DNA-binding sequence by random oligonucleotide selection. The consensus SATB1-binding sequence (CSBS) comprises a palindromic sequence in which two identical AT-rich half-sites are arranged as inverted repeats flanking a central cytosine or guanine. Strikingly, the CSBS half-site is identical to the conserved element ‘TAATA’ bound by the known homeodomains (HDs). Furthermore, we show that the high-affinity binding of SATB1 to DNA is dimerization-dependent and the HD also binds in similar fashion. Binding studies using HD-lacking SATB1 and binding target with increased spacer between the two half-sites led us to propose a model for SATB1–DNA complex in which the HDs bind in an antiparallel fashion to the palindromic consensus element via minor groove, bridged by the PDZ-like dimerization domain. CSBS-driven in vivo reporter analysis indicated that SATB1 acts as a repressor upon binding to the CSBS and most of its derivatives and the extent of repression is proportional to SATB1's binding affinity to these sequences. These studies provide mechanistic insights into the mode of DNA binding and its effect on the regulation of transcription by SATB1.

Phosphorylation-Dependent Interaction of SATB1 and PIAS1 Directs SUMO-Regulated Caspase Cleavage of SATB1

“…We further employed Position-Specific Iterated (PSI) BLAST to confirm that this N terminus is not homologous to the PDZ motif. In addition, many studies have failed to confirm homodimerization in vivo (40,48,69,70). Using in vivo coimmunoprecipitation of FLAG-SATB1(FL) with EGFP-SATB1(FL), and vice versa, or plate y2h assays, we failed to detect in vivo SATB1 homodimerization (Durrin, unpublished data).…”

“…Previously it was suggested that the N terminus of SATB1 facilitates SATB1 homodimerization and interaction with sumoylated PML and PML NBs, in addition to association with a number of transcription factors (26,46,48,49). Homodimerization of SATB1 is supposedly critical for SATB1 to localize to MAR elements.…”

“…Disruption of the dimer by apoptotic caspase cleavage leads to SATB1 dissociation from chromatin and concomitant chromatin condensation and fragmentation (11). These various functions of the N terminus are allegedly mediated through a PDZ domain (11,48). However, the Conserved Domain Database search used to identify this as a PDZ domain employed an E value that is of statistically low significance (i.e., E ϭ 1.8, which is similar to that of random sequences) (11).…”

Acetylation-Dependent Interaction of SATB1 and CtBP1 Mediates Transcriptional Repression by SATB1

“…Implications of SATB1 domains in its functional regulation impart unique properties to this chromatin organizer. The C-terminal homeodomain (HD) acts in concert with the Cut repeat-containing domain (CD) and directs high-affinity binding of SATB1 to its targets in a sequence-specific manner (35). The N-terminal PDZ-like domain aids in the formation of a homodimer that is essential for the DNA binding activity of SATB1 (15,35).…”

“…The C-terminal homeodomain (HD) acts in concert with the Cut repeat-containing domain (CD) and directs high-affinity binding of SATB1 to its targets in a sequence-specific manner (35). The N-terminal PDZ-like domain aids in the formation of a homodimer that is essential for the DNA binding activity of SATB1 (15,35). The N-terminal region harboring the PDZlike domain is a putative interface for its interaction with various cellular and viral proteins (25)(26)(27).…”