Identification of an N-terminal domain that is required
for the DNA-binding activity of SATB1. (A) Schematic representation of
SATB1 N- and C-terminal and internal deletions. Various truncated
versions of SATB1 cDNA that were used as templates for coupled in vitro
transcription and translation are shown. Construct 1 depicts all the
known functional domains in SATB1. All the constructs are named
according to the amino acids encoded by the full-length cDNA that they
represent. Black boxes, MAR-binding domain; gray boxes, homeodomain.
The result of DNA-binding studies using these constructs is summarized
in the “Activity” column. +, DNA-binding activity comparable to
that of the full-length protein; −, total lack of DNA binding. (B)
SDS-PAGE analysis of in vitro translation products. Coupled in vitro
transcription and translation of SATB1 with various terminal and
internal deletions as depicted in panel A were performed as described
in Materials and Methods. The 35S-labeled translation
products were resolved by SDS-10% PAGE and visualized by
autoradiography. The numbers above each lane correspond to the
constructs depicted in panel A. The dark patch at the bottom of the gel
indicates position of the dye front. (C) EMSA analysis. The DNA-binding
activity of each of the above constructs was monitored by EMSA analysis
using a 32P-labeled WT (25)7 probe as described
in Materials and Methods. Numbers on top of lanes correspond to those
of the constructs in panel A. Lane 14, control binding reaction using
vector (pBlueScript)-translated lysate. Free and bound, positions of
the unbound and protein-bound DNA probes, respectively.