Abstract
SATB1 (special AT-rich sequence-binding protein-1) provides a key link between DNA loop organization, chromatin modification/remodeling, and association of transcription factors at matrix attachment regions (MARs). To investigate the role of SATB1 in cellular events, we performed a yeast two-hybrid screen that identified SUMO-1, Ubc9, and protein inhibitor of activated STAT (PIAS) family members as SATB1 interaction partners. These proteins, working in concert, enhanced SUMO conjugation to lysine-744 of SATB1. Overexpression of SUMO or PIAS in Jurkat cells, which express high levels of endogenous SATB1, exhibited enhanced caspase cleavage of this MAR-associating protein. Sumoylation-deficient SATB1 (SATB1(K744R)) failed to display the characteristic caspase cleavage pattern; however, fusion of SUMO in-frame to SATB1(K744R) restored cleavage. A SUMO-independent interaction of inactive caspase-6 and SATB1 was noted. A subset of total cellular SATB1 localized into promyelocytic leukemia nuclear bodies where enhanced SATB1 cleavage was detected subsequent to caspase activation. These results reveal a novel sumoylation-directed caspase cleavage of this key regulatory molecule. The role of regulated proteolysis of SATB1 may be to control transcription in immune cells during normal cell functions or to assist in efficient and rapid clearance of nonfunctional or potentially damaging immune cells.
Highlights
Scaffold or matrix attachment regions (MARs)4 are AT-rich sequences in the DNA of eukaryotic chromosomes that bind
Caspase cleavage of SATB1 mediates its exclusion from DNA binding [9, 10], suggesting a mechanism that may lead to disassembly of higher order chromatin structure, proteolnuclear body; PIAS, protein inhibitor of activated STAT; STAT, signal transducers and activators of transcription; SUMO, small ubiquitin-like modifier; IL, interleukin; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; poly(ADPribose) polymerase (PARP), poly(ADP-ribose) polymerase; ATRA, all-trans retinoic acid; Ni-NTA, nickel-nitrilotriacetic acid; GST, glutathione S-transferase; Ab, antibody; FBS, fetal bovine serum; CPT, camptothecin; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein
Identification of SATB1-interacting Partners—SATB1 is important for assembling various nuclear factors and for the organization and regulation of region-specific chromatin structure at MAR elements [32, 33]
Summary
Plasmid Construction—SATB1 cDNA, encoding amino acids 58 –763, was excised from pECH-SATB1 [26] and inserted inframe with the cDNA encoding the binding domain of the LexA protein in the vector pLexA (Clontech Laboratories, Inc.). SATB1 cDNA and deletions/mutations were subsequently inserted into pBluescript II KS (Stratagene), pEGFP-C1 (Clontech), or pcDNA3.1/His (Invitrogen) for additional studies. Cells were transfected with the plasmid constructs described above by standard methods: Electroporation for Jurkat, Nalm-6, K562, and NB4; lipid technologies (Effectene; Qiagen) for HeLa and MCF-7. Proteins were transferred to Immobilon-P membranes, and proteins of interest were detected by Western blot technologies using antibodies to SATB1, EGFP (Clontech), and poly(ADPribose) polymerase (PARP), caspases-3 or -6, or -actin (Santa Cruz Biotechnology). Microscopy—For confocal microscopic studies, cells were transiently transfected with EGFP-SATB1 constructs on chamber slides, fixed for 10 min with 5% paraformaldehyde, washed three times in phosphate-buffered saline (PBS), and permeabilized in 0.5% Triton X-100 in PBS. 5-min washes in 3% bovine serum albumin-PBS, the secondary antibody, Alexa Fluor 594 rabbit antimouse IgG, was used as described by the supplier Laser excitation was 488 nm from an argon laser, 4% power for green fluorescence, and 543 nm from HeNe laser, 100% power for red fluorescence
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