Doxorubicin down-regulates Kruppel-associated box domain-associated protein 1 sumoylation that relieves its transcription repression on p21WAF1/CIP1 in breast cancer MCF-7 cells.
The role of post-translational modification, such as sumoylation, in modulating the efficacy of doxorubicin (Dox) treatment remains unclear. Transcriptional cofactor KRAB domain-associated protein 1 (KAP1) has been shown to complex with the KRAB zinc finger protein, ZBRK1, to repress the transcription of target genes. Through a combination of proteomic screening and site-directed mutagenesis approaches, we have identified lysines 554, 779, and 804 as the major sumoylation sites in KAP1. We then present evidence that Dox-mediated induction of cell cycle regulator p21 expression is differentially regulated by KAP1 sumoylation status. Moreover, the KAP1 sumoylation level was transiently decreased upon Dox exposure, and transfection with the KAP1 sumoylation mimetic, SUMO-1-KAP1, desensitizes breast cancer MCF-7 cells to Dox-elicited cell death. The sumoylation-dependent stimulation of KAP1 function is achieved by enhancing the methylation of H3-K9 and attenuating the acetylation of H3-K9 and H3-K14 at the p21 core promoter. We also show that occupancy of ZBRK1 response elements located at the p21 promoter by ZBRK1.KAP1 is independent of KAP1 sumoylation. Hence, sumoylation of KAP1 represses p21 transcription via a chromatin-silencing process without affecting interaction between KAP1.ZBRK1 and DNA, thus providing a novel mechanistic basis for the understanding of Dox-induced de-repression of p21 transcription. Taken together, our results suggest that Dox-induced decrease in KAP1 sumoylation is essential for Dox to induce p21 expression and subsequent cell growth inhibition in MCF-7 cells.
Lee
Yung-Kang
YK
Departments of Molecular Pharmacology and Toxicology, Norris Cancer Center, University of Southern California, Los Angeles, California 90033, USA.
Thomas
Stefani N
SN
Yang
Austin J
AJ
Ann
David K
DK
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism*; D016923:Cell Death; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology*; D020868:Gene Silencing; D006657:Histones / Q000378:metabolism; D006801:Humans; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000096:biosynthesis*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D000076183:Tripartite Motif-Containing Protein 28
D000903:Antibiotics, Antineoplastic; C493956:CDKN1A protein, human; D002843:Chromatin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D006657:Histones; D012097:Repressor Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D004317:Doxorubicin; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28