NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression.
Small ubiquitin-like modifier (SUMO) modification of transcription factors is generally associated with repression. Reverse genetic analysis of SUMO-1, and -2 conserved residues emphasized the importance of dual charge reversals in abrogating the critical role of SUMO-2 K33, K35, and K42 in repression. GST-SUMO-2-affinity chromatography followed by liquid chromatography (LC)-MS analysis identified proteins that appeared to bind preferentially to WT SUMO-2 versus SUMO-2 K33E and K35E. LSD1, NXP-2, KIAA0809 (ARIP4), SAE2, RanGAP1, PELP1, and SETDB1 bound to SUMO-2 and not to SUMO-2 K33E, K42E, or K35E and K42E. Although LSD1 is a histone lysine demethylase, and histone H3K4 was demethylated at a SUMO-2-repressed promoter, neither overexpression of a dominant-negative LSD1 nor LSD1 depletion with RNA interference affected SUMO-2-mediated repression, indicating that LSD1 is not essential for repression, in this context. When tethered to a promoter by fusion to Gal4, NXP-2 repressed transcription, consistent with a role for NXP-2 in SUMO-mediated repression. SUMO-2-associated proteins identified in this study may contribute to SUMO-dependent regulation of transcription or other processes.
Department of Medicine, Channing Laboratory, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Adam
A
Rosendorff
Shuhei
S
Sakakibara
Sixin
S
Lu
Elliott
E
Kieff
Yan
Y
Xuan
Alessandra
A
DiBacco
Yujiang
Y
Shi
Yang
Y
Shi
Grace
G
Gill
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D008745:Methylation; D008969:Molecular Sequence Data; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D034521:Nuclear Matrix-Associated Proteins; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; D004247:DNA