Cloning of SmNCoA-62, a novel nuclear receptor co-activator from Schistosoma mansoni: assembly of a complex with a SmRXR1/SmNR1 heterodimer, SmGCN5 and SmCBP1.
The Schistosoma mansoni nuclear receptors (NR) SmRXR1 and SmNR1 have recently been shown to form a heterodimer and to bind to canonic hormone response DNA elements. Recruitment of co-regulatory proteins to NRs is required for their transcriptional and biological activities. Here, we cloned a novel S. mansoni NR co-activator, SmNCoA-62. SmNCoA-62 is highly homologous to the human Vitamin D receptor co-activator NCoA62/SKIP. SmNCoA-62 contains the SNW nuclear receptor interaction domain and a putative C-terminus transactivation domain. By using in vitro pull-down assays, we fully mapped the interaction domains of S. mansoni NR co-activators, SmNCoA-62, SmGCN5 and SmCBP1 with SmRXR1 and SmNR1, as well as the domains that mediate interactions amongst the co-activators themselves. By mutagenesis analysis, we showed that SmCBP1 LxxLL motif 2 and LxxLL motif 3, but not LxxLL motif 1, were essential to mediate the interactions of SmCBP1 with the EF domains of SmRXR1 and SmNR1. Histone acetyltransferases SmGCN5 and SmCBP1 specifically acetylated the C/D domains of SmRXR1 and SmNR1. In addition, two acetylation sites of SmNR1 were identified. SmGCN5 and SmCBP1 also acetylated SmNCoA-62 but with significant differences in their acetylation activities. Using gel shift analysis, we were able to demonstrate, in vitro, the assembly of the co-activators on the SmRXR1/SmNR1 heterodimer bound to DNA. LxxLL motifs 2 and 3 of SmCBP1 seemed to play a crucial role for the assembly of the co-activators to the DNA-bound SmRXR1/SmNR1 heterodimer.
Fantappié
Marcelo Rosado
MR
Southwest Foundation for Biomedical Research, Department of Virology and Immunology, PO Box 78245-0594, San Antonio, TX, USA. fantappie@bioqmed.ufrj.br
Bastos de Oliveira
Francisco Meirelles
FM
de Moraes Maciel
Renata
R
Rumjanek
Franklin David
FD
Wu
Wenjie
W
Loverde
Philip T
PT
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D018507:Gene Expression Regulation, Developmental; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics* / Q000378:metabolism; D012550:Schistosoma mansoni / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D013045:Species Specificity
D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C459441:SmRXR-1 protein, Schistosoma mansoni