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Retinoic acid induced downregulation of MYCN is not mediated through changes in Sp1/Sp3
Author(s) -
Kanemaru Kelli K.,
Tuthill Matthew C.,
Takeuchi Kenneth K.,
Sidell Neil,
Wada Randal K.
Publication year - 2008
Publication title -
pediatric blood and cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.116
H-Index - 105
eISSN - 1545-5017
pISSN - 1545-5009
DOI - 10.1002/pbc.21273
Subject(s) - downregulation and upregulation , chromatin immunoprecipitation , immunoprecipitation , e2f , retinoic acid , biology , transcriptional regulation , microbiology and biotechnology , brd4 , promoter , cancer research , medicine , acetylation , gene , transcription factor , genetics , gene expression , bromodomain
Background Use of retinoic acid (RA) has become the standard of care in the treatment of high risk neuroblastoma (NB). In vitro, RA induces growth arrest and differentiation, an effect that likely underlies its activity in the clinical setting. An important event in differentiation is the transcriptional downregulation of the MYCN oncogene, which is frequently activated in aggressive tumors. While it is known that Sp1/Sp3 and E2F are necessary to drive basal MYCN expression, the mechanism for its downregulation by RA remains enigmatic. Changes in E2F binding have been reported, however these occurred after the actual transcriptional response. Here, post‐translational modifications of Sp proteins were examined as an alternate mechanism of RA‐mediated promoter regulation. Procedure Western blot was used to evaluate steady state levels of nuclear/cytoplasmic Sp1/Sp3. Promoter binding and DNA conformation were determined by gel shift, circular permutation, and chromatin immunoprecipitation assays. Immunoprecipitation/western and 32 P‐phosphoamino analyses were used to detect glycosylation, acetylation, sumoylation, and phosphorylation. Results RA did not affect the cellular level of Sp1/Sp3 proteins, their nuclear/cytoplasmic distribution, ability to bind the MYCN promoter, degree of Sp‐induced DNA bending, or post‐translational modifications. Conclusions MYCN RA response is not mediated solely though the region controlling basal activity. RA may be exerting its effects via multiple non‐adjacent regulatory regions, potentially including basal motifs, either within the MYCN promoter or distally, on the same or even different chromosomes. Such cooperative trans‐type DNA–protein interactions could explain the inaccessibility of this mechanism to the locus‐specific approaches employed up to this point. Pediatr Blood Cancer 2008;50:806–811. © 2007 Wiley‐Liss, Inc.