title: The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity.
abstract: BACKGROUND
  NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated.
  
  
  METHODS
  Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity.
  
  
  RESULTS
  Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex).
  
  
  CONCLUSIONS
  The present study shows that NRP/B acts as a transcriptional repressor by interacting with the co-repressors, HDAC1, providing new insight into the molecular mechanisms of NRP/B on tumor suppression.
authors.affiliation: CHA Cancer Institute, CHA University, Seoul, Korea.
authors.forename: Jina
authors.initials: J
authors.lastname: Choi
authors.affiliation: Cancer Cell Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Eun Sung
authors.initials: ES
authors.lastname: Yang
authors.affiliation: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Kiweon
authors.initials: K
authors.lastname: Cha
authors.affiliation: Cancer Cell Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: John
authors.initials: J
authors.lastname: Whang
authors.affiliation: CHA Cancer Institute, CHA University, Seoul, Korea.
authors.forename: Woo-Jung
authors.initials: WJ
authors.lastname: Choi
authors.affiliation: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Shalom
authors.initials: S
authors.lastname: Avraham
authors.affiliation: CHA Cancer Institute, CHA University, Seoul, Korea ; Cancer Cell Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Tae-Aug
authors.initials: TA
authors.lastname: Kim
keywords: Breast cancer; E2F transcription factor; NRP/B; Transcriptional activity