The rest repression of the neurosecretory phenotype is negatively modulated by BHC80, a protein of the BRAF/HDAC complex.
Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance.
Department of Molecular Biology and Functional Genomics, Vita-Salute San Raffaele University, 20132 Milan, Italy.
Andrijana
A
Klajn
Carmelo
C
Ferrai
Laura
L
Stucchi
Ilaria
I
Prada
Paola
P
Podini
Tadashi
T
Baba
Mariano
M
Rocchi
Jacopo
J
Meldolesi
Rosalba
R
D'Alessandro
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D053378:Chromogranin B / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes; D009489:Neurosecretion / Q000502:physiology*; D016716:PC12 Cells; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012097:Repressor Proteins / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D014162:Transfection; D000099229:RE1-Silencing Transcription Factor
D053378:Chromogranin B; D018076:DNA, Complementary; D006655:Histone Deacetylases; D012333:RNA, Messenger; D012097:Repressor Proteins; D050825:Synaptosomal-Associated Protein 25; D000099229:RE1-Silencing Transcription Factor; C506134:CHGB protein, human; C494208:SNAP25 protein, human; C494210:Snap25 protein, rat; C506140:chromogranin B, rat; C492634:PHF21A protein, human