Volume 15, Issue 1, Pages 43-56 (July 2004) Cyclin A Repression in Quiescent Cells Is Associated with Chromatin Remodeling of Its Promoter and Requires Brahma/SNF2α  Marjorie Coisy, Virginie Roure, Magali Ribot, Alexandre Philips, Christian Muchardt, Jean-Marie Blanchard, Jean-Christophe Dantonel  Molecular Cell  Volume 15, Issue 1, Pages 43-56 (July 2004) DOI: 10.1016/j.molcel.2004.06.022

Figure 1 Nucleosomal Structure of the Cyclin A Proximal Promoter Is Cell Cycle Regulated (A) DNA content analysis by flow cytometry of MEF cells. Time indicates hours after release from serum starvation (0, 6, 12, and 18 hr) or asynchronous cells in exponential growth (log). Lower table indicates percentages of cells in each phase of the cell cycle. (B) Northern blot analysis indicating RNA levels. (C) MNase sensitivity of cyclin A promoter. Ethidium bromide panel corresponds to 1 μg of each MNase-digested fraction. Lower panels correspond to autoradiography of PCR products. (D) PCR-mediated identification of mononucleosome limits in quiescent cells. Panels correspond to autoradiography of PCR products, from either mononucleosome template or genomic DNA template. (E) MNase sensitive sites mapping of cyclin A proximal promoter using oligos set 1 (see Experimental Procedures). (F) Ethidium bromide staining of the native chromatin fractions used for immunoprecipitation with anti-histone H4. (G) Autoradiography of PCR amplification products of extracted input fractions (input) and immunoprecipitated with anti-histone H4 fractions (ChIP αH4). (H) Overview of the micrococcal nuclease-sensitive sites within the cyclin A proximal promoter. Arrows correspond to the identified MNase-sensitive sites in quiescent/early G1 and S phase state. The two nucleosomes present in quiescent/early G1 state are indicated below. The lower diagram represents the loss of histones-DNA contacts occurring in late G1/S phase stage, upon gene activation. Molecular Cell 2004 15, 43-56DOI: (10.1016/j.molcel.2004.06.022)

Figure 2 Micrococcal Nuclease Sites Mapping of Exogenous Cyclin A Promoters (A) Overview of functionally identified sequences within the cyclin A promoter. (B) DNA content analysis by flow cytometry of 3T3 cells stably carrying a luciferase expression cassette under the control of the cyclin A wild-type, CCRE-mutated, and CHR-mutated promoters. Time indicates hours after release from serum starvation (0, 6, 12, and 18 hr) or asynchronous cells in exponential growth (log). (C) Northern blot analysis of RNA levels. (D) Mapping of MNase-sensitive sites within the exogenous cyclin A promoters. Molecular Cell 2004 15, 43-56DOI: (10.1016/j.molcel.2004.06.022)

Figure 3 Identification of Chromatin Remodeling Activity Recruited to the Cyclin A Promoter (A) Diagram of the nucleosome reconstitution procedure using biotinylated cyclin A promoters. (B) Western blot of complexes trapped by the cyclin A promoter assembled into a nucleosome. Antibodies used are indicated on the left. Input lane corresponds to 1/20 of the whole-cell extract fraction. (C) Western blot analysis of the expression of transfected HA-Brm and HA-Brg1. (D) Chromatin immunoprecipitation of HA-tagged Brm and Brg1 in quiescent (Quiescent) or in exponentially growing cells (Expo.). Endogenous (365 bp) and exogenous (320 bp) cyclin A promoters are indicated on the right. Molecular Cell 2004 15, 43-56DOI: (10.1016/j.molcel.2004.06.022)

Figure 4 Cyclin A Expression in Brm−/− Cells (A) FACS analysis of cyclin A expression in exponentially growing Brm+/+ and Brm−/− cells. (B) As in (A), but in serum-starved quiescent cells. (C) Immunofluorescence detection of cyclin A expression in quiescent Brm+/+ and Brm−/− cells. The percentage of nuclei showing cyclin A staining corresponds the mean of four independent experiments of 100 nuclei each. The SD is indicated in brackets. (D) Immunofluorescence detection of cyclin A and cyclin B in mitotic Brm+/+ and Brm−/− cells. Arrows point the different mitotic figures. Molecular Cell 2004 15, 43-56DOI: (10.1016/j.molcel.2004.06.022)

Figure 5 Lack of Cyclin A Repression and Nucleosomal Structure of Cyclin A Proximal Promoter in Brm-Deficient Cells (A) DNA content analysis by flow cytometry of Brm+/+ and Brm−/− cells. Time indicates hours after release from serum starvation (0, 6, 12, and 18 hr) or asynchronous cells in exponential growth (log). Lower table indicates percentages of cells in each phase of the cell cycle. (B) Western blot analysis of cyclin A expression. (C) Northern blot analysis of RNA levels. (D) MNase sensitivity of cyclin A promoter. Ethidium bromide panel corresponds to 1 μg of each MNase-digested fraction. Lower panels correspond to autoradiography of PCR products. (E) MNase sensitive sites mapping of cyclin A proximal promoter using oligos set 2 in quiescent Brm+/+ and Brm−/− cells. Molecular Cell 2004 15, 43-56DOI: (10.1016/j.molcel.2004.06.022)