The <i>Brahma</i> protein complex regulates circadian clock function in <i>Drosophila</i>.

<p><i>(A)</i> Western blots showing results of reciprocal co-IP assays to detect interaction of BRM with CLK, PER, and TIM in <i>Drosophila</i> S2 cells. Proteins extracted from S2 cells were either immunoprecipitated with α-FLAG to pull down BRM, α-HA to pull down TIM, and α-V5 to pull down CLK or PER. A negative control co-IP was performed for each pair of interactions using an antibody recognizing an epitope tag that is not present in the two proteins of interest. Immuno-complexes were subjected to western blotting to detect the bait protein or protein interactions. Input for the co-IP is indicated. All experiments were repeated at least three times. <i>(B)</i> Schematic of the core <i>Brahma</i> (SWI/SNF) chromatin-remodeling complex showing multiple subunits [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005307#pgen.1005307.ref029" target="_blank">29</a>]. All subunits, except Actin, were targeted for RNA<i>i</i> knockdown in behavioral assays. <i>(C)</i> Knockdown of individual <i>Brahma</i> complex subunits in <i>tim</i>-expressing neurons results in period lengthening. Shown are circadian periods for free running behavioral rhythms in constant dark conditions for males of the indicated genotypes. Analysis of period length was performed using Cycle P in the FaasX software package. Black bars represent control parental driver lines. Grey bars represent RNA<i>i</i> knockdown in <i>tim</i>-expressing neurons using the <i>tim-UAS-Gal4</i> driver (<i>w; UAS-dicer 2; TUG</i>). When available, multiple responder lines were used for RNA<i>i</i> knockdown for individual genes. Stock numbers for responder lines are indicated. Stocks starting with “v” were from VDRC, while stocks starting with “b” were from Bloomington Stock Center. Numbers within bars indicate sample size. Percentages above bars indicate percentage of flies displaying rhythmicity. Error bars = SEM. <i>(D)</i> Schematic of <i>brm</i> primer (b3) placement for quantitative PCR to assay extent of <i>brm</i> knockdown. <i>(E)</i> Expression of <i>brm</i> in control <i>TUG</i> parental driver line and <i>brm</i> RNA<i>i</i> mutant (v37720 X <i>TUG</i>) as assayed over a circadian day using quantitative PCR. Error bars = SEM of technical triplicates. Results are representative of two biological replicates. <i>(F)</i> Percent of <i>brm</i> knockdown in RNA<i>i</i> shown in (E) relative to <i>brm</i> expression in <i>TUG</i> parental control at corresponding time points. <i>(G)</i> Results of behavioral assays to determine circadian periodicity with similar format as described in (C). <i>brm</i> RNA<i>i</i> (v37720) was expressed in PDF neurons (<i>w; UAS-dicer 2</i>; <i>pdf-gal4</i> X v37720) as well as in <i>tim</i>-expressing neurons (grey bars). A dominant-negative <i>brahma</i> (<i>brm</i>^<i>K804R</i><i>)</i> transgene was expressed in <i>tim</i>-expressing cells (<i>TUG(II)</i>) or PDF neurons (<i>pdf-gal4</i>) (striped bars). Inhibition of <i>brm</i> RNAi expression during development using temperature-sensitive GAL80 system still resulted in a period-lengthening phenotype (white bar). <i>(H)</i> Three independent transgenic <i>BRM</i>^<i>RFL</i> transformants (BRM overexpression) display normal circadian phenotypes when crossed with the <i>TUG(II)</i> driver. Error bars = SEM.