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Drosophila Brahma complex remodels nucleosome organizations in multiple aspects.

Shi J, Zheng M, Ye Y, Li M, Chen X, Hu X, Sun J, Zhang X, Jiang C - Nucleic Acids Res. (2014)

Bottom Line: The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease.Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown.Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital of Tongji University, Shanghai Key Laboratory of Signaling and Disease Research, the School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

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Impact of Brm knockdown on gene expression. (A) qPCR results confirm the significantly reduced expression level of Brm through its knockdown (**P-value < 0.01, two-tailed t-test). (B) Western blotting analysis quantifies relative abundance of Brahma without (GFP37) and with (BrmIR37) Brm knockdown. (C) The screenshot of RNA-seq read density shows the high expression level of Brm inverted repeat (IR) transgene by heatshock induction. The blue bar indicates the position of Brm IR in Brm gene. (D) The volcano plot presents the significantly differentially expressed genes (red and green dots) with two or more folds change and P-value < 0.01.
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Figure 1: Impact of Brm knockdown on gene expression. (A) qPCR results confirm the significantly reduced expression level of Brm through its knockdown (**P-value < 0.01, two-tailed t-test). (B) Western blotting analysis quantifies relative abundance of Brahma without (GFP37) and with (BrmIR37) Brm knockdown. (C) The screenshot of RNA-seq read density shows the high expression level of Brm inverted repeat (IR) transgene by heatshock induction. The blue bar indicates the position of Brm IR in Brm gene. (D) The volcano plot presents the significantly differentially expressed genes (red and green dots) with two or more folds change and P-value < 0.01.

Mentions: We generated the control fly strain (UAS-mCD8GFP/Hs-Gal4) and the Brm RNAi fly strain (UAS-Brm-IR/Hs-Gal4). The Brm was knocked down by the expression of Brm IR transgene through heatshock (see ‘Materials and Methods’ section for the details). The qRT-PCR results showed that the expression level of Brm was significantly decreased by knockdown (Figure 1A). The western blotting result further confirmed the largely reduced level of Brahma complex in the knockdown sample (Figure 1B). The normalized read count on Brm gene body confirmed the high expression level of Brm IR transgene and low expression level of Brm in the Brm RNAi fly strain when compared to the control fly strain (Figure 1C). This suggested that the UAS-Gal4 system worked well to knockdown Brmin vivo in our study. To investigate the effect of Brm knockdown on global gene expression, we identified 872 significantly differentially expressed genes whose expression change was >2-fold after Brm knockdown (Figure 1D). The differentially expressed genes included the hormone-responsive Ecdysone-induced genes (Eig) and homeotic genes that were reported to be the targets of Brahma in the previous studies (26–28). Gene Ontology (GO) analysis found that the differentially expressed genes had diverse functions including molting cycles, de novo protein folding, immune response, sensory perception and more (Supplementary Figure S2). A previous study showed that the Brm mRNA level was significantly higher in 0–12 h Drosophila embryos than at later developmental stages (27). Consistent with it, knockdown of Brm in early embryos arrested the development of Drosophila embryos in our study. Moreover, Brm depletion from the zygote was lethal at late stages of embryonic development (29). Therefore, Brm has vital functions for the development of Drosophila embryos.


Drosophila Brahma complex remodels nucleosome organizations in multiple aspects.

Shi J, Zheng M, Ye Y, Li M, Chen X, Hu X, Sun J, Zhang X, Jiang C - Nucleic Acids Res. (2014)

Impact of Brm knockdown on gene expression. (A) qPCR results confirm the significantly reduced expression level of Brm through its knockdown (**P-value < 0.01, two-tailed t-test). (B) Western blotting analysis quantifies relative abundance of Brahma without (GFP37) and with (BrmIR37) Brm knockdown. (C) The screenshot of RNA-seq read density shows the high expression level of Brm inverted repeat (IR) transgene by heatshock induction. The blue bar indicates the position of Brm IR in Brm gene. (D) The volcano plot presents the significantly differentially expressed genes (red and green dots) with two or more folds change and P-value < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150808&req=5

Figure 1: Impact of Brm knockdown on gene expression. (A) qPCR results confirm the significantly reduced expression level of Brm through its knockdown (**P-value < 0.01, two-tailed t-test). (B) Western blotting analysis quantifies relative abundance of Brahma without (GFP37) and with (BrmIR37) Brm knockdown. (C) The screenshot of RNA-seq read density shows the high expression level of Brm inverted repeat (IR) transgene by heatshock induction. The blue bar indicates the position of Brm IR in Brm gene. (D) The volcano plot presents the significantly differentially expressed genes (red and green dots) with two or more folds change and P-value < 0.01.
Mentions: We generated the control fly strain (UAS-mCD8GFP/Hs-Gal4) and the Brm RNAi fly strain (UAS-Brm-IR/Hs-Gal4). The Brm was knocked down by the expression of Brm IR transgene through heatshock (see ‘Materials and Methods’ section for the details). The qRT-PCR results showed that the expression level of Brm was significantly decreased by knockdown (Figure 1A). The western blotting result further confirmed the largely reduced level of Brahma complex in the knockdown sample (Figure 1B). The normalized read count on Brm gene body confirmed the high expression level of Brm IR transgene and low expression level of Brm in the Brm RNAi fly strain when compared to the control fly strain (Figure 1C). This suggested that the UAS-Gal4 system worked well to knockdown Brmin vivo in our study. To investigate the effect of Brm knockdown on global gene expression, we identified 872 significantly differentially expressed genes whose expression change was >2-fold after Brm knockdown (Figure 1D). The differentially expressed genes included the hormone-responsive Ecdysone-induced genes (Eig) and homeotic genes that were reported to be the targets of Brahma in the previous studies (26–28). Gene Ontology (GO) analysis found that the differentially expressed genes had diverse functions including molting cycles, de novo protein folding, immune response, sensory perception and more (Supplementary Figure S2). A previous study showed that the Brm mRNA level was significantly higher in 0–12 h Drosophila embryos than at later developmental stages (27). Consistent with it, knockdown of Brm in early embryos arrested the development of Drosophila embryos in our study. Moreover, Brm depletion from the zygote was lethal at late stages of embryonic development (29). Therefore, Brm has vital functions for the development of Drosophila embryos.

Bottom Line: The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease.Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown.Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Laboratory Medicine, Shanghai Tenth People's Hospital of Tongji University, Shanghai Key Laboratory of Signaling and Disease Research, the School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.

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