The Drosophila Zinc Finger Proteins Aef1 and Cg10543 Are Co-Localized with SAGA, SWI/SNF and ORC Complexes on Gene Promoters and Involved in Transcription Regulation

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Abstract

In previous studies, we purified the DUB-module of the Drosophila SAGA complex and showed that a number of zinc proteins interact with it, including Aef1 and CG10543. In this work, we conducted a genome-wide study of the Aef1 and CG10543 proteins and showed that they are localized predominantly on the promoters of active genes. The binding sites of these proteins colocalize with the SAGA and dSWI/SNF chromatin modification and remodeling complexes, as well as with the ORC replication complex. It has been shown that the Aef1 and CG10543 proteins are involved in the regulation of the expression of some genes on the promoters of which they are located. Thus, the Aef1 and CG10543 proteins are new participants in the cell transcriptional network and colocalize with the main transcription and replication complexes of Drosophila.

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About the authors

J. V. Nikolenko

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: krasnov@genebiology.ru
Russian Federation, Moscow, 119991

M. M. Kurshakova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: krasnov@genebiology.ru
Russian Federation, Moscow, 119991

D. V. Kopytova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: krasnov@genebiology.ru
Russian Federation, Moscow, 119991

Y. A. Vdovina

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: krasnov@genebiology.ru
Russian Federation, Moscow, 119991

N. E. Vorobyova

Институт биологии гена Российской академии наук

Email: krasnov@genebiology.ru
Russian Federation, Москва, 119334

A. N. Krasnov

Институт биологии гена Российской академии наук

Author for correspondence.
Email: krasnov@genebiology.ru
Russian Federation, Москва, 119334

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Supplementary files

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2. Fig. 1. Testing the specificity of antibodies to Aef1 (a) and CG10543 (b) proteins using Western blot analysis. Panel a shows the protein extract from control cells (K) in two dilutions (1 and 1:5), as well as the protein extract from cells after Aef1 RNA interference (Aef1i), also taken in two dilutions. The Western blot (panel a) was incubated with the tested antibodies to the Aef1 protein, as well as with antibodies to α-tubulin as a control for loading. In panel b, each of the samples was applied in three dilutions (1, 1:3, 1:10). K is the extract from control cells, CG10543i is the extract from cells after CG10543 RNA interference. Western blot (panel b) was incubated with antibodies to the CG10543 protein, as well as with antibodies to α-tubulin as a loading control.

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3. Fig. 2. ChIP-Seq profiles of CG10543 (black) and Aef1 (gray) proteins in the wg gene region. The top panel shows the gene structure from the genome browser. The bottom panel shows the ChIP-Seq profiles in CPM units.

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4. Fig. 3. Distribution of Aef1 (upper left) and CG10543 (upper right) protein binding sites relative to annotated elements of the Drosophila genome. Relative abundance of all annotated elements in the genome (lower) is shown for comparison. TSS is the promoter region, TES is the end of the gene, Gene bodies are the region of the gene between the TSS and TES, Intergenic are the intergenic regions.

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5. Fig. 4. Consensus sequence of the potential binding site of the proteins CG10543 (a) and Aef1 (b).

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6. Fig. 5. Averaged ChIP-Seq profiles of ORC2 (ORC complex), OSA (SWI/SNF complex), and GCN5 (SAGA complex) proteins at the binding sites of Aef1, CG10543 proteins, and at random promoters. The subunits of these complexes are indicated on the left: ORC2 (ORC), OSA (SWI/SNF), GCN5 (SAGA). The average ChIP-Seq profile of proteins (indicated on the left) at three groups of sites (indicated at the top) is shown. The ordinate axis shows the profiles in CPM units, the abscissa axis shows the distance in kb relative to the peak center. Approximately 3500 sites were analyzed in each group. The horizontal bars indicate the signal level at random promoters. It is evident that the subunits of the SAGA, SWI/SNF, and ORC complexes are recruited to the Aef1 and CG10543 sites in greater quantities than to random promoters.

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7. Fig. 6. Transcription of some genes containing both proteins, CG10543 and Aef1, at their promoters upon knockdown of CG10543 and Aef1 in individual experiments. Gene names are indicated at the bottom. Light gray bars correspond to gene transcription upon Aef1 RNA interference, dark gray bars – upon CG10543 RNA interference. White bars correspond to gene transcription under normal conditions (control), taken as unity. The ordinate axis shows the ratio of transcription in the experiment and in the control. The ras64B gene was used as a normalization gene. The experiments were performed in triplicate. Error bars indicate the standard error of the mean. Statistically significant changes with p < 0.05 (Student's t-test was used) are indicated by an asterisk.

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