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. 2018 Apr 24;115(17):4435-4440.
doi: 10.1073/pnas.1719206115. Epub 2018 Apr 9.

New Class of Transcription Factors Controls Flagellar Assembly by Recruiting RNA Polymerase II in Chlamydomonas

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Free PMC article

New Class of Transcription Factors Controls Flagellar Assembly by Recruiting RNA Polymerase II in Chlamydomonas

Lili Li et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Cells have developed regulatory mechanisms that underlie flagellar assembly and maintenance, including the transcriptional regulation of flagellar genes, an initial step for making flagella. Although transcriptional regulation of flagellar gene expression is required for flagellar assembly in Chlamydomonas, no transcription factor that regulates the transcription of flagellar genes has been identified. We report that X chromosome-associated protein 5 (XAP5) acts as a transcription factor to regulate flagellar assembly in Chlamydomonas While XAP5 proteins are evolutionarily conserved across diverse organisms and play vital roles in diverse biological processes, nothing is known about the biochemical function of any member of this important protein family. Our data show that loss of XAP5 leads to defects in flagellar assembly. Posttranslational modifications of XAP5 track flagellar length during flagellar assembly, suggesting that cells possess a feedback system that modulates modifications to XAP5. Notably, XAP5 regulates flagellar gene expression via directly binding to a motif containing a CTGGGGTG-core. Furthermore, recruitment of RNA polymerase II (Pol II) machinery for transcriptional activation depends on the activities of XAP5. Our data demonstrate that, through recruitment of Pol II, XAP5 defines a class of transcription factors for transcriptional regulation of ciliary genes. This work provides insights into the biochemical function of the XAP5 family and the fundamental biology of the flagellar assembly, which enhance our understanding of the signaling and functions of flagella.

Keywords: Chlamydomonas; XAP5; cilia; transcription factor; transcriptional regulation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification and characterization of a xap5 mutant in Chlamydomonas. (A) Motile cells of the wild-type (WT) and rescued (xap5::XAP5-HA) strains were observed as long, winding tracks; conversely, palmelloid cells of af1-x were presented as bright focus. (Scale bar, 20 μm.) (B) Differential interference contrast (DIC) images of wild-type, af1-x, and rescued cells. Palmelloid af1-x cells were incubated with autolysin. (Scale bar, 5 μm.) (C) Immunofluorescence micrographs of wild-type and af1-x flagella labeled with acetylated tubulin antibodies after treatment with autolysin. (Scale bar, 10 μm.) (D) Flagellar length distribution of wild-type and af1-x cells after treatment with autolysin (n = 500). (E) Schematic view of the XAP5 gene; the foreign DNA insertion site in af1-x and the domain structure of the XAP5 protein are indicated.
Fig. 2.
Fig. 2.
XAP5 is phosphorylated in response to signals that induce flagellar assembly. (A) Immunoblot analysis of XAP5 during flagellar regeneration using an antibody against XAP5. Kinetics of flagellar regeneration upon shock-induced deflagellation is shown above the blot. Coomassie Brilliant Blue (CBB) showed an equal loading of all of the samples. H3, histone H3 antibody; pdf, cell sample before deflagellation. (B) Western blot analysis of whole-cell lysates from control and deflagellated xap5::XAP5-HA cells that were hatched with or without phosphatase (Ptase). (C) Control and deflagellated xap5::XAP5-HA cells were fractioned into whole-cell (WC), cytoplasm (Cyt), and nuclei (Nuc) fractions and were analyzed by immunoblotting. Antibodies against NAB1 and histone H3 were used to mark the cytoplasm and nuclei, respectively. (D) Immunoblot analysis of cell lysates from control and deflagellated xap5 cells expressing the HA-tagged wild-type, ΔNLS, and K106A-XAP5 protein. (E) Inhibition of flagellar regeneration by staurosporine. Cells were triggered to regenerate flagella by pH shock in the presence of actinomycin D (AD) (100 μg/mL), cycloheximide (CHX) (10 μg/mL), rapamycin (Rap) (50 μM), or staurosporine (Stau) (1 μM). Con, control. Data in A and E represent mean ± SD. One flagellum from at least 50 cells was measured at each time point in three independent experiments.
Fig. 3.
Fig. 3.
XAP5 modulates the transcription of ciliary genes via directly binding to the CTGGGGTG motif. (A) Analysis of the transcript abundance of flagellar-associated genes in wild-type and xap5 cells by real-time qPCR. (B) Quantification of the transcript abundance of ciliary genes in mutants defective in flagellar assembly. (C) Changes in the relative expression level of genes in wild-type and xap5 cells after pH shock-induced deflagellation. (D) DIC images of wild-type, ift70, and rescued (ift70::IFT70) cells. Palmelloid ift70 mutant cells were incubated with autolysin and released from the mother cell wall. (Scale bar, 5 μm.) (E) Schematic drawing representing the potential XAP5 target sites in the promoter regions of the ciliary genes. Data in AC represent the mean ± SD of three independent experiments. *P < 0.05. N.S., not significant (P > 0.05).
Fig. 4.
Fig. 4.
XAP5-mediated recruitment of the Pol II machinery to ciliary gene promoters. (A) ChIP-qPCR analysis of the indicated genes using three independently prepared samples. ChIP was performed in wild-type and xap5 cells with or without (Control) an antibody against RPB1. The regions amplified from chromatin immunoprecipitates by qPCR are indicated below each gene. (B) Detection of Pol II occupancy at ciliary genes in xap5::XAP5-HA and xap5 cells after pH shock-induced deflagellation (Defla). (C) Coimmunoprecipitation of XAP5 and Pol II machinery. Isolated nuclei from cells in the presence or absence of staurosporine were immunoprecipitated with antibody against HA or with preimmune IgG, followed by Western blotting using antibodies against HA and RPB1, respectively. (D) Detection of Pol II occupancy at ciliary genes by ChIP-qPCR in control and deflagellated wild-type cells in the presence or absence of staurosporine. Data in A, B, and D represent the mean ± SD of three independent experiments. *P < 0.05; N.S., not significant (P > 0.05).

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