The basic leucine zipper domain transcription factor Atf1 directly controls Cdc13 expression and regulates mitotic entry independently of Wee1 and Cdc25 in Schizosaccharomyces pombe

Abstract

Progression into mitosis is a major point of regulation in the Schizosaccharomyces pombe cell cycle, and its proper control is essential for maintenance of genomic stability. Investigation of the G(2)/M progression event in S. pombe has revealed the existence of a complex regulatory process that is responsible for making the decision to enter mitosis. Newer aspects of this regulation are still being revealed. In this paper, we report the discovery of a novel mode of regulation of G(2)/M progression in S. pombe. We show that the mitogen-activated protein kinase (MAPK)-regulated transcription factor Atf1 is a regulator of Cdc13 (mitotic cyclin) transcription and is therefore a prominent player in the regulation of mitosis in S. pombe. We have used genetic approaches to study the effect of overexpression or deletion of Atf1 on the cell length and G(2)/M progression of S. pombe cells. Our results clearly show that Atf1 overexpression accelerates mitosis, leading to an accumulation of cells with shorter lengths. The previously known major regulators of entry into mitosis are the Cdc25 phosphatase and the Wee1 kinase, which modulate cyclin-dependent kinase (CDK) activity. The significantly striking aspect of our discovery is that Atf1-mediated G(2)/M progression is independent of both Cdc25 and Wee1. We have shown that Atf1 binds to the Cdc13 promoter, leading to activation of Cdc13 expression. This leads to enhanced nuclear localization of CDK Cdc2, thereby promoting the G(2)/M transition.

FIG 1

Atf1 is important for controlling mitotic entry in S. pombe cells. (A) cdc25-22 cells transformed with pREP41 (φ) or pREP41+Atf1 were grown to log phase in the presence of thiamine, and then serial dilutions were spotted onto EMM-Leu-thiamine plates. cdc25-22, Δatf1cdc25-22, and Δatf1 cells were grown to log phase, and serial dilutions were then spotted onto YES plates. The plates were incubated at the indicated temperatures for 4 days before being photographed. Representative images of 3 independent experiments are shown. (B) Bright-field images of cdc25-22 cells transformed with pREP41 (φ) or pREP41Atf1, cdc25-22, and Δatf1cdc25-22 cells. For overexpression, cells were grown in EMM-leucine-thiamine for 24 h at 25°C, shifted to the indicated temperatures for 4 h, fixed, and then processed for imaging. cdc25-22 and Δatf1cdc25-22 cells were grown to log phase at 25°C and then shifted to indicated temperatures 37°C for 4 h, fixed, and then processed for imaging. Bar, 10 μm. (C) Quantification of the length of the cells shown in panel B using ImageJ software. Representative images of 3 independent experiments are shown.

FIG 2

Atf1-mediated mitotic acceleration is independent of Cdc25 activity and leads to abnormal mitosis. (A) Exponentially growing cdc25-22 and Δatf1cdc25-22 were synchronized at the G₂/M boundary by incubating at 37°C for 4 h and then released from the block by shifting to the permissive temperature of 25°C. Growth rate was then analyzed by determination of cell number at regular intervals. Data represent means of three independent experiments. (B) cdc25-22 cells overexpressing Atf1 or the empty vector were shifted to 37°C. Increase in cell number was then monitored for 4 h at this nonpermissive temperature. Data represent means of three independent experiments. (C) cdc25-22 cells overexpressing Atf1 were shifted to 25°C or 37°C for 4 h and fixed, and then their nuclei were stained with DAPI and images were taken. Bar, 10 μm. Representative images of 3 independent experiments are shown.

FIG 3

Atf1 regulates mitotic entry independent of Wee1. (A) Wild-type and wee1-50 cells transformed with pREP41 (φ) or pREP41+Atf1 were grown to log phase in the presence of thiamine, and then serial dilutions were spotted onto EMM-Leu-thiamine plates. The plates were incubated at the indicated temperatures for 4 days before being photographed. Representative images of 3 independent experiments are shown. (B) Growth rates of wee1-50 cells overexpressing Atf1 were determined at the indicated temperatures. Data represent means of three independent experiments.

FIG 4

Atf1 targets the activation of Cdc2 to control mitotic entry. Exponentially growing cdc2.33 (A) and cdc13-117 (B) cells overexpressing Atf1 at 25°C were shifted to the indicated temperatures for 4 h, and bright-field images of the cells were taken both at 25°C and at 37°C. Bar, 10 μm. Representative images of 3 independent experiments are shown.

FIG 5

Effect of Atf1 overexpression on the growth of Cdc2 mutants. Atf1 was overexpressed in wild-type (A), cdc2-1w (B), cdc2-3w (C), or cdc2.33 (D) cells for 24 h, and then the growth rates of these cells were measured and compared with those of control cells transformed with the empty vector. Data represent means of three independent experiments.

FIG 6

Atf1 overexpression leads to enhanced nuclear localization of Cdc2 through activation of Cdc13 transcription. (A) Images of wild-type S. pombe cells having a GFP-tagged genomic copy of Cdc2 and overexpressing Atf1 were taken. At least 100 cells were counted to determine the extent of nuclear localization of Cdc2. Bar, 10 μm. Representative images of 3 independent experiments are shown. (B) Real-time PCR analysis of Cdc13 expression in wild-type cells overexpressing Atf1 at 30°C and in cdc25-22 and Δatf1cdc25-22 cells (synchronized at the G₂/M boundary). 18S rRNA expression was used for normalization. Data represent means of three independent experiments. Statistical analysis was done using the Graph Pad Prism application. *, P < 0.05. (C) Atf1 was overexpressed in wild-type S. pombe cells, and the levels of the Cdc13 protein were detected by immunoblotting. (D) Bright-field images of cdc25-22 cells overexpressing Atf1bZIPΔ after incubation at the indicated temperatures for 4 h. Bar, 10 μm. Representative images of 3 independent experiments are shown.

FIG 7

Increase in Cdc13 expression can rescue the temperature sensitivity of Δatf1cdc25-22 cells. (A) Bright-field images of cdc2.33 and cdc25-22 cells overexpressing Cdc13 after incubation at the indicated temperatures for 4 h. Bar, 10 μm. (B) cdc25-22 and Δatf1cdc25-22 cells transformed with pREP41 (φ) or pREP41+Cdc13 were grown to log phase in the presence of thiamine, and then serial dilutions were spotted onto EMM-Leu plates with or without thiamine. The plates were incubated at the indicated temperatures for 4 days before being photographed. Representative images of 3 independent experiments are shown.

FIG 8

Chromatin immunoprecipitation analysis of Atf1-HA bound to Cdc13 promoter. (A) Location of the regions R1 and R2 on the genomic regions upstream of Cdc13 transcription start site (+1). These regions were tested for association with Atf1 using ChIP assays. (B) Exponentially growing wild-type S. pombe cells (no tag) or cells carrying a HA-6His-tagged genomic copy of Atf1 were harvested and lysed after formaldehyde cross-linking, and chromatin bound to Atf1-HA was obtained after immunoprecipitation with anti-HA antibodies (α-HA-IP). Recovered DNA was analyzed by PCR amplification with primers designed against the regions R1 and R2. WCE, whole-cell extracts before immunoprecipitation; -ve, no-template control for PCR.

FIG 9

Model for the role of Atf1 in mitotic entry. Atf1binding to Cdc13 promoter increases the levels of Cdc13 expression, which helps in increasing the nuclear concentration of the Cdc2-Cdc13 complex, resulting in accelerated mitotic entry.