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RNA pol II transcript abundance controls condensin accumulation at mitotically up-regulated and heat-shock-inducible genes in fission yeast.

Nakazawa N, Sajiki K, Xu X, Villar-Briones A, Arakawa O, Yanagida M - Genes Cells (2015)

Bottom Line: We found that condensin binds to RNA polymerase I-, II- and III-transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes.Pol II-mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA.However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin before proper chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, 904-0495, Japan.

No MeSH data available.


Related in: MedlinePlus

Loss of mitotically activated transcription in the Δsep1 mutant results in diminished association of condensin. (A) Expression of mitotically activated genes was greatly reduced in the Δsep1 mutant (Buck et al. 2004), but not in Δatf1. Transcript levels of four mitotically up-regulated ecm33+, gas1+, slp1+, and exg1+ genes were determined in mitotically arrested nda3 single, nda3 Δsep1, and nda3 Δatf1 double mutant cells by quantitative reverse transcription (RT)-PCR. Total extracted RNA was reverse-transcribed to cDNA, and resulting cDNA levels were quantified with error bars showing the standard deviation (n = 3). Relative RNA levels are shown as the ratio between the target gene and act1+ gene. (B) Enrichment of condensin Cut14-FLAG at ecm33+, gas1+, slp1+, and exg1+ genes. Cells were grown as shown in (A). ChIP-qPCR analysis of Cut14-FLAG was carried out in the nda3 single, nda3 Δsep1, and nda3 Δatf1 (control) double mutant background along with a no-tag strain. PCR primers at 6 regions (ecm33+, gas1+, slp1+, exg1+, act1+, and centromeric loci) were used. Relative enrichment was calculated as the ratio of IP to WCE with error bars showing standard deviations (n = 3). Condensin enrichment at these four mitotically transcribed genes requires the presence of Sep1.
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fig03: Loss of mitotically activated transcription in the Δsep1 mutant results in diminished association of condensin. (A) Expression of mitotically activated genes was greatly reduced in the Δsep1 mutant (Buck et al. 2004), but not in Δatf1. Transcript levels of four mitotically up-regulated ecm33+, gas1+, slp1+, and exg1+ genes were determined in mitotically arrested nda3 single, nda3 Δsep1, and nda3 Δatf1 double mutant cells by quantitative reverse transcription (RT)-PCR. Total extracted RNA was reverse-transcribed to cDNA, and resulting cDNA levels were quantified with error bars showing the standard deviation (n = 3). Relative RNA levels are shown as the ratio between the target gene and act1+ gene. (B) Enrichment of condensin Cut14-FLAG at ecm33+, gas1+, slp1+, and exg1+ genes. Cells were grown as shown in (A). ChIP-qPCR analysis of Cut14-FLAG was carried out in the nda3 single, nda3 Δsep1, and nda3 Δatf1 (control) double mutant background along with a no-tag strain. PCR primers at 6 regions (ecm33+, gas1+, slp1+, exg1+, act1+, and centromeric loci) were used. Relative enrichment was calculated as the ratio of IP to WCE with error bars showing standard deviations (n = 3). Condensin enrichment at these four mitotically transcribed genes requires the presence of Sep1.

Mentions: A forkhead transcription factor, Sep1, is essential for M-phase-specific transcription in fission yeast (Buck et al. 2004; Rustici et al. 2004). Using reverse transcription-PCR (RT-PCR), we confirmed that the deletion of sep1 gene abolishes mitotic transcription of C1 (ecm33+, gas1+, and slp1+) and C2 (exg1+) genes in mitotically arrested nda3 mutant cells (Fig.3A). Mitotic expression of these four genes was not significantly affected in a deletion mutant of the atf1 gene encoding the other transcription factor (Takeda et al. 1995). To examine whether condensin enrichment at mitotically activated genes depends on their transcripts, ChIP-qPCR assay was carried out using DNA probes corresponding to four C1 and C2 genes (Fig.3B). Cut14-FLAG proteins were immunoprecipitated with anti-FLAG antibodies in nda3 mitotic cells. Then, coprecipitated and WCE DNAs were amplified with real-time qPCR. The levels of precipitated DNAs were drastically diminished in Δsep1 mutant extracts, but not in Δatf1, showing their dependency on Sep1. In contrast, condensin enrichment at centromere DNA (Cen) did not decrease in Δsep1, suggesting that centromeric accumulation of condensin does not depend upon Sep1.


RNA pol II transcript abundance controls condensin accumulation at mitotically up-regulated and heat-shock-inducible genes in fission yeast.

Nakazawa N, Sajiki K, Xu X, Villar-Briones A, Arakawa O, Yanagida M - Genes Cells (2015)

Loss of mitotically activated transcription in the Δsep1 mutant results in diminished association of condensin. (A) Expression of mitotically activated genes was greatly reduced in the Δsep1 mutant (Buck et al. 2004), but not in Δatf1. Transcript levels of four mitotically up-regulated ecm33+, gas1+, slp1+, and exg1+ genes were determined in mitotically arrested nda3 single, nda3 Δsep1, and nda3 Δatf1 double mutant cells by quantitative reverse transcription (RT)-PCR. Total extracted RNA was reverse-transcribed to cDNA, and resulting cDNA levels were quantified with error bars showing the standard deviation (n = 3). Relative RNA levels are shown as the ratio between the target gene and act1+ gene. (B) Enrichment of condensin Cut14-FLAG at ecm33+, gas1+, slp1+, and exg1+ genes. Cells were grown as shown in (A). ChIP-qPCR analysis of Cut14-FLAG was carried out in the nda3 single, nda3 Δsep1, and nda3 Δatf1 (control) double mutant background along with a no-tag strain. PCR primers at 6 regions (ecm33+, gas1+, slp1+, exg1+, act1+, and centromeric loci) were used. Relative enrichment was calculated as the ratio of IP to WCE with error bars showing standard deviations (n = 3). Condensin enrichment at these four mitotically transcribed genes requires the presence of Sep1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig03: Loss of mitotically activated transcription in the Δsep1 mutant results in diminished association of condensin. (A) Expression of mitotically activated genes was greatly reduced in the Δsep1 mutant (Buck et al. 2004), but not in Δatf1. Transcript levels of four mitotically up-regulated ecm33+, gas1+, slp1+, and exg1+ genes were determined in mitotically arrested nda3 single, nda3 Δsep1, and nda3 Δatf1 double mutant cells by quantitative reverse transcription (RT)-PCR. Total extracted RNA was reverse-transcribed to cDNA, and resulting cDNA levels were quantified with error bars showing the standard deviation (n = 3). Relative RNA levels are shown as the ratio between the target gene and act1+ gene. (B) Enrichment of condensin Cut14-FLAG at ecm33+, gas1+, slp1+, and exg1+ genes. Cells were grown as shown in (A). ChIP-qPCR analysis of Cut14-FLAG was carried out in the nda3 single, nda3 Δsep1, and nda3 Δatf1 (control) double mutant background along with a no-tag strain. PCR primers at 6 regions (ecm33+, gas1+, slp1+, exg1+, act1+, and centromeric loci) were used. Relative enrichment was calculated as the ratio of IP to WCE with error bars showing standard deviations (n = 3). Condensin enrichment at these four mitotically transcribed genes requires the presence of Sep1.
Mentions: A forkhead transcription factor, Sep1, is essential for M-phase-specific transcription in fission yeast (Buck et al. 2004; Rustici et al. 2004). Using reverse transcription-PCR (RT-PCR), we confirmed that the deletion of sep1 gene abolishes mitotic transcription of C1 (ecm33+, gas1+, and slp1+) and C2 (exg1+) genes in mitotically arrested nda3 mutant cells (Fig.3A). Mitotic expression of these four genes was not significantly affected in a deletion mutant of the atf1 gene encoding the other transcription factor (Takeda et al. 1995). To examine whether condensin enrichment at mitotically activated genes depends on their transcripts, ChIP-qPCR assay was carried out using DNA probes corresponding to four C1 and C2 genes (Fig.3B). Cut14-FLAG proteins were immunoprecipitated with anti-FLAG antibodies in nda3 mitotic cells. Then, coprecipitated and WCE DNAs were amplified with real-time qPCR. The levels of precipitated DNAs were drastically diminished in Δsep1 mutant extracts, but not in Δatf1, showing their dependency on Sep1. In contrast, condensin enrichment at centromere DNA (Cen) did not decrease in Δsep1, suggesting that centromeric accumulation of condensin does not depend upon Sep1.

Bottom Line: We found that condensin binds to RNA polymerase I-, II- and III-transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes.Pol II-mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA.However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin before proper chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University, Onna-son, Okinawa, 904-0495, Japan.

No MeSH data available.


Related in: MedlinePlus