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Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.

Ignarski M, Singh A, Swart EC, Arambasic M, Sandoval PY, Nowacki M - Nucleic Acids Res. (2014)

Bottom Line: Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs.PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development.We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Freiestrasse 1, 3012 Bern, Switzerland.

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Effect of PTCAF-1 knockdown on cell survival, IES and transposon retention. (A) Effect of PTCAF-1 knockdown on cell survival. After going through autogamy in silencing medium the cells where recovered in WGP medium. The graph shows the survival after three days. Black: percentage of dead cells; grey: percentage of sick cells showing an altered number of divisions or behavior; white: percentage of cell growing at a normal rate. NOWA1 KD was used as a positive control. Silencing vector without insert was used as negative control. (B) Effect of PTCAF-1 knockdown on IES excision. IES retention was tested by PCR using primers flanking specific IESs sequences within the 51G and 51A genes as well as the mating-type locus (MT Locus). The excised form (IES−) is always detectable due to its presence in the fragments of the parental MAC. The unexcised form (IES+) is only detectable in case of retention in the newly developing macronuclei. Maternally controlled IESs: 51A712, 51A2591, 51A6649, 51G11, 51G2832, 51G4404, MT-Locus; non-maternally controlled IESs: 51A1835, 51A4404, 51A4578, 51A6435, 51G1413, 51G1832, 51G6447. (C) Effect of PTCAF-1 KD on Thon and Sardine transposons. Total DNA was extracted from vegetative (V), control and PTCAF-1 KD cultures at specific time points during sexual reproduction. E, M, L as in Figure 1C; 5, 15, 25 and 45 h after L. After running the DNA on a 1% agarose-gel and transfer to nylon membrane the samples were hybridized with Sardine and Thon transposon-specific probes. A mitochondrial DNA-specific probe was used as loading control. The graph shows the Thon signal quantification normalized to the loading control.
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Figure 2: Effect of PTCAF-1 knockdown on cell survival, IES and transposon retention. (A) Effect of PTCAF-1 knockdown on cell survival. After going through autogamy in silencing medium the cells where recovered in WGP medium. The graph shows the survival after three days. Black: percentage of dead cells; grey: percentage of sick cells showing an altered number of divisions or behavior; white: percentage of cell growing at a normal rate. NOWA1 KD was used as a positive control. Silencing vector without insert was used as negative control. (B) Effect of PTCAF-1 knockdown on IES excision. IES retention was tested by PCR using primers flanking specific IESs sequences within the 51G and 51A genes as well as the mating-type locus (MT Locus). The excised form (IES−) is always detectable due to its presence in the fragments of the parental MAC. The unexcised form (IES+) is only detectable in case of retention in the newly developing macronuclei. Maternally controlled IESs: 51A712, 51A2591, 51A6649, 51G11, 51G2832, 51G4404, MT-Locus; non-maternally controlled IESs: 51A1835, 51A4404, 51A4578, 51A6435, 51G1413, 51G1832, 51G6447. (C) Effect of PTCAF-1 KD on Thon and Sardine transposons. Total DNA was extracted from vegetative (V), control and PTCAF-1 KD cultures at specific time points during sexual reproduction. E, M, L as in Figure 1C; 5, 15, 25 and 45 h after L. After running the DNA on a 1% agarose-gel and transfer to nylon membrane the samples were hybridized with Sardine and Thon transposon-specific probes. A mitochondrial DNA-specific probe was used as loading control. The graph shows the Thon signal quantification normalized to the loading control.

Mentions: Upon PTCAF-1 knockdown (PTCAF-1 KD) we observed a strong decrease of PTCAF-1 mRNA level in the early stages of autogamy (E) compared to control (Figure 1C). To assess whether PtCAF-1 might play a role in Paramecium sexual development, we tested the survival of sexual progeny after PTCAF-1 KD (Figure 2A). Sixty-seven percent of the PTCAF-1 KD cells in our survival test died during a time course of 3 days (up to 12 cell divisions) after the progeny cells were refed and allowed to divide vegetatively (Figure 2A). Twenty-seven percent of the cells did not divide at the normal rate of four divisions per 24 h and 6% showed no growth defects. In the control culture, 94% of the cells grew at normal rates while 6% died. In the positive control (NOWA1 KD), 97% of the cells died and 3% survived with a reduced growth rate. This result was confirmed by using RNAi construct targeting an alternative region of PTCAF-1 (Supplementary Figure S2A). No cell death or reduced division was observed for Paramecium cells grown vegetatively in silencing medium for more than 12 divisions. PTCAF-1 KD did not result in premature termination of development at an early stage as we were able to observe all the developmental stages and the final formation of new developing MACs (data not shown). We saw no structural irregularities during development and there was no notable differences in DNA content between the PTCAF-1 KD cells and the control cells. Our deep sequencing results suggest that the overall scnRNA levels that are detected at the early time point during development are not affected in PTCAF-1 KD compared to control (Figure 4B). This suggests that the expression and functions of Dcl2/3 and Ptiwi01/09 (responsible for the production and protection of scnRNAs) were not affected by PTCAF-1 KD. We confirmed by northern blot that the expression of PTIWI01 is not affected by PTCAF-1 KD (Figure 1C), and that the localization of Ptiwi09-GFP is unchanged by this knockdown (data not shown).


Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.

Ignarski M, Singh A, Swart EC, Arambasic M, Sandoval PY, Nowacki M - Nucleic Acids Res. (2014)

Effect of PTCAF-1 knockdown on cell survival, IES and transposon retention. (A) Effect of PTCAF-1 knockdown on cell survival. After going through autogamy in silencing medium the cells where recovered in WGP medium. The graph shows the survival after three days. Black: percentage of dead cells; grey: percentage of sick cells showing an altered number of divisions or behavior; white: percentage of cell growing at a normal rate. NOWA1 KD was used as a positive control. Silencing vector without insert was used as negative control. (B) Effect of PTCAF-1 knockdown on IES excision. IES retention was tested by PCR using primers flanking specific IESs sequences within the 51G and 51A genes as well as the mating-type locus (MT Locus). The excised form (IES−) is always detectable due to its presence in the fragments of the parental MAC. The unexcised form (IES+) is only detectable in case of retention in the newly developing macronuclei. Maternally controlled IESs: 51A712, 51A2591, 51A6649, 51G11, 51G2832, 51G4404, MT-Locus; non-maternally controlled IESs: 51A1835, 51A4404, 51A4578, 51A6435, 51G1413, 51G1832, 51G6447. (C) Effect of PTCAF-1 KD on Thon and Sardine transposons. Total DNA was extracted from vegetative (V), control and PTCAF-1 KD cultures at specific time points during sexual reproduction. E, M, L as in Figure 1C; 5, 15, 25 and 45 h after L. After running the DNA on a 1% agarose-gel and transfer to nylon membrane the samples were hybridized with Sardine and Thon transposon-specific probes. A mitochondrial DNA-specific probe was used as loading control. The graph shows the Thon signal quantification normalized to the loading control.
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Figure 2: Effect of PTCAF-1 knockdown on cell survival, IES and transposon retention. (A) Effect of PTCAF-1 knockdown on cell survival. After going through autogamy in silencing medium the cells where recovered in WGP medium. The graph shows the survival after three days. Black: percentage of dead cells; grey: percentage of sick cells showing an altered number of divisions or behavior; white: percentage of cell growing at a normal rate. NOWA1 KD was used as a positive control. Silencing vector without insert was used as negative control. (B) Effect of PTCAF-1 knockdown on IES excision. IES retention was tested by PCR using primers flanking specific IESs sequences within the 51G and 51A genes as well as the mating-type locus (MT Locus). The excised form (IES−) is always detectable due to its presence in the fragments of the parental MAC. The unexcised form (IES+) is only detectable in case of retention in the newly developing macronuclei. Maternally controlled IESs: 51A712, 51A2591, 51A6649, 51G11, 51G2832, 51G4404, MT-Locus; non-maternally controlled IESs: 51A1835, 51A4404, 51A4578, 51A6435, 51G1413, 51G1832, 51G6447. (C) Effect of PTCAF-1 KD on Thon and Sardine transposons. Total DNA was extracted from vegetative (V), control and PTCAF-1 KD cultures at specific time points during sexual reproduction. E, M, L as in Figure 1C; 5, 15, 25 and 45 h after L. After running the DNA on a 1% agarose-gel and transfer to nylon membrane the samples were hybridized with Sardine and Thon transposon-specific probes. A mitochondrial DNA-specific probe was used as loading control. The graph shows the Thon signal quantification normalized to the loading control.
Mentions: Upon PTCAF-1 knockdown (PTCAF-1 KD) we observed a strong decrease of PTCAF-1 mRNA level in the early stages of autogamy (E) compared to control (Figure 1C). To assess whether PtCAF-1 might play a role in Paramecium sexual development, we tested the survival of sexual progeny after PTCAF-1 KD (Figure 2A). Sixty-seven percent of the PTCAF-1 KD cells in our survival test died during a time course of 3 days (up to 12 cell divisions) after the progeny cells were refed and allowed to divide vegetatively (Figure 2A). Twenty-seven percent of the cells did not divide at the normal rate of four divisions per 24 h and 6% showed no growth defects. In the control culture, 94% of the cells grew at normal rates while 6% died. In the positive control (NOWA1 KD), 97% of the cells died and 3% survived with a reduced growth rate. This result was confirmed by using RNAi construct targeting an alternative region of PTCAF-1 (Supplementary Figure S2A). No cell death or reduced division was observed for Paramecium cells grown vegetatively in silencing medium for more than 12 divisions. PTCAF-1 KD did not result in premature termination of development at an early stage as we were able to observe all the developmental stages and the final formation of new developing MACs (data not shown). We saw no structural irregularities during development and there was no notable differences in DNA content between the PTCAF-1 KD cells and the control cells. Our deep sequencing results suggest that the overall scnRNA levels that are detected at the early time point during development are not affected in PTCAF-1 KD compared to control (Figure 4B). This suggests that the expression and functions of Dcl2/3 and Ptiwi01/09 (responsible for the production and protection of scnRNAs) were not affected by PTCAF-1 KD. We confirmed by northern blot that the expression of PTIWI01 is not affected by PTCAF-1 KD (Figure 1C), and that the localization of Ptiwi09-GFP is unchanged by this knockdown (data not shown).

Bottom Line: Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs.PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development.We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Freiestrasse 1, 3012 Bern, Switzerland.

Show MeSH
Related in: MedlinePlus