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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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Comparison of H3K27me3 signals between control, PTCAF-1 KD and NOWA1 KD cells. (A) H3K27me3 signal in the parental MAC. (B) H3K27me3 signal in the parental MAC after fragmentation. (C) H3K27me3 signal in the developing MACs. Green: H3K27me3; blue: DAPI; m: parental macronucleus; dotted circles: developing macronucleus; scale bar: 10 μm. Control: cells silenced with empty L4440 vector, NOWA1 KD: NOWA1 knockdown, PTCAF-1 KD: PTCAF-1 knockdown.
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Figure 6: Comparison of H3K27me3 signals between control, PTCAF-1 KD and NOWA1 KD cells. (A) H3K27me3 signal in the parental MAC. (B) H3K27me3 signal in the parental MAC after fragmentation. (C) H3K27me3 signal in the developing MACs. Green: H3K27me3; blue: DAPI; m: parental macronucleus; dotted circles: developing macronucleus; scale bar: 10 μm. Control: cells silenced with empty L4440 vector, NOWA1 KD: NOWA1 knockdown, PTCAF-1 KD: PTCAF-1 knockdown.

Mentions: As in T. thermophila, where H3K9me3 and H3K27me3 are required for heterochromatin formation prior to programmed DNA elimination (38,42) we observed the appearance of both these histone modifications during development in P. tetraurelia. Immunofluorescent labelling of control cells with H3K9me3- and H3K27me3-specific antibodies showed very low levels of these modifications in the vegetative MAC (Figures 5A and 6A). Higher levels of both modifications were detected in the fragments of the parental MAC at the beginning of sexual development (Figures 5B and 6B) and even higher levels were observed in the developing MACs (Figures 5C and 6C). Since PtCAF-1 has a histone binding domain, we were interested to see if it might be involved in the setting of these histone modifications. At the same time we were also interested to see if NOWA1 KD would affect H3K9me3 and H3K27me3. No significant difference in H3K9me3/K27me3 level was detected in the parental MAC between control, PTCAF-1 KD and NOWA1 KD (Figures 5A and 6A). In the fragments of the old MAC the signals for H3K9me3/K27me3 were strongly reduced in the PTCAF-1 KD and NOWA1 KD cells (Figures 5B and 6B). In the developing macronuclei, where both modifications are normally present at the highest level, the signals in both knockdown experiments were also strongly reduced (Figures 5C and 6C). Although both knockdowns did not lead to 100% lethality in progeny cells, the changes of the levels of H3K9me3 and H3K27me3 were visible in all cells at the corresponding life stages. These observations suggest that the absence of either protein may lead to the reduction of H3K9me3/K27me3 in both parental fragments and the developing MACs.


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)

Comparison of H3K27me3 signals between control, PTCAF-1 KD and NOWA1 KD cells. (A) H3K27me3 signal in the parental MAC. (B) H3K27me3 signal in the parental MAC after fragmentation. (C) H3K27me3 signal in the developing MACs. Green: H3K27me3; blue: DAPI; m: parental macronucleus; dotted circles: developing macronucleus; scale bar: 10 μm. Control: cells silenced with empty L4440 vector, NOWA1 KD: NOWA1 knockdown, PTCAF-1 KD: PTCAF-1 knockdown.
© Copyright Policy - creative-commons
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Figure 6: Comparison of H3K27me3 signals between control, PTCAF-1 KD and NOWA1 KD cells. (A) H3K27me3 signal in the parental MAC. (B) H3K27me3 signal in the parental MAC after fragmentation. (C) H3K27me3 signal in the developing MACs. Green: H3K27me3; blue: DAPI; m: parental macronucleus; dotted circles: developing macronucleus; scale bar: 10 μm. Control: cells silenced with empty L4440 vector, NOWA1 KD: NOWA1 knockdown, PTCAF-1 KD: PTCAF-1 knockdown.
Mentions: As in T. thermophila, where H3K9me3 and H3K27me3 are required for heterochromatin formation prior to programmed DNA elimination (38,42) we observed the appearance of both these histone modifications during development in P. tetraurelia. Immunofluorescent labelling of control cells with H3K9me3- and H3K27me3-specific antibodies showed very low levels of these modifications in the vegetative MAC (Figures 5A and 6A). Higher levels of both modifications were detected in the fragments of the parental MAC at the beginning of sexual development (Figures 5B and 6B) and even higher levels were observed in the developing MACs (Figures 5C and 6C). Since PtCAF-1 has a histone binding domain, we were interested to see if it might be involved in the setting of these histone modifications. At the same time we were also interested to see if NOWA1 KD would affect H3K9me3 and H3K27me3. No significant difference in H3K9me3/K27me3 level was detected in the parental MAC between control, PTCAF-1 KD and NOWA1 KD (Figures 5A and 6A). In the fragments of the old MAC the signals for H3K9me3/K27me3 were strongly reduced in the PTCAF-1 KD and NOWA1 KD cells (Figures 5B and 6B). In the developing macronuclei, where both modifications are normally present at the highest level, the signals in both knockdown experiments were also strongly reduced (Figures 5C and 6C). Although both knockdowns did not lead to 100% lethality in progeny cells, the changes of the levels of H3K9me3 and H3K27me3 were visible in all cells at the corresponding life stages. These observations suggest that the absence of either protein may lead to the reduction of H3K9me3/K27me3 in both parental fragments and the developing MACs.

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