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RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition.

Newhart A, Powers SL, Shastrula PK, Sierra I, Joo LM, Hayden JE, Cohen AR, Janicki SM - Mol. Biol. Cell (2016)

Bottom Line: Rpp29 is a protein subunit of RNase P.Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly.Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, Wistar Institute, Philadelphia, PA 19104.

No MeSH data available.


Related in: MedlinePlus

Rpp29 is not required for maintenance of transcriptional silencing. (A) Strand-specific qRT-PCR analysis of sense and antisense transgene RNA levels in inactive and activated HeLa HI 1-1 and U2OS 2-6-3 cells after shRNA knockdown of Rpp29 and ATRX and ICP0 expression. The pLKO.1 vector was used as a control. A primer pair in rabbit β-globin exon 3 was used for PCR. Results are the average of at least three independent experiments. SDs are shown in the form of error bars; p values were calculated using unpaired t test. (B) Graphs of the average FRAP of H3.3-YFP in HeLa HI 1-1/H3.3-YFP cells over the course of 10 min of imaging. ShRNA constructs were expressed for 72 h before photobleaching: control pLKO.1 (n = 14) and Rpp29+ATRX double knockdown (n = 14). (C) Model of Rpp29 function in histone H3.3 chromatin assembly at a DAXX-ATRX–regulated site, showing two steps: 1) H3.3 recruitment through a transcriptional signal that Rpp29 functions to down-regulate, and 2) nucleosomal deposition of H3.3 by DAXX and ATRX.
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Figure 9: Rpp29 is not required for maintenance of transcriptional silencing. (A) Strand-specific qRT-PCR analysis of sense and antisense transgene RNA levels in inactive and activated HeLa HI 1-1 and U2OS 2-6-3 cells after shRNA knockdown of Rpp29 and ATRX and ICP0 expression. The pLKO.1 vector was used as a control. A primer pair in rabbit β-globin exon 3 was used for PCR. Results are the average of at least three independent experiments. SDs are shown in the form of error bars; p values were calculated using unpaired t test. (B) Graphs of the average FRAP of H3.3-YFP in HeLa HI 1-1/H3.3-YFP cells over the course of 10 min of imaging. ShRNA constructs were expressed for 72 h before photobleaching: control pLKO.1 (n = 14) and Rpp29+ATRX double knockdown (n = 14). (C) Model of Rpp29 function in histone H3.3 chromatin assembly at a DAXX-ATRX–regulated site, showing two steps: 1) H3.3 recruitment through a transcriptional signal that Rpp29 functions to down-regulate, and 2) nucleosomal deposition of H3.3 by DAXX and ATRX.

Mentions: To decipher the link between H3.3 chromatin incorporation and transcriptional silencing, we knocked Rpp29 down in the HeLa cell line and measured S and AS transgene RNA levels 3 h after activation (Figure 9A). Compared to control cells (pLKO.1), Rpp29 knockdown did not significantly increase S and AS RNA expression, which suggests that Rpp29 is not required to maintain silencing. In fact, the increase in H3.3 deposition that accompanies Rpp29 knockdown (Figure 8E) may suppress array activation, because H3.3 chromatin assembly is required for the establishment of heterochromatic histone PTMs, including H3K27me3 at HIRA-regulated bivalent genes (Banaszynski et al., 2013) and H3K9me3 at DAXX-ATRX–regulated sites (Elsasser et al., 2015; He et al., 2015; Jang et al., 2015; Udugama et al., 2015; Voon et al., 2015).


RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition.

Newhart A, Powers SL, Shastrula PK, Sierra I, Joo LM, Hayden JE, Cohen AR, Janicki SM - Mol. Biol. Cell (2016)

Rpp29 is not required for maintenance of transcriptional silencing. (A) Strand-specific qRT-PCR analysis of sense and antisense transgene RNA levels in inactive and activated HeLa HI 1-1 and U2OS 2-6-3 cells after shRNA knockdown of Rpp29 and ATRX and ICP0 expression. The pLKO.1 vector was used as a control. A primer pair in rabbit β-globin exon 3 was used for PCR. Results are the average of at least three independent experiments. SDs are shown in the form of error bars; p values were calculated using unpaired t test. (B) Graphs of the average FRAP of H3.3-YFP in HeLa HI 1-1/H3.3-YFP cells over the course of 10 min of imaging. ShRNA constructs were expressed for 72 h before photobleaching: control pLKO.1 (n = 14) and Rpp29+ATRX double knockdown (n = 14). (C) Model of Rpp29 function in histone H3.3 chromatin assembly at a DAXX-ATRX–regulated site, showing two steps: 1) H3.3 recruitment through a transcriptional signal that Rpp29 functions to down-regulate, and 2) nucleosomal deposition of H3.3 by DAXX and ATRX.
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Related In: Results  -  Collection

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Figure 9: Rpp29 is not required for maintenance of transcriptional silencing. (A) Strand-specific qRT-PCR analysis of sense and antisense transgene RNA levels in inactive and activated HeLa HI 1-1 and U2OS 2-6-3 cells after shRNA knockdown of Rpp29 and ATRX and ICP0 expression. The pLKO.1 vector was used as a control. A primer pair in rabbit β-globin exon 3 was used for PCR. Results are the average of at least three independent experiments. SDs are shown in the form of error bars; p values were calculated using unpaired t test. (B) Graphs of the average FRAP of H3.3-YFP in HeLa HI 1-1/H3.3-YFP cells over the course of 10 min of imaging. ShRNA constructs were expressed for 72 h before photobleaching: control pLKO.1 (n = 14) and Rpp29+ATRX double knockdown (n = 14). (C) Model of Rpp29 function in histone H3.3 chromatin assembly at a DAXX-ATRX–regulated site, showing two steps: 1) H3.3 recruitment through a transcriptional signal that Rpp29 functions to down-regulate, and 2) nucleosomal deposition of H3.3 by DAXX and ATRX.
Mentions: To decipher the link between H3.3 chromatin incorporation and transcriptional silencing, we knocked Rpp29 down in the HeLa cell line and measured S and AS transgene RNA levels 3 h after activation (Figure 9A). Compared to control cells (pLKO.1), Rpp29 knockdown did not significantly increase S and AS RNA expression, which suggests that Rpp29 is not required to maintain silencing. In fact, the increase in H3.3 deposition that accompanies Rpp29 knockdown (Figure 8E) may suppress array activation, because H3.3 chromatin assembly is required for the establishment of heterochromatic histone PTMs, including H3K27me3 at HIRA-regulated bivalent genes (Banaszynski et al., 2013) and H3K9me3 at DAXX-ATRX–regulated sites (Elsasser et al., 2015; He et al., 2015; Jang et al., 2015; Udugama et al., 2015; Voon et al., 2015).

Bottom Line: Rpp29 is a protein subunit of RNase P.Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly.Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, Wistar Institute, Philadelphia, PA 19104.

No MeSH data available.


Related in: MedlinePlus