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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

Histone H3.3 forms a complex with Rpp29, fibrillarin, and RPL23a. (A) Localization of YFP-Rpp29, YFP-fibrillarin, and YFP-RPL23a in relation to the inactive transgene array in U2OS 2-6-3 cells marked by Cherry-lac repressor (arrows). Scale bar, 5 μm, 1 μm (enlarged inset). (B) Localization of YFP-Rpp29 (a–d), YFP-Fibrillarin (e–h) and YFP-RPL23a (i–l) in relation to H3.3-CFP and the activator, Cherry-tTA-ER, which is shown in the top enlarged insets in c, g, and k. Yellow lines in enlarged merge insets (bottom inset, c, g, and k) show the path through which the red, green, and blue intensities were measured in the intensity profiles (d, h, and l). Asterisks mark the start of the measured line. (C) Pearson’s r analysis of the overlap between YFP-tagged proteins and Cherry-tTA-ER (white bars) and H3.3-CFP (gray bars) at the activated transgene array. The correlation between Cherry-tTA-ER and YFP-tTA-ER (n = 10) was analyzed as a positive control. Cherry-tTA-ER and H3.3-YFP (n = 11) were analyzed as a negative control. Rpp29 (n = 11), fibrillarin (n = 10), and RPL23a (n = 13) were compared with both Cherry-tTA-ER and H3.3-CFP. (D) Analyses of interactions between YFP-tagged proteins, detected with α-GFP antibody, and the bacterially expressed GST proteins, GST, GST-H3.3 (N-tail-αN) wild type (WT), and the 4-PTM construct (diagram in Figure 1B), detected by colloidal blue staining. Right, analysis of the effects of RNase A, RNase III, and DNase I treatments on binding.
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Figure 3: Histone H3.3 forms a complex with Rpp29, fibrillarin, and RPL23a. (A) Localization of YFP-Rpp29, YFP-fibrillarin, and YFP-RPL23a in relation to the inactive transgene array in U2OS 2-6-3 cells marked by Cherry-lac repressor (arrows). Scale bar, 5 μm, 1 μm (enlarged inset). (B) Localization of YFP-Rpp29 (a–d), YFP-Fibrillarin (e–h) and YFP-RPL23a (i–l) in relation to H3.3-CFP and the activator, Cherry-tTA-ER, which is shown in the top enlarged insets in c, g, and k. Yellow lines in enlarged merge insets (bottom inset, c, g, and k) show the path through which the red, green, and blue intensities were measured in the intensity profiles (d, h, and l). Asterisks mark the start of the measured line. (C) Pearson’s r analysis of the overlap between YFP-tagged proteins and Cherry-tTA-ER (white bars) and H3.3-CFP (gray bars) at the activated transgene array. The correlation between Cherry-tTA-ER and YFP-tTA-ER (n = 10) was analyzed as a positive control. Cherry-tTA-ER and H3.3-YFP (n = 11) were analyzed as a negative control. Rpp29 (n = 11), fibrillarin (n = 10), and RPL23a (n = 13) were compared with both Cherry-tTA-ER and H3.3-CFP. (D) Analyses of interactions between YFP-tagged proteins, detected with α-GFP antibody, and the bacterially expressed GST proteins, GST, GST-H3.3 (N-tail-αN) wild type (WT), and the 4-PTM construct (diagram in Figure 1B), detected by colloidal blue staining. Right, analysis of the effects of RNase A, RNase III, and DNase I treatments on binding.

Mentions: Histone H3.3 is enriched in nucleoli before being incorporated into chromatin. (A) Diagram of the inducible transgene drawn to scale. Cherry-lac repressor allows the transgene integration site to be visualized. Transcription is induced from the minimal CMV promoter by the activators Cherry-tTA-ER (+)4-OHT and rtTA (+)Dox. The transcribed RNA encodes CFP fused to a peroxisomal targeting signal (SKL). The RNA is visualized by YFP-MS2, which binds to the stem-loops in the transcript. The 3′ end of the transcription unit is composed of the intron 2 splicing unit from the rabbit β-globin gene. The recruitment of YFP-tagged factors to the array can be monitored by coexpression with the DNA- and RNA-binding proteins. (B) Diagram of the H3.3 constructs expressed as YFP- or GST-fusion proteins in the recruitment and in vitro binding assays (Figure 3D). The amino acid differences between H3.3 and H3.2/H3.21 are shown in red. The red asterisks in the 4-PTM construct represent K37A, R42A, R49A, and R52A. (C) Localization of H3.3-YFP, expressed in U2OS 2-6-3 cells for 18 (a–d) and 48 h (e–h), in relation to the activated transgene array, marked by Cherry-tTA-ER. Localization of H3.3 N-tail-αN-YFP expressed for 18 (i–l) and 48 h (m–p). Arrows indicate the transgene array, and arrowheads indicate nucleoli. Yellow lines in enlarged merge insets (c, g, k, and o) show the path through which the red and green signals were measured in the intensity profiles (d, h, l, and p). Asterisks mark the start of the measured line. Scale bar, 5 μm, 1 μm (enlarged inset). (D) Percentage of transcriptionally activated cells with enrichment of H3.3-YFP and H3.3 N-tail-αN-YFP at the active transcription site and in nucleoli 18 and 48 h posttransfection. For each time point, 100 cells were counted from three independent transfections. SDs are shown in the form of error bars; p values were calculated using the unpaired t test.


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)

Histone H3.3 forms a complex with Rpp29, fibrillarin, and RPL23a. (A) Localization of YFP-Rpp29, YFP-fibrillarin, and YFP-RPL23a in relation to the inactive transgene array in U2OS 2-6-3 cells marked by Cherry-lac repressor (arrows). Scale bar, 5 μm, 1 μm (enlarged inset). (B) Localization of YFP-Rpp29 (a–d), YFP-Fibrillarin (e–h) and YFP-RPL23a (i–l) in relation to H3.3-CFP and the activator, Cherry-tTA-ER, which is shown in the top enlarged insets in c, g, and k. Yellow lines in enlarged merge insets (bottom inset, c, g, and k) show the path through which the red, green, and blue intensities were measured in the intensity profiles (d, h, and l). Asterisks mark the start of the measured line. (C) Pearson’s r analysis of the overlap between YFP-tagged proteins and Cherry-tTA-ER (white bars) and H3.3-CFP (gray bars) at the activated transgene array. The correlation between Cherry-tTA-ER and YFP-tTA-ER (n = 10) was analyzed as a positive control. Cherry-tTA-ER and H3.3-YFP (n = 11) were analyzed as a negative control. Rpp29 (n = 11), fibrillarin (n = 10), and RPL23a (n = 13) were compared with both Cherry-tTA-ER and H3.3-CFP. (D) Analyses of interactions between YFP-tagged proteins, detected with α-GFP antibody, and the bacterially expressed GST proteins, GST, GST-H3.3 (N-tail-αN) wild type (WT), and the 4-PTM construct (diagram in Figure 1B), detected by colloidal blue staining. Right, analysis of the effects of RNase A, RNase III, and DNase I treatments on binding.
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Figure 3: Histone H3.3 forms a complex with Rpp29, fibrillarin, and RPL23a. (A) Localization of YFP-Rpp29, YFP-fibrillarin, and YFP-RPL23a in relation to the inactive transgene array in U2OS 2-6-3 cells marked by Cherry-lac repressor (arrows). Scale bar, 5 μm, 1 μm (enlarged inset). (B) Localization of YFP-Rpp29 (a–d), YFP-Fibrillarin (e–h) and YFP-RPL23a (i–l) in relation to H3.3-CFP and the activator, Cherry-tTA-ER, which is shown in the top enlarged insets in c, g, and k. Yellow lines in enlarged merge insets (bottom inset, c, g, and k) show the path through which the red, green, and blue intensities were measured in the intensity profiles (d, h, and l). Asterisks mark the start of the measured line. (C) Pearson’s r analysis of the overlap between YFP-tagged proteins and Cherry-tTA-ER (white bars) and H3.3-CFP (gray bars) at the activated transgene array. The correlation between Cherry-tTA-ER and YFP-tTA-ER (n = 10) was analyzed as a positive control. Cherry-tTA-ER and H3.3-YFP (n = 11) were analyzed as a negative control. Rpp29 (n = 11), fibrillarin (n = 10), and RPL23a (n = 13) were compared with both Cherry-tTA-ER and H3.3-CFP. (D) Analyses of interactions between YFP-tagged proteins, detected with α-GFP antibody, and the bacterially expressed GST proteins, GST, GST-H3.3 (N-tail-αN) wild type (WT), and the 4-PTM construct (diagram in Figure 1B), detected by colloidal blue staining. Right, analysis of the effects of RNase A, RNase III, and DNase I treatments on binding.
Mentions: Histone H3.3 is enriched in nucleoli before being incorporated into chromatin. (A) Diagram of the inducible transgene drawn to scale. Cherry-lac repressor allows the transgene integration site to be visualized. Transcription is induced from the minimal CMV promoter by the activators Cherry-tTA-ER (+)4-OHT and rtTA (+)Dox. The transcribed RNA encodes CFP fused to a peroxisomal targeting signal (SKL). The RNA is visualized by YFP-MS2, which binds to the stem-loops in the transcript. The 3′ end of the transcription unit is composed of the intron 2 splicing unit from the rabbit β-globin gene. The recruitment of YFP-tagged factors to the array can be monitored by coexpression with the DNA- and RNA-binding proteins. (B) Diagram of the H3.3 constructs expressed as YFP- or GST-fusion proteins in the recruitment and in vitro binding assays (Figure 3D). The amino acid differences between H3.3 and H3.2/H3.21 are shown in red. The red asterisks in the 4-PTM construct represent K37A, R42A, R49A, and R52A. (C) Localization of H3.3-YFP, expressed in U2OS 2-6-3 cells for 18 (a–d) and 48 h (e–h), in relation to the activated transgene array, marked by Cherry-tTA-ER. Localization of H3.3 N-tail-αN-YFP expressed for 18 (i–l) and 48 h (m–p). Arrows indicate the transgene array, and arrowheads indicate nucleoli. Yellow lines in enlarged merge insets (c, g, k, and o) show the path through which the red and green signals were measured in the intensity profiles (d, h, l, and p). Asterisks mark the start of the measured line. Scale bar, 5 μm, 1 μm (enlarged inset). (D) Percentage of transcriptionally activated cells with enrichment of H3.3-YFP and H3.3 N-tail-αN-YFP at the active transcription site and in nucleoli 18 and 48 h posttransfection. For each time point, 100 cells were counted from three independent transfections. SDs are shown in the form of error bars; p values were calculated using the unpaired t test.

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