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. 2010 Feb 15;109(3):553-63.
doi: 10.1002/jcb.22432.

Methylation of histone H3 and H4 by PRMT5 regulates ribosomal RNA gene transcription (V体育平台登录)

Sarmila Majumder  1 , Lapo Alinari, Satavisha Roy, Tyler Miller, Jharna Datta, Said Sif, Robert Baiocchi, Samson T Jacob
Affiliations

Methylation of histone H3 and H4 by PRMT5 regulates ribosomal RNA gene transcription

Sarmila Majumder et al. J Cell Biochem. .

Abstract

In an effort to understand the epigenetic regulation of ribosomal RNA gene (rDNA) expression we have previously demonstrated the role of DNA methyltransferases and methyl CpG binding proteins in rRNA synthesis. Here, we studied the role of protein arginine methyltransferase PRMT5 and the two methylated histones H3R8Me2 and H4R3Me2, in rDNA expression in Epstein Barr virus- transformed primary B-cells (LCLs) and in HeLa cells responding to serum-regulated growth. Chromatin immunoprecipitation assay showed that histones H3 and H4 associated with rRNA promoters were differentially methylated at arginine residues 8 and 3, respectively, depending on its transcriptional activity. Association of PRMT5 and methylated H3 with the unmethylated promoters in resting B-cells was significantly reduced in rapidly growing LCLs. Unlike PRMT5 and H3R8Me2, histone H4 associated with both methylated and unmethylated rRNA promoters in resting B-cells was methylated at the R3 residue. However, a dramatic decrease in R3 methylation of H4 recruited to the unmethylated rRNA promoters was observed in LCLs while it remained unaltered in the fraction bound to the methylated promoters. Differential interaction of PRMT5 and methylation of H3 and H4 associated with the rRNA promoters was also observed when serum starved HeLa cells were allowed to grow in serum replenished media VSports手机版. Ectopic expression of PRMT5 suppressed activity of both unmethylated and methylated rRNA promoter in transient transfection assay whereas siRNA mediated knockdown of PRMT5 increased rRNA synthesis in HeLa cells. These data suggest a key role of PRMT5 and the two methylated histones in regulating rRNA promoter activity. .

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Figures

Fig. 1
Fig. 1
EBV transformation of B-cells results in increased ribosomal RNA synthesis compared to resting B-cells. A: Total RNA was isolated from EBV infected B-cells harvested on Days 0, 4, 8, 16, and 35 and ribosomal gene transcription was measured by real-time PCR. Fold increase in 47S rRNA synthesis was calculated after normalization to β-actin. B: Freshly isolated B-cells from two individual donors were infected with EBV and total RNA was isolated on day 0 and Day 35. 47S rRNA synthesis was analyzed by real-time PCR in B-cells from two individual donors (INF1 and INF2) normalized to β-actin and compared between day 0 and EBV infected B-cell populations on Day 35. C: Total RNA isolated from EBV transformed B-cell lines was subjected to Real-time PCR. 47S rRNA synthesis normalized to b-actin in resting B-cells (B) was compared with that in two different EBV transformed lymphoblastoid cell lines (LCL1 and LCL2). Error bars represents standard deviation of triplicate measurements.
Fig. 2
Fig. 2
UBF expression and association with unmethylated (active) rRNA promoter are not altered during increased rRNA synthesis in EBV infected B-cells. UBF mRNA levels in resting B cells and LCLs (Panel A), and EBV-infected cells (Panel C) were monitored by real-time PCR and protein levels were analyzed by Western blot (Panels B,D). Copy number of UBF was normalized to b-actin and UBF protein level was normalized to GAPDH. E: Schematic diagram of PCR amplified rRNA promoter. F: Resting B-cells and exponentially growing EBV-transformed cells (LCL1, LCL2) were cross-linked with formaldehyde and chromatin prepared from these cells was immunoprecipitated with anti-UBF antibody. DNA pulled down by the antibody as well as input DNA were divided into three identical fractions that were either mock-digested or digested with Hpa II (H) or Msp I (M). An aliquot of the product from each digestion was subjected to QPCR. The reaction products were separated on polyacrylamide and Kodak software was used to quantify the PCR products. Association with methylated promoter = Hpa II signal in ChIP DNA/Hpa II signal in input (1:200 dilution). Association with unmethylated promoter = signal in undigested minus signal in Hpa II digested ChIP DNA/ input signal in undigested minus signal in Hpa II digested DNA (1:200 dilution).
Fig. 3
Fig. 3
Association of PRMT5 with rRNA promoter is reduced in EBV transformed B-cells. A: PRMT5 level was monitored by Western blot analysis in EBV transformed cells (LCLs), EBV-infected and resting B cells. B: Resting B-cells and exponentially growing EBV transformed cells (LCL1, LCL2) were cross-linked with formaldehyde and chromatin from these cells was immunoprecipitated with anti-PRMT5 antibody. The pulled down DNA was either left uncut (U) or digested with Hpa II (H) or Msp I (M). rRNA promoter was amplified from all three sets of precipitated and digested DNA and analyzed by semi-quantitative PCR. The bar diagram represents association of PRMT5 with unmethylated and methylated rRNA promoter (as described in Fig. 2F).
Fig. 4
Fig. 4
A: H3R8Me2 associates preferentially with unmethylated rRNA promoter in resting B-cells, which is reduced in EBV transformed B-cells. Formaldehyde cross-linked chromatin prepared from resting B-cells and exponentially growing EBV transformed cells (LCL1, LCL2) was immunoprecipitated with anti-H3R8Me2 antibody. The pulled down DNA was either left uncut (U) or digested with Hpa II (H) or Msp I (M). rRNA promoter was amplified from all three sets of precipitated and digested DNA and analyzed by semi-quantitative PCR. Association of H3R8Me2 with unmethylated and methylated rRNA promoter is represented in the bar diagram (as described in Fig. 2F). B: H4R3Me2 associates preferentially with methylated rRNA promoter in the EBV transformed B-lymphocytes. Chromatin from resting B-cells and exponentially growing EBV transformed cells (LCL1, LCL2) was immunoprecipitated with anti-H4R3Me2 antibody, and digested with Hpa II (H) or Msp I (M). rRNA promoter was amplified from uncut (U) and restriction enzyme digested DNA by semi-quantitative PCR. H4R3Me2 binding to unmethylated and methylated rRNA promoter is represented in the bar diagram (as described in Fig. 2F).
Fig. 5
Fig. 5
PRMT5 expression remains unaltered in serum starved and serum replenished HeLa cells. A: Exponentially growing HeLa cells were serum starved (0%) for 72 h (−FBS) and then allowed to grow in presence of 10% serum for 3 h. 47S rRNA synthesis in these cells was compared by real time PCR and normalized to β-actin. B: Whole cell extracts from HeLa cells (−FBS and +FBS) were subjected to Western blot analysis with anti-PRMT5 antibody. The blot was reprobed with anti-GAPDH antibody for protein normalization. C: PRMT5 co-localizes with nucleolin in HeLa cell nucleolus. HeLa cells were stained with TRITC tagged mouse monoclonal antibody against nucleolin and with FITC-tagged rabbit polyclonal antibody against PRMT5. The cells were also stained with DAPI and visualized under fluorescence microscope. D: PRMT5 preferentially associates with unmethylated rRNA promoter in serum starved HeLa cells. Formaldehyde crosslinked chromatin prepared from serum starved (−FBS) and serum replenished (+FBS) HeLa cells was immunoprecipitated with anti-PRMT5 antibody (PRMT5) or preimmune sera (Preimn), and rRNA promoter was amplified from immunoprecipitated DNA and input DNA (Input). E: The immunoprecipitated DNA was either left undigested, or digested with Hpa II or Msp I. rRNA promoter was amplified from uncut, Hpa II and Msp I digested DNA by semi-quantitative PCR. The bar diagram represents PRMT5 binding to unmethylated and methylated rRNA promoter (as described in Fig. 2F).
Fig. 6
Fig. 6
H3R8Me2 preferentially associates with the unmethylated rRNA promoter in serum starved HeLa cells and its association is reduced upon serum supplementation. A: Serum starved and serum supplemented HeLa cells were cross-linked with formaldehyde and chromatin prepared from these cells was immunoprecipitated with anti-H3R8Me2 antibody. The pulled down DNA was either left uncut (U) or digested with Hpa II (H) or Msp I (M). rRNA promoter was amplified from all three sets of precipitated and digested DNA and analyzed by semi-quantitative PCR. H3R8Me2 binding to unmethylated and methylated rRNA promoter is represented in the bar diagram (as described in Fig. 2F). B: H4R3Me2 preferentially associates with unmethylated promoter in serum starved and methylated promoter in serum-supplemented cells. Chromatin prepared from serum starved and serum supplemented HeLa cells was immunoprecipitated with anti-H4R3Me2 antibody. The pulled down DNA was either left uncut (U) or digested with Hpa II (H) or Msp I (M). rRNA promoter was amplified from all three sets of precipitated and digested DNA and analyzed by semi-quantitative PCR. H4R3Me2 binding to unmethylated and methylated rRNA promoter is represented after normalization to input DNA (as described in Fig. 2F).
Fig. 7
Fig. 7
Ectopic expression of PRMT5 in HeLa cells inhibits the activity of both unmethylated and methylated rRNA promoter. A: Schematic representation of the rRNA promoter/luciferase reporter plasmid (pHrD-IRES-Luc) used in the transfection study. B: HeLa cells were transiently transfected with Flag tagged PRMT5 expression vector. The expression of PRMT5 protein was monitored by Western blot analysis using anti-Flag M2-monoclonal antibody. C: HeLa cells were transiently transfected with empty vector (0) or 2 and 4 µg of PRMT5 expression vector along with pHrD-IRES-Luc and the internal control pRL-TK. Cells harvested 48 h after transfection were analyzed for luciferase activity and normalized to pRL-TK. D: HeLa cells were transiently transfected with empty vector (0) and 4 µg of PRMT5 expression vector along with unmethylated or methylated pHrD-IRES-Luc and pRL-TK. Cells harvested 48 h after transfection were analyzed for luciferase activity and normalized to pRL-TK. E: HeLa cells were transfected with PRMT5 siRNA or control siRNA and expression of PRMT5 was monitored by western blot analysis at 24, 48, and 72 h after transfection. F: HeLa cells transfected with PRMT5 siRNA or control siRNA were harvested 24 h posttransfection and rRNA synthesis was monitored by real time PCR. Error bars represents standard deviation of triplicate measurements.
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V体育2025版 - References

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