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Human fetal globin gene expression is regulated by LYAR.

Ju J, Wang Y, Liu R, Zhang Y, Xu Z, Wang Y, Wu Y, Liu M, Cerruti L, Zou F, Ma C, Fang M, Tan R, Jane SM, Zhao Q - Nucleic Acids Res. (2014)

Bottom Line: We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent.We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells.Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China.

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LYAR interacts with PRMT5. (A) SimplyBlue Safestain staining of an SDS-PAGE gel of 3x FLAG eluates of anti-Flag antibody immunoprecipitates from K562 cells stably overexpressing PRMT5-Flag. Bands corresponding to Nucleolin, PRMT5, LYAR and MEP50 are indicated. (B) Co-immunoprecipitation of endogenous PRMT5 and LYAR from K562 cells. Asterisk indicates immunoglobulin heavy chains. (C) GST pull-down assay. Purified GST and GST-PRMT5 fusion proteins (bottom panel, Coomassie stain) pre-adsorbed to glutathione-sepharose beads were incubated with 35S-labeled in vitro transcribed and translated LYAR. Specifically bound protein was eluted from washed beads and visualized by autoradiography (top panel) after SDS-PAGE. (D) Purified GST and GST fusion proteins containing amino acids 1–67, 68–153, 154–239 and 240–379 of LYAR (bottom panel, Coomassie stain) pre-adsorbed to glutathione-sepharose beads were incubated with PRMT5-overexpressing K562 cell lysate. Specifically bound protein was visualized by western blot using anti-PRMT5 antibody after SDS-PAGE. (E) Cellular localization of LYAR and PRMT5 in K562 cells was shown by immunofluorescence with anti-LYAR, anti-PRMT5 antibodies and DAPI nuclear counterstaining. Scale bar, 50 μM. (F) ChIP-reChIP (anti-LYAR antibody ChIP followed by anti-PRMT5 antibody ChIP) analysis of LYAR and PRMT5 on the γ-globin promoter or MyoD promoter. Normal rabbit IgG served as the control. Data show mean ± SD from three independent experiments. #P > 0.05, **P< 0.01 compared to the IgG control.
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Figure 1: LYAR interacts with PRMT5. (A) SimplyBlue Safestain staining of an SDS-PAGE gel of 3x FLAG eluates of anti-Flag antibody immunoprecipitates from K562 cells stably overexpressing PRMT5-Flag. Bands corresponding to Nucleolin, PRMT5, LYAR and MEP50 are indicated. (B) Co-immunoprecipitation of endogenous PRMT5 and LYAR from K562 cells. Asterisk indicates immunoglobulin heavy chains. (C) GST pull-down assay. Purified GST and GST-PRMT5 fusion proteins (bottom panel, Coomassie stain) pre-adsorbed to glutathione-sepharose beads were incubated with 35S-labeled in vitro transcribed and translated LYAR. Specifically bound protein was eluted from washed beads and visualized by autoradiography (top panel) after SDS-PAGE. (D) Purified GST and GST fusion proteins containing amino acids 1–67, 68–153, 154–239 and 240–379 of LYAR (bottom panel, Coomassie stain) pre-adsorbed to glutathione-sepharose beads were incubated with PRMT5-overexpressing K562 cell lysate. Specifically bound protein was visualized by western blot using anti-PRMT5 antibody after SDS-PAGE. (E) Cellular localization of LYAR and PRMT5 in K562 cells was shown by immunofluorescence with anti-LYAR, anti-PRMT5 antibodies and DAPI nuclear counterstaining. Scale bar, 50 μM. (F) ChIP-reChIP (anti-LYAR antibody ChIP followed by anti-PRMT5 antibody ChIP) analysis of LYAR and PRMT5 on the γ-globin promoter or MyoD promoter. Normal rabbit IgG served as the control. Data show mean ± SD from three independent experiments. #P > 0.05, **P< 0.01 compared to the IgG control.

Mentions: In previous studies, we have shown that PRMT5 interacts with DNMT3A, thereby coupling DNA methylation and histone arginine methylation on the human γ-promoter to silence γ-globin gene expression (27). To identify other potential proteins interacting with PRMT5, we performed immunoprecipitation experiments using anti-Flag antibody conjugated to sepharose beads to precipitate PRMT5 and associated proteins from cellular extracts of K562 cells stably overexpressing Flag-tagged PRMT5. Proteins were eluted with 3× Flag peptide and subjected to mass spectrometry analysis. We found that, in addition to previously identified PRMT5-interaction partner proteins, such as MEP50 and Nucleolin (33,34), peptides corresponding to a new protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) were also present (Figure 1A) (29). To confirm the interaction between LYAR and PRMT5, endogenous proteins from K562 cells were analyzed by immunoprecipitation and western blot. We found that PRMT5 was co-immunoprecipitated with LYAR protein from K562 cellular extract by anti-LYAR antibody (Figure 1B). Furthermore, GST pull-down assays demonstrated that these proteins interacted directly (Figure 1C), and their interaction was mediated by amino acids 154–239 of LYAR containing a stretch of mostly charged residues (Figure 1D and boxed sequence in Supplementary Figure S3). Immunofluorescence staining demonstrated that LYAR co-localized with PRMT5 in the nucleus (Figure 1E). As PRMT5 was previously demonstrated to bind the γ-globin promoter, we determined if LYAR also bound the γ-globin promoter. ChIP-reChIP experiments showed that LYAR and PRMT5 together bound the γ globin promoter, but did not bind the MyoD promoter (Figure 1F). Furthermore, enriched binding of LYAR occurred around the proximal promoter and the first exon region of the γ-globin gene (Supplementary Figure S4).


Human fetal globin gene expression is regulated by LYAR.

Ju J, Wang Y, Liu R, Zhang Y, Xu Z, Wang Y, Wu Y, Liu M, Cerruti L, Zou F, Ma C, Fang M, Tan R, Jane SM, Zhao Q - Nucleic Acids Res. (2014)

LYAR interacts with PRMT5. (A) SimplyBlue Safestain staining of an SDS-PAGE gel of 3x FLAG eluates of anti-Flag antibody immunoprecipitates from K562 cells stably overexpressing PRMT5-Flag. Bands corresponding to Nucleolin, PRMT5, LYAR and MEP50 are indicated. (B) Co-immunoprecipitation of endogenous PRMT5 and LYAR from K562 cells. Asterisk indicates immunoglobulin heavy chains. (C) GST pull-down assay. Purified GST and GST-PRMT5 fusion proteins (bottom panel, Coomassie stain) pre-adsorbed to glutathione-sepharose beads were incubated with 35S-labeled in vitro transcribed and translated LYAR. Specifically bound protein was eluted from washed beads and visualized by autoradiography (top panel) after SDS-PAGE. (D) Purified GST and GST fusion proteins containing amino acids 1–67, 68–153, 154–239 and 240–379 of LYAR (bottom panel, Coomassie stain) pre-adsorbed to glutathione-sepharose beads were incubated with PRMT5-overexpressing K562 cell lysate. Specifically bound protein was visualized by western blot using anti-PRMT5 antibody after SDS-PAGE. (E) Cellular localization of LYAR and PRMT5 in K562 cells was shown by immunofluorescence with anti-LYAR, anti-PRMT5 antibodies and DAPI nuclear counterstaining. Scale bar, 50 μM. (F) ChIP-reChIP (anti-LYAR antibody ChIP followed by anti-PRMT5 antibody ChIP) analysis of LYAR and PRMT5 on the γ-globin promoter or MyoD promoter. Normal rabbit IgG served as the control. Data show mean ± SD from three independent experiments. #P > 0.05, **P< 0.01 compared to the IgG control.
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Figure 1: LYAR interacts with PRMT5. (A) SimplyBlue Safestain staining of an SDS-PAGE gel of 3x FLAG eluates of anti-Flag antibody immunoprecipitates from K562 cells stably overexpressing PRMT5-Flag. Bands corresponding to Nucleolin, PRMT5, LYAR and MEP50 are indicated. (B) Co-immunoprecipitation of endogenous PRMT5 and LYAR from K562 cells. Asterisk indicates immunoglobulin heavy chains. (C) GST pull-down assay. Purified GST and GST-PRMT5 fusion proteins (bottom panel, Coomassie stain) pre-adsorbed to glutathione-sepharose beads were incubated with 35S-labeled in vitro transcribed and translated LYAR. Specifically bound protein was eluted from washed beads and visualized by autoradiography (top panel) after SDS-PAGE. (D) Purified GST and GST fusion proteins containing amino acids 1–67, 68–153, 154–239 and 240–379 of LYAR (bottom panel, Coomassie stain) pre-adsorbed to glutathione-sepharose beads were incubated with PRMT5-overexpressing K562 cell lysate. Specifically bound protein was visualized by western blot using anti-PRMT5 antibody after SDS-PAGE. (E) Cellular localization of LYAR and PRMT5 in K562 cells was shown by immunofluorescence with anti-LYAR, anti-PRMT5 antibodies and DAPI nuclear counterstaining. Scale bar, 50 μM. (F) ChIP-reChIP (anti-LYAR antibody ChIP followed by anti-PRMT5 antibody ChIP) analysis of LYAR and PRMT5 on the γ-globin promoter or MyoD promoter. Normal rabbit IgG served as the control. Data show mean ± SD from three independent experiments. #P > 0.05, **P< 0.01 compared to the IgG control.
Mentions: In previous studies, we have shown that PRMT5 interacts with DNMT3A, thereby coupling DNA methylation and histone arginine methylation on the human γ-promoter to silence γ-globin gene expression (27). To identify other potential proteins interacting with PRMT5, we performed immunoprecipitation experiments using anti-Flag antibody conjugated to sepharose beads to precipitate PRMT5 and associated proteins from cellular extracts of K562 cells stably overexpressing Flag-tagged PRMT5. Proteins were eluted with 3× Flag peptide and subjected to mass spectrometry analysis. We found that, in addition to previously identified PRMT5-interaction partner proteins, such as MEP50 and Nucleolin (33,34), peptides corresponding to a new protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) were also present (Figure 1A) (29). To confirm the interaction between LYAR and PRMT5, endogenous proteins from K562 cells were analyzed by immunoprecipitation and western blot. We found that PRMT5 was co-immunoprecipitated with LYAR protein from K562 cellular extract by anti-LYAR antibody (Figure 1B). Furthermore, GST pull-down assays demonstrated that these proteins interacted directly (Figure 1C), and their interaction was mediated by amino acids 154–239 of LYAR containing a stretch of mostly charged residues (Figure 1D and boxed sequence in Supplementary Figure S3). Immunofluorescence staining demonstrated that LYAR co-localized with PRMT5 in the nucleus (Figure 1E). As PRMT5 was previously demonstrated to bind the γ-globin promoter, we determined if LYAR also bound the γ-globin promoter. ChIP-reChIP experiments showed that LYAR and PRMT5 together bound the γ globin promoter, but did not bind the MyoD promoter (Figure 1F). Furthermore, enriched binding of LYAR occurred around the proximal promoter and the first exon region of the γ-globin gene (Supplementary Figure S4).

Bottom Line: We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent.We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells.Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China.

Show MeSH
Related in: MedlinePlus