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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 represses γ-globin gene expression in K562 cells. (A) Western blot analysis with indicated antibodies of cell lysate from LYAR-knockdown 1 (KD1) and LYAR-knockdown 2 (KD2) or scrambled (Scr) control K562 cells. (B) LYAR gene expression analysis by Q-RT-PCR of RNA extracted from LYAR-KD1, LYAR-KD2 and scrambled control (Scr) K562 cells normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (C) γ-globin gene expression analysis by Q-RT-PCR of RNA extracted from LYAR-KD1, LYAR-KD2 and scrambled control (Scr) K562 cells normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (D) LYAR, PRMT5 and histone H4R3me2s ChIP analyses at the γ-globin promoter were performed in Scr or LYAR-knockdown (KD) K562 cells. Results are shown as mean ± SD from three independent experiments. (E) Histone H3K9ac ChIP analysis as in D. *P< 0.05, **P< 0.01 compared to the scrambled control. (F) Western blot analysis of LYAR and PRMT5 from cell lysates of LYAR-KD and Scramble K562 cells.
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Figure 4: LYAR represses γ-globin gene expression in K562 cells. (A) Western blot analysis with indicated antibodies of cell lysate from LYAR-knockdown 1 (KD1) and LYAR-knockdown 2 (KD2) or scrambled (Scr) control K562 cells. (B) LYAR gene expression analysis by Q-RT-PCR of RNA extracted from LYAR-KD1, LYAR-KD2 and scrambled control (Scr) K562 cells normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (C) γ-globin gene expression analysis by Q-RT-PCR of RNA extracted from LYAR-KD1, LYAR-KD2 and scrambled control (Scr) K562 cells normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (D) LYAR, PRMT5 and histone H4R3me2s ChIP analyses at the γ-globin promoter were performed in Scr or LYAR-knockdown (KD) K562 cells. Results are shown as mean ± SD from three independent experiments. (E) Histone H3K9ac ChIP analysis as in D. *P< 0.05, **P< 0.01 compared to the scrambled control. (F) Western blot analysis of LYAR and PRMT5 from cell lysates of LYAR-KD and Scramble K562 cells.

Mentions: To determine the role of LYAR in γ-globin gene regulation, we generated two stable LYAR knockdown K562 cell lines using lentiviral vectors containing specific shRNAs. LYAR protein levels were assessed by western blot (Figure 4A), and γ-globin gene expression was quantified by Q-RT-PCR from total RNA in these cells. The cell growth rate was not changed when LYAR was knocked down in K562 cells (Supplementary Figure S6). As assessed by Q-RT-PCR, LYAR expression levels were reduced in these two cell lines to about 40% of the Scramble control (Figure 4B), and γ-globin expression was significantly increased compared to the scramble (Figure 4C). Of note, other markers of erythroid differentiation of K562 cells, such as CD71 and v-myb, were not increased in LYAR knockdown cells (Supplementary Figure S9A). To test whether PRMT5 binding to the γ-globin promoter was associated with LYAR binding on the γ-globin promoter, we performed ChIP experiments in LYAR knock-down cells. We found that the levels of PRMT5 binding on the promoter were significantly reduced when LYAR protein was reduced (Figure 4D). The histone mark H4R3me2s, which is induced by PRMT5, was consistently decreased in LYAR knockdown cells (Figure 4D). We also observed an increased level of histone mark H3K9 acetylation on the γ-globin promoter in LYAR knockdown cells (Figure 4E). Of note, the level of PRMT5 was not affected in LYAR knockdown cells compared to the Scramble control (Figure 4F). These data indicate that LYAR repressed expression of human γ-globin, and contributed to PRMT5 binding to the γ-globin promoter in K562 cells.


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 represses γ-globin gene expression in K562 cells. (A) Western blot analysis with indicated antibodies of cell lysate from LYAR-knockdown 1 (KD1) and LYAR-knockdown 2 (KD2) or scrambled (Scr) control K562 cells. (B) LYAR gene expression analysis by Q-RT-PCR of RNA extracted from LYAR-KD1, LYAR-KD2 and scrambled control (Scr) K562 cells normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (C) γ-globin gene expression analysis by Q-RT-PCR of RNA extracted from LYAR-KD1, LYAR-KD2 and scrambled control (Scr) K562 cells normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (D) LYAR, PRMT5 and histone H4R3me2s ChIP analyses at the γ-globin promoter were performed in Scr or LYAR-knockdown (KD) K562 cells. Results are shown as mean ± SD from three independent experiments. (E) Histone H3K9ac ChIP analysis as in D. *P< 0.05, **P< 0.01 compared to the scrambled control. (F) Western blot analysis of LYAR and PRMT5 from cell lysates of LYAR-KD and Scramble K562 cells.
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Figure 4: LYAR represses γ-globin gene expression in K562 cells. (A) Western blot analysis with indicated antibodies of cell lysate from LYAR-knockdown 1 (KD1) and LYAR-knockdown 2 (KD2) or scrambled (Scr) control K562 cells. (B) LYAR gene expression analysis by Q-RT-PCR of RNA extracted from LYAR-KD1, LYAR-KD2 and scrambled control (Scr) K562 cells normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (C) γ-globin gene expression analysis by Q-RT-PCR of RNA extracted from LYAR-KD1, LYAR-KD2 and scrambled control (Scr) K562 cells normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (D) LYAR, PRMT5 and histone H4R3me2s ChIP analyses at the γ-globin promoter were performed in Scr or LYAR-knockdown (KD) K562 cells. Results are shown as mean ± SD from three independent experiments. (E) Histone H3K9ac ChIP analysis as in D. *P< 0.05, **P< 0.01 compared to the scrambled control. (F) Western blot analysis of LYAR and PRMT5 from cell lysates of LYAR-KD and Scramble K562 cells.
Mentions: To determine the role of LYAR in γ-globin gene regulation, we generated two stable LYAR knockdown K562 cell lines using lentiviral vectors containing specific shRNAs. LYAR protein levels were assessed by western blot (Figure 4A), and γ-globin gene expression was quantified by Q-RT-PCR from total RNA in these cells. The cell growth rate was not changed when LYAR was knocked down in K562 cells (Supplementary Figure S6). As assessed by Q-RT-PCR, LYAR expression levels were reduced in these two cell lines to about 40% of the Scramble control (Figure 4B), and γ-globin expression was significantly increased compared to the scramble (Figure 4C). Of note, other markers of erythroid differentiation of K562 cells, such as CD71 and v-myb, were not increased in LYAR knockdown cells (Supplementary Figure S9A). To test whether PRMT5 binding to the γ-globin promoter was associated with LYAR binding on the γ-globin promoter, we performed ChIP experiments in LYAR knock-down cells. We found that the levels of PRMT5 binding on the promoter were significantly reduced when LYAR protein was reduced (Figure 4D). The histone mark H4R3me2s, which is induced by PRMT5, was consistently decreased in LYAR knockdown cells (Figure 4D). We also observed an increased level of histone mark H3K9 acetylation on the γ-globin promoter in LYAR knockdown cells (Figure 4E). Of note, the level of PRMT5 was not affected in LYAR knockdown cells compared to the Scramble control (Figure 4F). These data indicate that LYAR repressed expression of human γ-globin, and contributed to PRMT5 binding to the γ-globin promoter in K562 cells.

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