Limits...
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.

Show MeSH

Related in: MedlinePlus

Role of LYAR in developmental globin gene silencing. (A) Western blot analysis using indicated antibodies of cell lysate from human erythroid progenitor cells of CB and adult peripheral blood (AE). (B) Q-RT-PCR analysis of α-globin, β-globin, γ-globin and LYAR gene expression from CB erythroid progenitor cells overexpressing LYAR (LYAR-OE) or containing vector normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (C) Western blot analysis using indicated antibodies of cell lysate from human erythroid progenitor cells of CB overexpressing LYAR or containing vector. (D) Q-RT-PCR analysis of α-globin, γ-globin, β-globin or LYAR gene expression from human AE progenitor cells of LYAR-knockdown (KD) or scrambled control (AE) normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (E) Western blot analysis of LYAR and Hsp70 from cell lysates of Scramble and LYAR-KD erythroid progenitor cells. (F) Hemoglobin analysis shows percentage of HbF from Scramble and LYAR-KD erythroid progenitor cells. (G) Wright-Giemsa-stained AE progenitor cells at day 12 of differentiation. Scale bar, 50 μM. (H) Flow cytometric analysis for CD71 and GPA expression of Scramble and LYAR-KD erythroid progenitor cells on days 6 and 12 of differentiation. (I) Localization of LYAR across the β-globin locus measured by ChIP in chromatin fractions from AE progenitors. The precipitated DNA was amplified with primers specific for the indicated regions of the β-globin locus. HS, hypersensitive site; pro, promoter; G/Aγ, intergenic region between Gγ- and Aγ-globin genes. Results are shown as mean ± SD from three independent experiments. #P> 0.05, *P< 0.05, **P< 0.01 compared to IgG control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150809&req=5

Figure 6: Role of LYAR in developmental globin gene silencing. (A) Western blot analysis using indicated antibodies of cell lysate from human erythroid progenitor cells of CB and adult peripheral blood (AE). (B) Q-RT-PCR analysis of α-globin, β-globin, γ-globin and LYAR gene expression from CB erythroid progenitor cells overexpressing LYAR (LYAR-OE) or containing vector normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (C) Western blot analysis using indicated antibodies of cell lysate from human erythroid progenitor cells of CB overexpressing LYAR or containing vector. (D) Q-RT-PCR analysis of α-globin, γ-globin, β-globin or LYAR gene expression from human AE progenitor cells of LYAR-knockdown (KD) or scrambled control (AE) normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (E) Western blot analysis of LYAR and Hsp70 from cell lysates of Scramble and LYAR-KD erythroid progenitor cells. (F) Hemoglobin analysis shows percentage of HbF from Scramble and LYAR-KD erythroid progenitor cells. (G) Wright-Giemsa-stained AE progenitor cells at day 12 of differentiation. Scale bar, 50 μM. (H) Flow cytometric analysis for CD71 and GPA expression of Scramble and LYAR-KD erythroid progenitor cells on days 6 and 12 of differentiation. (I) Localization of LYAR across the β-globin locus measured by ChIP in chromatin fractions from AE progenitors. The precipitated DNA was amplified with primers specific for the indicated regions of the β-globin locus. HS, hypersensitive site; pro, promoter; G/Aγ, intergenic region between Gγ- and Aγ-globin genes. Results are shown as mean ± SD from three independent experiments. #P> 0.05, *P< 0.05, **P< 0.01 compared to IgG control.

Mentions: Human γ-globin gene expression is developmentally regulated, and is gradually silenced after birth. To examine the possibility that LYAR plays a role in this process, western blot analysis was performed using an anti-LYAR antibody in human CB and AE progenitor cells. High levels of LYAR were observed in AE progenitor cells (γ-globin ‘off’ state), whereas barely detectable levels of LYAR were found in CB cells (γ-globin ‘on’ state) (Figure 6A). This is consistent with the putative role of LYAR as a repressor of γ-globin expression.


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)

Role of LYAR in developmental globin gene silencing. (A) Western blot analysis using indicated antibodies of cell lysate from human erythroid progenitor cells of CB and adult peripheral blood (AE). (B) Q-RT-PCR analysis of α-globin, β-globin, γ-globin and LYAR gene expression from CB erythroid progenitor cells overexpressing LYAR (LYAR-OE) or containing vector normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (C) Western blot analysis using indicated antibodies of cell lysate from human erythroid progenitor cells of CB overexpressing LYAR or containing vector. (D) Q-RT-PCR analysis of α-globin, γ-globin, β-globin or LYAR gene expression from human AE progenitor cells of LYAR-knockdown (KD) or scrambled control (AE) normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (E) Western blot analysis of LYAR and Hsp70 from cell lysates of Scramble and LYAR-KD erythroid progenitor cells. (F) Hemoglobin analysis shows percentage of HbF from Scramble and LYAR-KD erythroid progenitor cells. (G) Wright-Giemsa-stained AE progenitor cells at day 12 of differentiation. Scale bar, 50 μM. (H) Flow cytometric analysis for CD71 and GPA expression of Scramble and LYAR-KD erythroid progenitor cells on days 6 and 12 of differentiation. (I) Localization of LYAR across the β-globin locus measured by ChIP in chromatin fractions from AE progenitors. The precipitated DNA was amplified with primers specific for the indicated regions of the β-globin locus. HS, hypersensitive site; pro, promoter; G/Aγ, intergenic region between Gγ- and Aγ-globin genes. Results are shown as mean ± SD from three independent experiments. #P> 0.05, *P< 0.05, **P< 0.01 compared to IgG control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150809&req=5

Figure 6: Role of LYAR in developmental globin gene silencing. (A) Western blot analysis using indicated antibodies of cell lysate from human erythroid progenitor cells of CB and adult peripheral blood (AE). (B) Q-RT-PCR analysis of α-globin, β-globin, γ-globin and LYAR gene expression from CB erythroid progenitor cells overexpressing LYAR (LYAR-OE) or containing vector normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (C) Western blot analysis using indicated antibodies of cell lysate from human erythroid progenitor cells of CB overexpressing LYAR or containing vector. (D) Q-RT-PCR analysis of α-globin, γ-globin, β-globin or LYAR gene expression from human AE progenitor cells of LYAR-knockdown (KD) or scrambled control (AE) normalized to β-actin mRNA. Results are shown as mean ± SD from three independent experiments. (E) Western blot analysis of LYAR and Hsp70 from cell lysates of Scramble and LYAR-KD erythroid progenitor cells. (F) Hemoglobin analysis shows percentage of HbF from Scramble and LYAR-KD erythroid progenitor cells. (G) Wright-Giemsa-stained AE progenitor cells at day 12 of differentiation. Scale bar, 50 μM. (H) Flow cytometric analysis for CD71 and GPA expression of Scramble and LYAR-KD erythroid progenitor cells on days 6 and 12 of differentiation. (I) Localization of LYAR across the β-globin locus measured by ChIP in chromatin fractions from AE progenitors. The precipitated DNA was amplified with primers specific for the indicated regions of the β-globin locus. HS, hypersensitive site; pro, promoter; G/Aγ, intergenic region between Gγ- and Aγ-globin genes. Results are shown as mean ± SD from three independent experiments. #P> 0.05, *P< 0.05, **P< 0.01 compared to IgG control.
Mentions: Human γ-globin gene expression is developmentally regulated, and is gradually silenced after birth. To examine the possibility that LYAR plays a role in this process, western blot analysis was performed using an anti-LYAR antibody in human CB and AE progenitor cells. High levels of LYAR were observed in AE progenitor cells (γ-globin ‘off’ state), whereas barely detectable levels of LYAR were found in CB cells (γ-globin ‘on’ state) (Figure 6A). This is consistent with the putative role of LYAR as a repressor of γ-globin expression.

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