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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 binds the 5′UTR of the γ-globin gene. (A) Schematic representation of a luciferase reporter construct containing either WT or mutant LYAR-binding site. HS2, DNase hypersensitive site 2. (B) Relative luciferase activity assay from 293T cells containing either vector, overexpressed HA-tagged LYAR, FLAG-tagged PRMT5 or both. Histograms show mean ± SD, n = 3. (C) Relative luciferase activity assay as in (B) with mutant promoter. Histograms show mean ± SD, n = 3. **P< 0.01, #P> 0.05 compared to the empty vector. (D) Western blot analysis of HA-tagged LYAR and FLAG-tagged PRMT5 from cell lysates of transfected 293T cells. GADPH was used as a loading control. (E) EMSA competition analysis with the indicated amounts (10-, 50- or 100-fold molar excess) of WT cold probe or mutant (Mut) cold probe competitors of K562 nuclear extract. DNA binding bands are indicated by arrows. Sequences of probes are shown below. (F) EMSA of K562 nuclear extract using anti-LYAR antibody or control rabbit IgG. Super shifted band is indicated.
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Figure 3: LYAR binds the 5′UTR of the γ-globin gene. (A) Schematic representation of a luciferase reporter construct containing either WT or mutant LYAR-binding site. HS2, DNase hypersensitive site 2. (B) Relative luciferase activity assay from 293T cells containing either vector, overexpressed HA-tagged LYAR, FLAG-tagged PRMT5 or both. Histograms show mean ± SD, n = 3. (C) Relative luciferase activity assay as in (B) with mutant promoter. Histograms show mean ± SD, n = 3. **P< 0.01, #P> 0.05 compared to the empty vector. (D) Western blot analysis of HA-tagged LYAR and FLAG-tagged PRMT5 from cell lysates of transfected 293T cells. GADPH was used as a loading control. (E) EMSA competition analysis with the indicated amounts (10-, 50- or 100-fold molar excess) of WT cold probe or mutant (Mut) cold probe competitors of K562 nuclear extract. DNA binding bands are indicated by arrows. Sequences of probes are shown below. (F) EMSA of K562 nuclear extract using anti-LYAR antibody or control rabbit IgG. Super shifted band is indicated.

Mentions: In order to test whether LYAR together with PRMT5 functions to transcriptionally regulate gene activity by binding to GGTTAT, we constructed a luciferase reporter gene driven by DNase hypersensitive site 2 (HS2) of the β-globin locus and a minimal γ-promoter (nucleotide −130 to +40) with either wild-type (WT) or a mutant LYAR-binding site (Figure 3A). When the WT reporter was co-transfected with expression vector containing HA-tagged LYAR or FLAG-tagged PRMT5 or both into 293T cells, a significant decrease in relative luciferase activity was observed compared to the empty vector (Figure 3B). However, no significant change was found when the mutant LYAR-binding site reporter was used (Figure 3C). The expression of the exogenous protein was verified by western blot analysis (Figure 3D). This result demonstrated that LYAR could regulate transcription through binding GGTTAT.


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 binds the 5′UTR of the γ-globin gene. (A) Schematic representation of a luciferase reporter construct containing either WT or mutant LYAR-binding site. HS2, DNase hypersensitive site 2. (B) Relative luciferase activity assay from 293T cells containing either vector, overexpressed HA-tagged LYAR, FLAG-tagged PRMT5 or both. Histograms show mean ± SD, n = 3. (C) Relative luciferase activity assay as in (B) with mutant promoter. Histograms show mean ± SD, n = 3. **P< 0.01, #P> 0.05 compared to the empty vector. (D) Western blot analysis of HA-tagged LYAR and FLAG-tagged PRMT5 from cell lysates of transfected 293T cells. GADPH was used as a loading control. (E) EMSA competition analysis with the indicated amounts (10-, 50- or 100-fold molar excess) of WT cold probe or mutant (Mut) cold probe competitors of K562 nuclear extract. DNA binding bands are indicated by arrows. Sequences of probes are shown below. (F) EMSA of K562 nuclear extract using anti-LYAR antibody or control rabbit IgG. Super shifted band is indicated.
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Related In: Results  -  Collection

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Figure 3: LYAR binds the 5′UTR of the γ-globin gene. (A) Schematic representation of a luciferase reporter construct containing either WT or mutant LYAR-binding site. HS2, DNase hypersensitive site 2. (B) Relative luciferase activity assay from 293T cells containing either vector, overexpressed HA-tagged LYAR, FLAG-tagged PRMT5 or both. Histograms show mean ± SD, n = 3. (C) Relative luciferase activity assay as in (B) with mutant promoter. Histograms show mean ± SD, n = 3. **P< 0.01, #P> 0.05 compared to the empty vector. (D) Western blot analysis of HA-tagged LYAR and FLAG-tagged PRMT5 from cell lysates of transfected 293T cells. GADPH was used as a loading control. (E) EMSA competition analysis with the indicated amounts (10-, 50- or 100-fold molar excess) of WT cold probe or mutant (Mut) cold probe competitors of K562 nuclear extract. DNA binding bands are indicated by arrows. Sequences of probes are shown below. (F) EMSA of K562 nuclear extract using anti-LYAR antibody or control rabbit IgG. Super shifted band is indicated.
Mentions: In order to test whether LYAR together with PRMT5 functions to transcriptionally regulate gene activity by binding to GGTTAT, we constructed a luciferase reporter gene driven by DNase hypersensitive site 2 (HS2) of the β-globin locus and a minimal γ-promoter (nucleotide −130 to +40) with either wild-type (WT) or a mutant LYAR-binding site (Figure 3A). When the WT reporter was co-transfected with expression vector containing HA-tagged LYAR or FLAG-tagged PRMT5 or both into 293T cells, a significant decrease in relative luciferase activity was observed compared to the empty vector (Figure 3B). However, no significant change was found when the mutant LYAR-binding site reporter was used (Figure 3C). The expression of the exogenous protein was verified by western blot analysis (Figure 3D). This result demonstrated that LYAR could regulate transcription through binding GGTTAT.

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