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Histone demethylase LSD1-mediated repression of GATA-2 is critical for erythroid differentiation.

Guo Y, Fu X, Jin Y, Sun J, Liu Y, Huo B, Li X, Hu X - Drug Des Devel Ther (2015)

Bottom Line: Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells.Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the GATA-2 locus and suppresses GATA-2 expression, via histone demethylation.Our data revealed that LSD1 mediates erythroid differentiation, via epigenetic modification of the GATA-2 locus.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jilin University, Changchun, People's Republic of China.

ABSTRACT

Background: The transcription factor GATA-2 is predominantly expressed in hematopoietic stem and progenitor cells and counteracts the erythroid-specific transcription factor GATA-1, to modulate the proliferation and differentiation of hematopoietic cells. During hematopoietic cell differentiation, GATA-2 exhibits dynamic expression patterns, which are regulated by multiple transcription factors.

Methods: Stable LSD1-knockdown cell lines were established by growing murine erythroleukemia (MEL) or mouse embryonic stem cells together with virus particles, in the presence of Polybrene(®) at 4 μg/mL, for 24-48 hours followed by puromycin selection (1 μg/mL) for 2 weeks. Real-time polymerase chain reaction (PCR)-based quantitative chromatin immunoprecipitation (ChIP) analysis was used to test whether the TAL1 transcription factor is bound to 1S promoter in the GATA-2 locus or whether LSD1 colocalizes with TAL1 at the 1S promoter. The sequential ChIP assay was utilized to confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation. Western blot analysis was employed to detect the protein expression. The alamarBlue(®) assay was used to examine the proliferation of the cells, and the absorbance was monitored at optical density (OD) 570 nm and OD 600 nm.

Results: In this study, we showed that LSD1 regulates the expression of GATA-2 during erythroid differentiation. Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells. Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the GATA-2 locus and suppresses GATA-2 expression, via histone demethylation.

Conclusion: Our data revealed that LSD1 mediates erythroid differentiation, via epigenetic modification of the GATA-2 locus.

No MeSH data available.


Related in: MedlinePlus

LSD1-knockdown causes increased H3K4me2 at the GATA-2 locus.Notes: (A) MEL cells were treated with DMSO for the indicated number of days. Cross-linked chromatin DNA was immunoprecipitated with the anti-H3K4me2 antibody and analyzed by PCR with primers specific to the indicated sites in the GATA-2 locus. (B) Cross-linked chromatin DNA from MEL cells with or without LSD1-knockdown was immunoprecipitated with an anti-H3K4me2 antibody and analyzed by PCR with primers specific to the indicated sites in the GATA-2 locus. #P<0.05 represents the relative fold enrichment on day 3 compared with that on day 0. *P<0.05 represents the relative fold enrichment in LSD1-knockdown cells compared with the blank vector cells. This experiment was repeated at least for three times.Abbreviations: DMSO, dimethyl sulfoxide; LSD1-kd, LSD1-knockdown; MEL, murine erythroleukemia; PCR, polymerase chain reaction.
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f4-dddt-9-3153: LSD1-knockdown causes increased H3K4me2 at the GATA-2 locus.Notes: (A) MEL cells were treated with DMSO for the indicated number of days. Cross-linked chromatin DNA was immunoprecipitated with the anti-H3K4me2 antibody and analyzed by PCR with primers specific to the indicated sites in the GATA-2 locus. (B) Cross-linked chromatin DNA from MEL cells with or without LSD1-knockdown was immunoprecipitated with an anti-H3K4me2 antibody and analyzed by PCR with primers specific to the indicated sites in the GATA-2 locus. #P<0.05 represents the relative fold enrichment on day 3 compared with that on day 0. *P<0.05 represents the relative fold enrichment in LSD1-knockdown cells compared with the blank vector cells. This experiment was repeated at least for three times.Abbreviations: DMSO, dimethyl sulfoxide; LSD1-kd, LSD1-knockdown; MEL, murine erythroleukemia; PCR, polymerase chain reaction.

Mentions: LSD1 is an important histone demethylase.34 Our results showed that the enrichment of LSD1 was increased at the 1S promoter in the GATA-2 locus (Figure 3D), suggesting that LSD1 might decrease H3K4me2 at the 1S promoter via its lysine demethylases activity. Thus, we investigated the alteration of histone methylation levels at the GATA-2 locus during erythroid differentiation. The result of ChIP assays demonstrated that H3K4me2 was significantly decreased at −2.8 kb, 1S promoter, and 1G promoter in the GATA-2 locus in DMSO-treated MEL cells on day 3 compared with that on day 0 (P<0.05) (Figure 4A). The decreased H3K4me2 at the GATA-2 locus, particularly at the 1S promoter, was correlated with the decreased expression of GATA-2 during erythroid cell differentiation, suggesting that LSD1 might inhibit the expression of GATA-2 via histone modification. To confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation, we performed ChIP analysis for H3K4me2 enrichment at the GATA-2 locus in LSD1-knockdown MEL cells. H3K4me2 levels were significantly increased in the LSD1-knockdown cells compared with the control vector cells (P<0.05) (Figure 4B), demonstrating that LSD1 is required for the decreased H3K4me2 at the GATA-2 locus during erythroid differentiation.


Histone demethylase LSD1-mediated repression of GATA-2 is critical for erythroid differentiation.

Guo Y, Fu X, Jin Y, Sun J, Liu Y, Huo B, Li X, Hu X - Drug Des Devel Ther (2015)

LSD1-knockdown causes increased H3K4me2 at the GATA-2 locus.Notes: (A) MEL cells were treated with DMSO for the indicated number of days. Cross-linked chromatin DNA was immunoprecipitated with the anti-H3K4me2 antibody and analyzed by PCR with primers specific to the indicated sites in the GATA-2 locus. (B) Cross-linked chromatin DNA from MEL cells with or without LSD1-knockdown was immunoprecipitated with an anti-H3K4me2 antibody and analyzed by PCR with primers specific to the indicated sites in the GATA-2 locus. #P<0.05 represents the relative fold enrichment on day 3 compared with that on day 0. *P<0.05 represents the relative fold enrichment in LSD1-knockdown cells compared with the blank vector cells. This experiment was repeated at least for three times.Abbreviations: DMSO, dimethyl sulfoxide; LSD1-kd, LSD1-knockdown; MEL, murine erythroleukemia; PCR, polymerase chain reaction.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482369&req=5

f4-dddt-9-3153: LSD1-knockdown causes increased H3K4me2 at the GATA-2 locus.Notes: (A) MEL cells were treated with DMSO for the indicated number of days. Cross-linked chromatin DNA was immunoprecipitated with the anti-H3K4me2 antibody and analyzed by PCR with primers specific to the indicated sites in the GATA-2 locus. (B) Cross-linked chromatin DNA from MEL cells with or without LSD1-knockdown was immunoprecipitated with an anti-H3K4me2 antibody and analyzed by PCR with primers specific to the indicated sites in the GATA-2 locus. #P<0.05 represents the relative fold enrichment on day 3 compared with that on day 0. *P<0.05 represents the relative fold enrichment in LSD1-knockdown cells compared with the blank vector cells. This experiment was repeated at least for three times.Abbreviations: DMSO, dimethyl sulfoxide; LSD1-kd, LSD1-knockdown; MEL, murine erythroleukemia; PCR, polymerase chain reaction.
Mentions: LSD1 is an important histone demethylase.34 Our results showed that the enrichment of LSD1 was increased at the 1S promoter in the GATA-2 locus (Figure 3D), suggesting that LSD1 might decrease H3K4me2 at the 1S promoter via its lysine demethylases activity. Thus, we investigated the alteration of histone methylation levels at the GATA-2 locus during erythroid differentiation. The result of ChIP assays demonstrated that H3K4me2 was significantly decreased at −2.8 kb, 1S promoter, and 1G promoter in the GATA-2 locus in DMSO-treated MEL cells on day 3 compared with that on day 0 (P<0.05) (Figure 4A). The decreased H3K4me2 at the GATA-2 locus, particularly at the 1S promoter, was correlated with the decreased expression of GATA-2 during erythroid cell differentiation, suggesting that LSD1 might inhibit the expression of GATA-2 via histone modification. To confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation, we performed ChIP analysis for H3K4me2 enrichment at the GATA-2 locus in LSD1-knockdown MEL cells. H3K4me2 levels were significantly increased in the LSD1-knockdown cells compared with the control vector cells (P<0.05) (Figure 4B), demonstrating that LSD1 is required for the decreased H3K4me2 at the GATA-2 locus during erythroid differentiation.

Bottom Line: Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells.Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the GATA-2 locus and suppresses GATA-2 expression, via histone demethylation.Our data revealed that LSD1 mediates erythroid differentiation, via epigenetic modification of the GATA-2 locus.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Jilin University, Changchun, People's Republic of China.

ABSTRACT

Background: The transcription factor GATA-2 is predominantly expressed in hematopoietic stem and progenitor cells and counteracts the erythroid-specific transcription factor GATA-1, to modulate the proliferation and differentiation of hematopoietic cells. During hematopoietic cell differentiation, GATA-2 exhibits dynamic expression patterns, which are regulated by multiple transcription factors.

Methods: Stable LSD1-knockdown cell lines were established by growing murine erythroleukemia (MEL) or mouse embryonic stem cells together with virus particles, in the presence of Polybrene(®) at 4 μg/mL, for 24-48 hours followed by puromycin selection (1 μg/mL) for 2 weeks. Real-time polymerase chain reaction (PCR)-based quantitative chromatin immunoprecipitation (ChIP) analysis was used to test whether the TAL1 transcription factor is bound to 1S promoter in the GATA-2 locus or whether LSD1 colocalizes with TAL1 at the 1S promoter. The sequential ChIP assay was utilized to confirm the role of LSD1 in the regulation of H3K4me2 at the GATA-2 locus during erythroid differentiation. Western blot analysis was employed to detect the protein expression. The alamarBlue(®) assay was used to examine the proliferation of the cells, and the absorbance was monitored at optical density (OD) 570 nm and OD 600 nm.

Results: In this study, we showed that LSD1 regulates the expression of GATA-2 during erythroid differentiation. Knockdown of LSD1 results in increased GATA-2 expression and inhibits the differentiation of MEL and embryonic stem cells. Furthermore, we demonstrated that LSD1 binds to the 1S promoter of the GATA-2 locus and suppresses GATA-2 expression, via histone demethylation.

Conclusion: Our data revealed that LSD1 mediates erythroid differentiation, via epigenetic modification of the GATA-2 locus.

No MeSH data available.


Related in: MedlinePlus