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Regulatory role of G9a and LSD1 in the Transcription of Olfactory Receptors during Leukaemia Cell Differentiation

View Article: PubMed Central - PubMed

ABSTRACT

Recent studies have reported the ectopic expression of olfactory receptors (ORs) in non-olfactory tissues, however, their physiological roles were not well elucidated. ORs are expressed in and function in different types of cancers. Here, we identified that the H3K9me2 levels of several OR promoters decreased during differentiation in the HL-60, human myeloid leukaemia cell line, by all-trans-retinoic acid (ATRA). We found that the differential OR promoters H3K9me2 levels were regulated by G9a and LSD1, resulting in the decrease of ORs transcription during HL-60 differentiation. G9a and LSD1 could regulate the expression of ORs in several non-olfactory cells via the methylation and demethylation of H3K9me2. In addition, we demonstrated that knockdown of OR significantly reduced cell proliferation. Therefore, the epigenetic regulation of ORs transcription is critical for carcinogenesis.

No MeSH data available.


G9a and LSD1 regulate OR expression through the methylation and demethylation of H3K9.(A) ChIP analyses of the OR1N1 and OR10G2 promoters in ATRA-treated HL-60 cells were conducted using anti-G9a, anti-LSD1, anti-H3K4me2, anti-H3K9me2, and anti-IgG antibodies and were examined via qRT-PCR analyses. (B,C) HL-60 cells were treated with BIX01294 (5 μM) or GSK-LSD1 (500 nM) for 48 or 24 h, respectively. (B) ChIP analyses of the OR1N1 and OR10G2 promoters were performed using anti-G9a, anti-H3K9me2, and anti-IgG antibodies and examined by qRT-PCR analyses. (C) Using anti-LSD1, anti-H3K4me2, anti-H3K9me2, and anti-IgG antibodies, ChIP analyses were performed. The results were analysed by qRT-PCR. (A–C) These results are shown as mean ± SDs. (n = 3).
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f3: G9a and LSD1 regulate OR expression through the methylation and demethylation of H3K9.(A) ChIP analyses of the OR1N1 and OR10G2 promoters in ATRA-treated HL-60 cells were conducted using anti-G9a, anti-LSD1, anti-H3K4me2, anti-H3K9me2, and anti-IgG antibodies and were examined via qRT-PCR analyses. (B,C) HL-60 cells were treated with BIX01294 (5 μM) or GSK-LSD1 (500 nM) for 48 or 24 h, respectively. (B) ChIP analyses of the OR1N1 and OR10G2 promoters were performed using anti-G9a, anti-H3K9me2, and anti-IgG antibodies and examined by qRT-PCR analyses. (C) Using anti-LSD1, anti-H3K4me2, anti-H3K9me2, and anti-IgG antibodies, ChIP analyses were performed. The results were analysed by qRT-PCR. (A–C) These results are shown as mean ± SDs. (n = 3).

Mentions: During ATRA-mediated HL-60 differentiation, we showed that H3K9me2 levels on the promoters of each ORs increased (Fig. 1D). We further examined whether G9a or LSD1 regulate OR expression via the methylation or demethylation of H3K9 respectively, by ChIP-qPCR. The recruitment of G9a to OR promoters increased 48 h after ATRA treatment, while the amount of LSD1 at OR promoters decreased (Fig. 3A and Supplementary Figure 3A). Furthermore, H3K9me2 levels were increased at OR promoters. However, during HL-60 differentiation, the level of H3K4me2 at OR1N1, OR4F6, OR7A17, and OR10G2 promoters decreased, consistent with the low ORs mRNA expression levels. To further confirm the roles of G9a and LSD1 in HL-60 differentiation, we treated HL-60 cells with BIX01294 and GSK-LSD1, respectively. Treatment with BIX01294 inhibited the recruitment of G9a to OR promoter regions and resulted in decreased H3K9me2 levels (Fig. 3B and Supplementary Figure 3B). In contrast, treatment with GSK-LSD1 resulted in decreased LSD1 recruitment to the OR promoters and inhibited the demethylation of H3K9me2 (Fig. 3C and Supplementary Figure 3C). Unexpectedly, GSK-LSD1 treatment also increased H3K4me2 level, suggesting that LSD1 might demethylate H3K4me2 and H3K9me2 on OR promoter regions. Taken together, these data suggest that G9a and LSD1 regulate H3K9 methylation levels during ATRA-mediated HL-60 differentiation.


Regulatory role of G9a and LSD1 in the Transcription of Olfactory Receptors during Leukaemia Cell Differentiation
G9a and LSD1 regulate OR expression through the methylation and demethylation of H3K9.(A) ChIP analyses of the OR1N1 and OR10G2 promoters in ATRA-treated HL-60 cells were conducted using anti-G9a, anti-LSD1, anti-H3K4me2, anti-H3K9me2, and anti-IgG antibodies and were examined via qRT-PCR analyses. (B,C) HL-60 cells were treated with BIX01294 (5 μM) or GSK-LSD1 (500 nM) for 48 or 24 h, respectively. (B) ChIP analyses of the OR1N1 and OR10G2 promoters were performed using anti-G9a, anti-H3K9me2, and anti-IgG antibodies and examined by qRT-PCR analyses. (C) Using anti-LSD1, anti-H3K4me2, anti-H3K9me2, and anti-IgG antibodies, ChIP analyses were performed. The results were analysed by qRT-PCR. (A–C) These results are shown as mean ± SDs. (n = 3).
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f3: G9a and LSD1 regulate OR expression through the methylation and demethylation of H3K9.(A) ChIP analyses of the OR1N1 and OR10G2 promoters in ATRA-treated HL-60 cells were conducted using anti-G9a, anti-LSD1, anti-H3K4me2, anti-H3K9me2, and anti-IgG antibodies and were examined via qRT-PCR analyses. (B,C) HL-60 cells were treated with BIX01294 (5 μM) or GSK-LSD1 (500 nM) for 48 or 24 h, respectively. (B) ChIP analyses of the OR1N1 and OR10G2 promoters were performed using anti-G9a, anti-H3K9me2, and anti-IgG antibodies and examined by qRT-PCR analyses. (C) Using anti-LSD1, anti-H3K4me2, anti-H3K9me2, and anti-IgG antibodies, ChIP analyses were performed. The results were analysed by qRT-PCR. (A–C) These results are shown as mean ± SDs. (n = 3).
Mentions: During ATRA-mediated HL-60 differentiation, we showed that H3K9me2 levels on the promoters of each ORs increased (Fig. 1D). We further examined whether G9a or LSD1 regulate OR expression via the methylation or demethylation of H3K9 respectively, by ChIP-qPCR. The recruitment of G9a to OR promoters increased 48 h after ATRA treatment, while the amount of LSD1 at OR promoters decreased (Fig. 3A and Supplementary Figure 3A). Furthermore, H3K9me2 levels were increased at OR promoters. However, during HL-60 differentiation, the level of H3K4me2 at OR1N1, OR4F6, OR7A17, and OR10G2 promoters decreased, consistent with the low ORs mRNA expression levels. To further confirm the roles of G9a and LSD1 in HL-60 differentiation, we treated HL-60 cells with BIX01294 and GSK-LSD1, respectively. Treatment with BIX01294 inhibited the recruitment of G9a to OR promoter regions and resulted in decreased H3K9me2 levels (Fig. 3B and Supplementary Figure 3B). In contrast, treatment with GSK-LSD1 resulted in decreased LSD1 recruitment to the OR promoters and inhibited the demethylation of H3K9me2 (Fig. 3C and Supplementary Figure 3C). Unexpectedly, GSK-LSD1 treatment also increased H3K4me2 level, suggesting that LSD1 might demethylate H3K4me2 and H3K9me2 on OR promoter regions. Taken together, these data suggest that G9a and LSD1 regulate H3K9 methylation levels during ATRA-mediated HL-60 differentiation.

View Article: PubMed Central - PubMed

ABSTRACT

Recent studies have reported the ectopic expression of olfactory receptors (ORs) in non-olfactory tissues, however, their physiological roles were not well elucidated. ORs are expressed in and function in different types of cancers. Here, we identified that the H3K9me2 levels of several OR promoters decreased during differentiation in the HL-60, human myeloid leukaemia cell line, by all-trans-retinoic acid (ATRA). We found that the differential OR promoters H3K9me2 levels were regulated by G9a and LSD1, resulting in the decrease of ORs transcription during HL-60 differentiation. G9a and LSD1 could regulate the expression of ORs in several non-olfactory cells via the methylation and demethylation of H3K9me2. In addition, we demonstrated that knockdown of OR significantly reduced cell proliferation. Therefore, the epigenetic regulation of ORs transcription is critical for carcinogenesis.

No MeSH data available.