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. 2022 Sep 13:10:993525.
doi: 10.3389/fcell.2022.993525. eCollection 2022.

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7

Zhuoqi Li  1 , Juanli Qiao  1 , Wanru Ma  1 , Jing Zhou  1 , Liankun Gu  1 , Dajun Deng  1 , Baozhen Zhang  1
Affiliations

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7

Zhuoqi Li et al. Front Cell Dev Biol. .

Erratum in

Abstract

Background: It is well known that P16 INK4A , P14 ARF , P15 INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14 ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16 INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16 INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16 INK4A expression is mediated with CBX7. Results: In this study, we found that the upregulation of P16 INK4A , P14 ARF , P15 INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p < 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16 INK4A , P14 ARF and P15 INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells. Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16 INK4A , P14 ARF , P15 INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes' expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters.

Keywords: CBX7; P14AS; lncRNA; p14ARF; p15INK4B; p16INK4A.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The status of basal CBX7, P16 INK4A , P14 ARF , P15 INK4B , P14AS and ANRIL expression in human cell lines. (A) the results of Western blot; (B) qRT-PCR analyses. The relative mRNA levels are presented as mean ± SD. N.S: not significant. *: p < 0.05. **: p < 0.01 in Student’s t-test.
FIGURE 2
FIGURE 2
Effect of transient P14AS overexpression on the levels of P14 ARF , P16 INK4A and P15 INK4B expression in qTR-PCR analyses.
FIGURE 3
FIGURE 3
Increasing CBX7 expression enables P14AS to upregulate the P14 ARF , P15 INK4B , P16 INK4A expression levels in MGC803 and HepG2 cells with weak basal CBX7 expression in qRT-PCR (left charts for the relative RNA level) and Western blot analyses (right image and chart for the amounts of proteins). (A) MGC803; (B) HepG2 cells.
FIGURE 4
FIGURE 4
Downregulation of CBX7 expression disables P14AS to upregulate the P14 ARF , P15 INK4B , P16 INK4A expression levels in HEK293T and HCT116 cells with active basal CBX7 expression in qRT-PCR and Western blot analyses. (A) HEK293T; (B) HCT116 cells; (C) the efficiency of CBX7 knockdown in HEK293T and HCT116.
FIGURE 5
FIGURE 5
Coexpression of P14AS with P14 ARF , P15 INK4B and P16 INK4a genes in human colon cancer tissues with high CBX7 expression. (A) Colon cancer tissues; (B) Surgical margin tissues. N.S: not significant. *: p < 0.05. **: p < 0.01.
FIGURE 6
FIGURE 6
P14AS competitively binds to CBX7 and prevents the binding of CBX7 to ANRIL and to P14 ARF , P15 INK4B , P16 INK4A promoters. (A) Levels of P14AS and ANRIL binding to endogenous CBX7, as determined by RIP-qPCR analyses; and (B) binding of gene promoters to endogenous CBX7, as determined by ChIP-qPCR analyses. The experiments were repeated using both HEK293T and HCT116 cell lines.
FIGURE 7
FIGURE 7
Inhibition of repressive histone modifications at promoters of P14 ARF , P15 INK4B , and P16 INK4A by P14AS is CBX7-dependent, as determined by ChIP-qPCR analyses. (A) The ChIP-qPCR fragments and histone modifications within the locus, a screenshot from UCSC Genome Browser; (B) The levels of H3K9m3 and H3K27m3 at the three target genes promoters in HCT116 cells stably transfected with the P14AS or empty vector; (C) The levels of H3K9m3 and H3K27m3 at the three genes’ promoters in MGC803 cells cotransfected with P14AS and CBX7 or empty control vectors. The gel pictures show the size and specificity of qPCR products.
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