C/EBPα is indispensable for PML/RARα-mediated suppression of long non-coding RNA NEAT1 in acute promyelocytic leukemia cells

Abstract

Better understanding of the transcriptional regulatory network in acute promyelocytic leukemia (APL) cells is critical to illustrate the pathogenesis of other types of acute myeloid leukemia. Previous studies have primarily focused on the retinoic acid signaling pathway and how it is interfered with by promyelocytic leukemia/retinoic acid receptor-α (PML/RARα) fusion protein. However, this hardly explains how APL cells are blocked at the promyelocytic stage. Here, we demonstrated that C/EBPα bound and transactivated the promoter of long non-coding RNA NEAT1, an essential element for terminal differentiation of APL cells, through C/EBP binding sites. More importantly, PML/RARα repressed C/EBPα-mediated transactivation of NEAT1 through binding to NEAT1 promoter. Consistently, mutation of the C/EBP sites or deletion of retinoic acid responsive elements (RAREs) and RARE half motifs abrogated the PML/RARα-mediated repression. Moreover, silencing of C/EBPα attenuated ATRA-induced NEAT1 upregulation and APL cell differentiation. Finally, simultaneous knockdown of C/EBPα and C/EBPβ reduces ATRA-induced upregulation of C/EBPε and dramatically impaired NEAT1 activation and APL cell differentiation. In sum, C/EBPα binds and transactivates NEAT1 whereas PML/RARα represses this process. This study describes an essential role for C/EBPα in PML/RARα-mediated repression of NEAT1 and suggests that PML/RARα could contribute to the pathogenesis of APL through suppressing C/EBPα targets.

Keywords: APL; C/EBPα; NEAT1; PML/RARα; transcriptional regulation.

Figure 1

C/EBPα directly binds and transactivates the promoter region of NEAT1. (A) Upper panel: Schematic representation of putative C/EBP binding sites in the NEAT1 promoter. Lower panel: C/EBPα ChIP-qPCR showing the enrichment of C/EBPα in each putative binding site, the negative control and positive control (SPI1 promoter) in NB4 cells that were untreated or treated with ATRA at 1μM for 24 h (RA 24h). (B) ChIP was performed on two APL patient samples with anti-C/EBPα antibody. DNA fragments at NEAT1 promoter were subsequently measured with qPCR. (C) The 1656 bp NEAT1 promoter reporter construct (125 ng) was transfected into 293T cells along with pcDNA3.1 vector (empty) or pcDNA3.1-C/EBPα (C/EBPα) expression plasmid (500 ng). The data represent the mean ± S.E.M from 3 replicates.

Figure 2

C/EBPα transactivates NEAT1 through the -1453 and -54 sites in the NEAT1 promoter. (A) Schema of the NEAT1 promoter region shows the different mutation/truncation constructs used in this study. □ represents the wild-type C/EBP binding site and ⊠ represents the mutated site. (B) The wild-type (wt) or double mutated (co-mut) promoter construct (125 ng) was co-transfected into 293T cells along with the C/EBPα expression construct (500 ng). (C) Different mutation/truncation luciferase promoter plasmids were co-transfected with 500ng of the pcDNA3.1 (empty) or pcDNA3.1-C/EBPα (C/EBPα) vector into 293T cells. The data represent the mean ± S.E.M from three replicates. * indicates p<0.05.

Figure 3

PML/RARα binds to NEAT1 promoter and represses the C/EBPα-mediated transactivation of NEAT1. (A) ChIP was performed in NB4 cells with anti-PML, anti-RARα, or nonspecific (normal immunoglobulin G (IgG)) antibodies. The immunoprecipitated DNA was amplified by PCR, followed by agarose electrophoresis. (B) The promoter of NEAT1 was co-transfected into 293T cells along with pcDNA3.1 vector or pcDNA3.1-PML/RARα expression plasmid in the absence or presence of C/EBPα. (-) and (+) represent the absence or presence of the indicated plasmid. (C, D) NEAT1 promoter truncation plasmids that contain (trunc1 and trunc2) or do not contain RARE and RARE half motifs (-54F and -1453F) were co-transfected with pcDNA3.1 vector or pcDNA3.1-C/EBPα and with or without PML/RARα-expression construct. Luciferase activity was detected 24 h after transfection. (E) The wild-type (wt) or double mutated (co-mut) NEAT1 promoter construct was co-transfected into U937 cells along with pcDNA3.1 vector or pcDNA3.1-PML/RARα expression plasmid. (F, G) NEAT1 promoter truncation plasmids in the presence (trunc1 and trunc2) or absence of RARE and RARE half motifs (-54F and -1453F) were co-transfected with pcDNA3.1 vector or pcDNA3.1-PML/RARα expression construct. (H) The wild-type (wt) or double mutated (co-mut) NEAT1 promoter construct was transfected into NB4 cells. Six hours later, cells were treated with ATRA and tested at the indicated time points. The error bar represents the standard error of the mean (S.E.M.) (n=3). * indicates p<0.05.

Figure 4

Knockdown of C/EBPα attenuates ATRA-induced upregulation of NEAT1 and NB4 cell differentiation. (A) NB4 cells were transfected with 3 μg siRNA targeting C/EBPα (siC/EBPα) or negative control siRNA (NC). Six hours later, cells were treated with 1μM ATRA for 24 h. Expression of C/EBPα was subsequently determined by qRT-PCR. (B, C) Expression of NEAT1 and NEAT1_2 isoform in C/EBPα-silenced NB4 cells was detected both before and after ATRA treatment. (D, E) The granulocytic differentiation marker CD11b, CD18, and CD11c in C/EBPα-silenced NB4 cells were tested after ATRA treatment for 24 h. The data represent the mean ± S.E.M from three replicates. * indicates p<0.05. (F, G) The expression of NEAT1 and NEAT1_2 isoform in C/EBPα-silenced primary APL bone marrow cells was measured after ATRA treatment for 24 h.

Figure 5

Double knockdown of C/EBPα and C/EBPβ reduces ATRA-induced upregulation of C/EBPε and markedly impairs NEAT1 upregulation and NB4 cell differentiation. (A) C/EBPβ knockdown (kd-C/EBPβ) or control (NC) NB4 cells were transfected with C/EBPα siRNA (kd-C/EBPα) or negative control siRNA (NC). The protein levels of C/EBPα, C/EBPβ, C/EBPε, and GAPDH were determined in NB4 cells before and after ATRA treatment (1 μM for 24 h). (B) Expression of NEAT1 and NEAT1_2 isoform in C/EBPα and C/EBPβ double-silenced (double-KD) NB4 cells was analyzed after ATRA treatment for 24 h. (C) Flow cytometric analysis of CD11b, CD18, and CD11c expression in NB4 cells with or without C/EBPα and C/EBPβ double knockdown (double-KD) following ATRA treatment for 24h. The data represent the mean ± S.E.M. from three replicates. * indicates p<0.05.