miR-21 Gene Expression Triggered by AP-1 Is Sustained through a Double-Negative Feedback Mechanism

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Abstract

miR-21 has been reported to be highly expressed in various cancers and to be inducible in a human promyelocytic cell line, HL-60, after phorbol 12-myristate 13-acetate (PMA) treatment. To examine molecular mechanisms involved in miR-21 expression, we analyzed the structure of the miR-21 gene by determining its promoter and primary transcripts. We show that activation protein 1 (AP-1) activates the miR-21 transcription in conjugation with the SWI/SNF complex, after PMA stimulation, through the conserved AP-1 and PU.1 binding sites in the promoter identified here. The previous findings of enhanced miR-21 expression in several cancers may therefore reflect the elevated AP-1 activity in these carcinomas. A single precursor RNA containing miR-21 was transcribed just downstream from the TATA box in this promoter, which is located in an intron of a coding gene, TMEM49. More important, expression of this overlapping gene is completely PMA-independent and all its transcripts are polyadenylated before reaching the miR-21 hairpin embedding region, indicating that miRNAs could have their own promoter even if overlapped with other genes. By available algorithms that predict miRNA target using a conservation of sequence complementary to the miRNA seed sequence, we next predicted and confirmed that the NFIB mRNA is a target of miR-21. NFIB protein usually binds the miR-21 promoter in HL-60 cells as a negative regulator and is swept off from the miR-21 promoter during PMA-induced macrophage differentiation of HL-60. The translational repression of NFIB mRNA by miR-21 accelerates clearance of NFIB in parallel with the simultaneous miR-21-independent transcriptional repression of NFIB after PMA stimulation. Since exogenous miR-21 expression moderately induced endogenous miR-21, an evolutionarily conserved double-negative feedback regulation would be operating as a mechanism to sustain miR-21 expression.

Introduction

Tissue-specific gene expression in multicellular organisms has been previously considered to be principally regulated at the transcriptional level, but currently the posttranscriptional regulation in this process is considered to be more universal and diverse than before.1 An example of this is the activity of the phylogenetically conserved 20- to 24-nucleotide RNA, designated microRNAs (miRNA).2 More than 500 species of miRNAs have now been identified in humans3, 4 and mediate the repression of target mRNAs by suppressing translation or promoting mRNA decay.5 Whereas the vertebrate miRNA genes are thought to be generally transcribed by RNA polymerase II to produce a pri-miRNA containing a 5′-cap structure and poly(A) tail,6, 7 it still remains largely unknown, however, how human miRNA expression itself is regulated at the transcriptional level. Several reports have now shown examples of mammalian miRNA gene regulation by known transcription factors.8, 9

Cloning-based assay shows that there are high copies of miR-21 molecules in several human normal tissues.10, 11, 12 Interestingly, miR-21 expression is inducible in a human promyelocytic cell line, HL-60, after phorbol 12-myristate 13-acetate (PMA) treatment, which induces macrophage-like differentiation.13 miR-21 is also highly expressed in human glioblastoma14 and in various other solid tumors such as lung, breast, stomach, prostate, colon, and pancreatic cancers.15

These reports suggest that miR-21 would play important roles in both cell differentiation and carcinogenesis. Here we have analyzed the structure of the miR-21 gene in detail by characterizing its promoter, transcriptional initiation site, and transcripts. We show how a single primary transcript containing miR-21(pri-miR-21) is transcribed from an evolutionarily conserved promoter that resides in an intron of an overlapping coding gene, TMEM49. We have further identified NFIB mRNA as a target of miR-21 and finally present how this miRNA gene is regulated via miR-21 promoter, miR-21 molecule, and its target during macrophage differentiation of HL-60.

Section snippets

The promoter and transcriptional initiation site of miR-21 gene

We speculated that the large number of important regulatory systems that utilize miRNAs would be highly conserved across different vertebrate species and recently developed an algorithm to search for putative promoter regions of miRNAs (miPPRs) on the basis of two critical assumptions.16 First, these miRNAs not only would be derived from pre-miRNA sequences that are conserved among vertebrates, but would also have a conserved promoter sequence. Second, such a conserved promoter harbors at least

Discussion

In this study, we intensively analyzed the gene structure of miR-21 biochemically. Unlike in the previous study that reported the TSS of miR-21, authentic TSS was present 30 bp downstream from the conserved TATA box that was located in miPPR-21 (Fig. 1c), which is predicted by our recently developed promoter prediction algorithm. Very recently, it was reported that miR-21 is induced by two Stat3 binding sites that locate about 900 bp upstream from the previously reported TSS after IL-3

Primer extension and sequencing

For the determination of the TSSs of pri-miR-21, primer extension analyses were performed in parallel with the sequencing of the same region. Primer extension products were obtained by reverse transcription of 12 μg total RNA (pri-miR-21) with the corresponding 5′-32P-labeled primer (Supplementary Table S1) using SuperScript III (Invitrogen). DNA sequence ladders were obtained using a dideoxy chain-termination reaction. As the DNA templates for this reaction, the flanking regions of the primers

Acknowledgements

We thank Dr. K. Semba for his advice on the luciferase assay techniques and Drs. K. Miyake and S. Akashi for their advice on the electroporation techniques. We also thank Drs. A. Dutta and Y.-S. Lee for their advice on the primer extension of miRNA. The cDNA of PU.1 and NFIB were kind gifts from Dr. H. Sakano and Drs. M. Imagawa and S. Osada, respectively. We thank Dr. Y.-B. Shi for the anti-NFIB serum. The HL-60 cells were obtained from the Cell Resource Center for Biomedical Research,

References (47)

  • M. Ui et al.

    Endogenous AP-1 levels necessary for oncogenic activity are higher than those sufficient to support normal growth

    Biochem. Biophys. Res. Commun.

    (2000)
  • S.N. Ho et al.

    Site-directed mutagenesis by overlap extension using the polymerase chain reaction

    Gene

    (1989)
  • S.M. Gopalan et al.

    Nuclear factor-1-X regulates astrocyte-specific expression of the alpha1-antichymotrypsin and glial fibrillary acidic protein genes

    J. Biol. Chem.

    (2006)
  • M. Puzianowska-Kuznicka et al.

    Nuclear factor I as a potential regulator during postembryonic organ development

    J. Biol. Chem.

    (1996)
  • C.S. Chan et al.

    Revealing posttranscriptional regulatory elements through network-level conservation

    PLoS Comput. Biol.

    (2005)
  • I. Bentwich et al.

    Identification of hundreds of conserved and nonconserved human microRNAs

    Nat. Genet.

    (2005)
  • S. Griffiths-Jones et al.

    miRBase: microRNA sequences, targets and gene nomenclature

    Nucleic Acids Res.

    (2006)
  • P.D. Zamore et al.

    Ribo-gnome: the big world of small RNAs

    Science

    (2005)
  • Y. Lee et al.

    MicroRNA genes are transcribed by RNA polymerase II

    EMBO J.

    (2004)
  • X. Cai et al.

    Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs

    RNA

    (2004)
  • Y. Zhao et al.

    Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis

    Nature

    (2005)
  • K.A. O'Donnell et al.

    c-Myc-regulated microRNAs modulate E2F1 expression

    Nature

    (2005)
  • M. Lagos-Quintana et al.

    Identification of novel genes coding for small expressed RNAs

    Science

    (2001)
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