p53-repressed miRNAs are involved with E2F in a feed-forward loop promoting proliferation

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Ran Brosh^1,a, Reut Shalgi^1,2,a, Atar Liran¹, Gilad Landan¹, Katya Korotayev³, Giang Huong Nguyen^4,5, Espen Enerly^6,7, Hilde Johnsen⁶, Yosef Buganim¹, Hilla Solomon¹, Ido Goldstein¹, Shalom Madar¹, Naomi Goldfinger¹, Anne-Lise Børresen-Dale^6,7, Doron Ginsberg³, Curtis C Harris⁴, Yitzhak Pilpel², Moshe Oren¹ & Varda Rotter¹

^aThese authors contributed equally to this work

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Synopsis

Precise regulation of gene expression is crucial for maintaining homeostasis in healthy tissues and for the execution of cellular programs such as proliferation, differentiation and cell death. In the last decade, microRNAs (miRNAs) have been uncovered as an expanding family of gene expression regulators. These short non-coding RNAs regulate gene expression at the post-transcriptional level by promoting translational inhibition or mRNA degradation (Bartel, 2004). Similar to protein-coding genes, the expression of miRNAs is also regulated by transcription factors (TFs), and induction or repression of miRNAs has been demonstrated to play a role in physiological processes such as immune response (Thai et al, 2007) and apoptosis (Chang et al, 2007; Raver-Shapira et al, 2007). Accordingly, deregulation of miRNAs is associated with diverse types of diseases, including a variety of cancers (Esquela-Kerscher and Slack, 2006; Volinia et al, 2006).

In an earlier computational study, we predicted the presence of several types of regulatory network motifs that involve TFs and miRNAs (Shalgi et al, 2007), and may provide a mechanism for fine-tuned coordination between transcriptional and post-transcriptional regulation of gene expression. Here, we describe and experimentally demonstrate one such regulatory motif, termed feed-forward loop (FFL), which involves the TF E2F1, a set of miRNAs, and their common targets (Figure 8). In this FFL, E2F1, a key regulator of cell-cycle progression, transcriptionally activates a family of 15 miRNAs that are organized in three paralogous polycistrons on three different chromosomes. These miRNAs silence a group of antiproliferative regulators including the pocket proteins pRb and p130 and the CDK inhibitors p21 and p57. Importantly, these genes are themselves transcriptional targets of E2F1. Thus, a TF activates a set of genes as well as a set of miRNAs, which in turn post-transcriptionally regulate that set of genes. Increasing the complexity of this regulatory FFL, many of the shared targets of E2F1 and the miRNAs function as regulators of the cell cycle; some negatively regulate E2F itself. For example, the pocket proteins pRB and p130 are the major components that regulate the activity of E2F family members throughout the phases of the cell cycle through direct protein–protein interaction.

Figure 8

A schematic model for the cell-cycle regulatory network comprising E2F, p53, miRs and other cell-cycle regulators. Arrows correspond to direct transcriptional activation, whereas bar-headed lines represent direct or indirect inhibition mediated by the following mechanisms: post-transcription gene silencing (miRNAs and their targets), protein binding and inactivation (pocket proteins and E2Fs; as well as CDK inhibitors and CDKs, that in turn inhibit pocket proteins by phosphorylation). The circular arrow represents E2F self-activating ability. Possible mechanisms underlying the repression of E2F by p53 are detailed in the discussion.

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The TF p53 is regarded as one of the key proteins that prevent malignant transformation (Ryan et al, 2001), and deactivating mutations of this tumor suppressor are highly common in a wide variety of tumors (Hussain and Harris, 1999). A hallmark activity of p53 is the inhibition of proliferation and the induction of cellular senescence on diverse types of stress signals with oncogenic potential, including DNA damage, telomere shortening and oncogene activation. There are several known mechanisms by which p53 negatively regulates proliferation, the key one being the transcriptional activation of the CDK inhibitor p21, which indirectly inhibits the activity of E2F family members. Another recently discovered mechanism for inhibiting proliferation by p53 is the induction of miRNAs from the miR-34 family, which also modulate the E2F pathway (He et al, 2007; Tarasov et al, 2007; Tazawa et al, 2007; Kumamoto et al, 2008). Additionally, direct and indirect transcriptional repression by p53 is considered important for its ability to inhibit proliferation (Ho and Benchimol, 2003).

Using miRNA microarrays, we discovered that p53 activation during cellular senescence in primary human fibroblasts leads to a decrease in the expression of the above-mentioned family of miRNAs, including members of the miR-17-92, miR-106b/93/25 and miR-106a-92 polycistronic miRNA clusters. A similar decrease in miRNA expression was observed in human breast cancer specimens that harbor wild-type p53 as compared with those that harbor mutant forms of p53. We further investigated the mechanism by which p53 represses the expression of this group of miRNAs, and found that activation of p53 leads to a dramatic reduction of E2F1 mRNA, protein and activity levels, which in turn leads to a decrease in the E2F1-dependent transcriptional activation of these miRNAs.

To study the consequence of deregulation of this FFL and importance of its inhibition by p53, we ectopically expressed representative members from the set of p53-repressed miRNAs, namely the miR-106b/93/25 polycistron, in primary human fibroblasts. Consequently, these cells acquired an enhanced proliferative phenotype manifested by increased growth rate, increased colony formation efficiency and delayed entry into replicative senescence. These results position the repression of this set of miRNAs as a novel mechanism by which p53 inhibits proliferation and controls cell fate.

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