Reut Shalgi

Global and Local Architecture of the Mammalian microRNA–Transcription Factor Regulatory Network

microRNAs (miRs) are small RNAs that regulate gene expression at the posttranscriptional level. It is anticipated that, in combination with transcription factors (TFs), they span a regulatory network that controls thousands of mammalian genes. Here we set out to uncover local and global architectural features of the mammalian miR regulatory network. Using evolutionarily conserved potential binding sites of miRs in human targets, and conserved binding sites of TFs in promoters, we uncovered two regulation networks. The first depicts combinatorial interactions between pairs of miRs with many shared targets. The network reveals several levels of hierarchy, whereby a few miRs interact with many other lowly connected miR partners. We revealed hundreds of “target hubs” genes, each potentially subject to massive regulation by dozens of miRs. Interestingly, many of these target hub genes are transcription regulators and they are often related to various developmental processes. The second network consists of miR–TF pairs that coregulate large sets of common targets. We discovered that the network consists of several recurring motifs. Most notably, in a significant fraction of the miR–TF coregulators the TF appears to regulate the miR, or to be regulated by the miR, forming a diversity of feed-forward loops. Together these findings provide new insights on the architecture of the combined transcriptional–post transcriptional regulatory network.

p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC

Aberrant oncogene activation induces cellular senescence, an irreversible growth arrest that acts as a barrier against tumorigenesis. To identify microRNAs (miRNAs) involved in oncogene-induced senescence, we examined the expression of miRNAs in primary human TIG3 fibroblasts after constitutive activation of B-RAF. Among the regulated miRNAs, both miR-34a and miR-146a were strongly induced during senescence. Although members of the miR-34 family are known to be transcriptionally regulated by p53, we find that miR-34a is regulated independently of p53 during oncogene-induced senescence. Instead, upregulation of miR-34a is mediated by the ETS family transcription factor, ELK1. During senescence, miR-34a targets the important proto-oncogene MYC and our data suggest that miR-34a thereby coordinately controls a set of cell cycle regulators. Hence, in addition to its integration in the p53 pathway, we show that alternative cancer-related pathways regulate miR-34a, emphasising its significance as a tumour suppressor.

Differentially Regulated Micro-RNAs and Actively Translated Messenger RNA Transcripts by Tumor Suppressor p53 in Colon Cancer

Purpose: The aim of this study was to investigate the role of p53 in regulating micro-RNA (miRNA) expression due to its function as a transcription factor. In addition, p53 may also affect other cellular mRNA gene expression at the translational level either via its mediated miRNAs or due to its RNA-binding function. Experimental Design: The possible interaction between p53 and miRNAs in regulating gene expression was investigated using human colon cancer HCT-116 (wt-p53) and HCT-116 (null-p53) cell lines. The effect of p53 on the expression of miRNAs was investigated using miRNA expression array and real-time quantitative reverse transcription-PCR analysis. Results: Our investigation indicated that the expression levels of a number of miRNAs were affected by wt-p53. Down-regulation of wt-p53 via small interfering RNA abolished the effect of wt-p53 in regulating miRNAs in HCT-116 (wt-p53) cells. Global sequence analysis revealed that over 46% of the 326 miRNA putative promoters contain potential p53-binding sites, suggesting that some of these miRNAs were potentially regulated directly by wt-p53. In addition, the expression levels of steady-state total mRNAs and actively translated mRNA transcripts were quantified by high-density microarray gene expression analysis. The results indicated that nearly 200 cellular mRNA transcripts were regulated at the posttranscriptional level, and sequence analysis revealed that some of these mRNAs may be potential targets of miRNAs, including translation initiation factor eIF-5A, eIF-4A, and protein phosphatase 1. Conclusion: To the best of our knowledge, this is the first report demonstrating that wt-p53 and miRNAs interact in influencing gene expression and providing insights of how p53 regulates genes at multiple levels via unique mechanisms.

Widespread regulation of translation by elongation pausing in heat shock

Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation, although this mechanism may not always explain the full extent of repression. Here, using ribosome footprinting, we show that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells. The genome-wide nature of the phenomenon, its location, and features of protein N termini suggested the involvement of ribosome-associated chaperones. After severe heat shock, Hsp70s interactions with the translational machinery were markedly altered and its association with ribosomes was reduced. Pretreatment with mild heat stress or overexpression of Hsp70 protected cells from heat shock-induced elongation pausing, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses.

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

Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild‐type p53. These miRNAs are repressed by p53 in an E2F1‐mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi‐gene transcriptional and post‐transcriptional feed‐forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network.

EGF Decreases the Abundance of MicroRNAs That Restrain Oncogenic Transcription Factors

Some cancers showed decreased abundance of a subset of EGF-regulated microRNAs, which allows the production of oncogenic transcription factors. A Loss of Restraint Growth factors, such as epidermal growth factor (EGF), bind to receptors to stimulate cell proliferation, a process critical during development and in wound healing. Dysregulation of the signaling pathways initiated by the EGF receptor (EGFR) has been implicated in cancer. Noting that aberrant expression of microRNAs, small noncoding RNAs that inhibit the expression of target genes, is common in human malignancies, Avraham et al. explored the role of microRNAs in regulating EGFR signaling. They found that EGF elicited a rapid—and transient—decrease in the abundance of a group of 23 microRNAs, thereby enabling the induction of potentially oncogenic transcription factor targets. Moreover, the abundance of this group of microRNAs was decreased in breast cancers and brain cancers with molecular lesions consistent with increased EGFR signaling. The authors conclude that, under basal conditions, this group of microRNAs restrains potentially oncogenic signaling pathways downstream of the EGFR. Their decreased abundance in cancer thus enables the dysregulated activity of oncogenic transcription factors and signaling pathways transiently activated by EGF signaling, thereby promoting the aberrant cellular behaviors associated with cancer. Epidermal growth factor (EGF) stimulates cells by launching gene expression programs that are frequently deregulated in cancer. MicroRNAs, which attenuate gene expression by binding complementary regions in messenger RNAs, are broadly implicated in cancer. Using genome-wide approaches, we showed that EGF stimulation initiates a coordinated transcriptional program of microRNAs and transcription factors. The earliest event involved a decrease in the abundance of a subset of 23 microRNAs. This step permitted rapid induction of oncogenic transcription factors, such as c-FOS, encoded by immediate early genes. In line with roles as suppressors of EGF receptor (EGFR) signaling, we report that the abundance of this early subset of microRNAs is decreased in breast and in brain tumors driven by the EGFR or the closely related HER2. These findings identify specific microRNAs as attenuators of growth factor signaling and oncogenesis.

Repression of transposable-elements – a microRNA anti-cancer defense mechanism?

MicroRNAs (miRNAs) appear to be key players in the maintenance of genomic integrity. Recent evidence implies that cancers often avoid miRNA-mediated regulation, and global repression of miRNAs is associated with increased tumorigenicity. Here we suggest that miRNAs are directly involved in the maintenance of genomic integrity through global repression of transposable elements (TEs), whose expression and transposition are well-documented causes of genomic instability in mammalian somatic tissues. Hence, one outcome of the tumors ability to avoid miRNA-mediated regulation might be the enhancement of genomic instability and mutability due to derepression of TEs. We outline possible mechanisms underlying TE repression by miRNAs, including post-transcriptional silencing and transcriptional silencing through DNA and histone methylation. This hypothesis calls into consideration the need to study the role of miRNAs and the RNAi machinery in the nucleus, and specifically their impact on the maintenance of genomic integrity in the context of cancer.

CpG Islands as a Putative Source for Animal miRNAs: Evolutionary and Functional Implications

MicroRNAs (miRs) are considered major contributors to the evolution of animal morphological complexity. Multiple bursts of novel miR families were documented throughout animal evolution, yet, their evolutionary origins are not understood. Here, we discuss two alternative genomic sources for novel miR families, namely, transposable elements, which were previously described, and a newly proposed origin: CpG islands. We show that these two origins are evolutionarily distinct and that they correspond to marked differences in several functional and genomic characteristics. Together, our results shed light on the intriguing origin of one of the major constituents of regulatory networks in animals, miRs.