New Books April 2004

Background: Two distinct classes of regulators have been implicated in regulating neuronal gene expression and mediating neuronal identity: transcription factors such as REST/NRSF (RE1 silencing transcription factor) and CREB (cAMP response element-binding protein), and microRNAs (miRNAs). How these two classes of regulators act together to mediate neuronal gene expression is unclear. Results: Using comparative sequence analysis, here we report the identification of 895 sites (NRSE) as the putative targets of REST. A set of the identified NRSE sites is present in the vicinity of the miRNA genes that are specifically expressed in brain-related tissues, suggesting the transcriptional regulation of these miRNAs by REST. We have further identified target genes of these miRNAs, and discovered that REST and its cofactor complex are targets of multiple brainrelated miRNAs including miR-124a, miR-9 and miR-132. Given the role of both REST and miRNA as repressors, these findings point to a double-negative feedback loop between REST and the miRNAs in stabilizing and maintaining neuronal gene expression. Additionally, we find that the brain-related miRNA genes are highly enriched with evolutionarily conserved cAMP response elements (CRE) in their regulatory regions, implicating the role of CREB in the positive regulation of these miRNAs. Conclusion: The expression of neuronal genes and neuronal identity are controlled by multiple factors, including transcriptional regulation through REST and post-transcriptional modification by several brain-related miRNAs. We demonstrate that these different levels of regulation are coordinated through extensive feedbacks, and propose a network among REST, CREB proteins and the brain-related miRNAs as a robust program for mediating neuronal gene expression. Background Regulation of gene expression is critical for nervous system development and function. The nervous system relies on a complex network of signaling molecules and regulators to orchestrate a robust gene expression program that leads to the orderly acquisition and maintenance of neuronal identity. Identifying these regulators and their target genes is essential for understanding the regulation of neuronal genes and Published: 26 September 2006 Genome Biology 2006, 7:R85 (doi:10.1186/gb-2006-7-9-r85) Received: 12 May 2006 Revised: 1 August 2006 Accepted: 26 September 2006 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/9/R85 Genome Biology 2006, 7:R85 R85.2 Genome Biology 2006, Volume 7, Issue 9, Article R85 Wu and Xie http://genomebiology.com/2006/7/9/R85 elucidating the role of these regulators in neural development and function. The transcriptional repressor REST (RE1 silencing transcription factor, also called neuron-restrictive silencer factor or NRSF) plays a fundamental role in regulating neuronal gene expression and promoting neuronal fate [1,2]. REST contains a zinc-finger DNA-binding domain and two repressor domains interacting with corepressors CoREST and mSin3a. The corepressors additionally recruit the methyl DNA-binding protein MeCP2, histone deacetylases (HDAC), and other silencing machinery, which alter the conformation of chromatin resulting in a compact and inactive state [3-6]. REST is known to target many neuronal genes, and is pivotal in restricting their expression exclusively in neuronal tissues by repressing their expression in cells outside the nervous system. Recent work also points to REST as a key regulator in the transition from embryonic stem cells to neural progenitors and from neural progenitors to neurons [7]. The role of REST in nervous system development is intriguingly manifested by its expression, which is lower in neural stem/progenitor cells than in pluripotent stem cells, and becomes minimal in postmitotic neurons [7]. The expression of REST is shown to be regulated by retinoic acid; however, other forms of regulatory mechanisms are unknown. Another important class of regulators implicated in neuronal gene expression control and neuronal fate determination is the microRNA (miRNA) [8-10]. MiRNAs are an abundant class of endogenous approximately 22-nucleotide RNAs that repress gene expression post-transcriptionally. Hundreds of miRNAs have been identified in almost all metazoans including worm, fly, and mammals, and are believed to regulate thousands of genes by virtue of base pairing to 3' untranslated regions (3'UTRs) of the messages. Many of the characterized miRNAs are involved in developmental regulation, including the timing and neuronal asymmetry in worm; growth control and apoptosis in fly; brain morphogenesis in zebrafish; and hematopoetic and adipocyte differentiation, cardiomyocyte development, and dendritic spine development in mammals [8,11,12]. Based on data from a recent survey [13], we note that the human genome contains about 326 miRNA genes, many of which are highly or specifically expressed in neural tissues [14]. The function of the brain-related miRNAs and the mechanisms underlying their transcriptional control are beginning to emerge [12,15-17]. In addition to REST and miRNAs, many other classes of regulators might also be involved in controlling neuronal gene expression. This control could be carried out through a variety of mechanisms, such as changing chromatin state, affecting mRNA stability and transport, and post-translational modifications. Here we focus specifically on regulation through REST and miRNAs. To gain a better understanding of how REST and miRNAs regulate neuronal gene expression, we took the initial step of producing a reliable list of genes targeted by REST and several brain-related miRNAs using computational approaches. A list of these target genes should be informative in unraveling the function of these regulators. Moreover, we anticipate that a global picture of the target genes may provide a clue as to how REST and miRNAs act together to coordinate neuronal gene expression programs and promote neuronal identity. REST represses target genes by binding to an approximately 21-nucleotide binding site known as NRSE (neuron-restrictive silencer element, also called RE1), which is present in the regulatory regions of target genes. Previously, several genome-wide analyses of NRSE sites have been carried out [6,18,19]. These analyses used pattern-matching algorithms to search for sequences matching a consensus derived from known REST binding sites. The most recent work identified 1,892 sites in the human genome [19]. However, there are several factors limiting the utilities of the pattern-matching algorithms. Most notably, transcriptional factors can bind with variable affinities to sequences that are allowed to vary at certain positions. Consequently, methods based on consensus sequence matching are likely to miss target sites with weaker binding affinities. Indeed, it has been noted that both L1CAM and SNAP25 genes contain an experimentally validated NRSE site that diverges from the NRSE consensus [19], and was not identified in the previous analyses. In addition, even sequences perfectly matching the NRSE consensus could occur purely by chance, and therefore do not necessarily imply that they are functional. Given the vast size of the human genome, random matches could significantly add to the false positive rate of a prediction. For example, in the most recent analysis, it was estimated that 41% of the 1,892 predicted sites occur purely by chance, and likely represent false positives [19]. We have developed a method to systematically identify candidate NRSE sites in the human genome without these two main limitations of the previous methods. To address the first limitation, we utilized a profile-based approach, which computes the overall binding affinity of a site to REST without requiring strict matching of each base to the NRSE consensus. To reduce false positives, we rely on comparative sequence analysis to identify only sites that are conserved in orthologous human, mouse, rat and dog regions [20-23]. MiRNAs repress gene expression by base-pairing to the messages of protein-coding genes for translational repression or message degradation. The pairing of miRNA seeds (nucleotides 2 to 7 of the miRNAs) to messages is necessary and appears sufficient for miRNA regulation [24-26]. This enables the prediction of miRNA targets by searching for evolutionarily conserved 7-nucleotide matches to miRNA seeds in the 3'UTRs of the protein-coding genes [21,27-30]. We have Genome Biology 2006, 7:R85 http://genomebiology.com/2006/7/9/R85 Genome Biology 2006, Volume 7, Issue 9, Article R85 Wu and Xie R85.3