Published online 23 November 2008 Nucleic Acids Research, 2009, Vol. 37, No. 1 123–128 doi:10.1093/nar/gkn920 Twist-1 regulates the miR-199a/214 cluster during development Youn-Bok Lee1, Ioannis Bantounas 1 , Do-Young Lee 1 , Leonidas Phylactou 2 , Maeve A. Caldwell1 and James B. Uney1,* 1The Henry-Wellcome Laboratories for Integrated Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY, UK and 2The Cyprus Institute of Neurology and Genetics, 6 International Airport Avenue PO Box 3462, 1683 Nicosia, Cyprus Received July 20, 2008; Revised October 10, 2008; Accepted November 1, 2008 ABSTRACT MicroRNAs are known to regulate developmental processes but their mechanism of regulation remains largely uncharacterized. We show the transcription factor Twist-1 drives the expression of a 7.9-kb noncoding RNA transcript (from the Dynamin-3 gene intron) that encodes a miR-199a and miR-214 cluster. We also show that knocking down Twist-1 with shRNAs decreased miR-199a/214 levels and that Twist-1 bound an E-Box promoter motif to developmentally regulate the expression of these miRNAs. The expression of HIF-1 (known to mediate Twist-1 transcription), miR-199a and miR-214 was maximal at E12.5 and the miRNAs were expressed specifically in mouse cerebellum, midbrain, nasal process and fore- and hindlimb buds. This study shows the expression of the miR199a/214 cluster is controlled by Twist-1 via an E-Box promoter element and supports a role for these miRNAs as novel inter- mediates in the pathways controlling the develop- ment of specific neural cell populations. INTRODUCTION MicroRNAs (miRNA) bind the 30-untranslated region (UTR) of mRNAs and elicit translational repression or mRNA cleavage and this action represents an entirely new level of post-transcriptional regulation. More than 500 human miRNAs together with some of their mRNA targets have now been identiﬁed empirically and it is clear that they act to govern fundamental cellular pathways controlling development, cancer and apoptosis (1,2). Although miRNAs are now known to be key regulators of gene expression, the pathways controlling the expres- sion of individual miRNA genes are only beginning to be elucidated. For example, recently, the transcription of miR-146a during development was shown to be sup- pressed by promyelocytic leukemia zinc-ﬁnger (PLZF) protein (3,4); the transcription factor GATA-1 was shown to drive the expression of a single transcript encoding miR-144 and miR-451 during zebra ﬁsh devel- opment (5). While investigating the control of miR-214 expression we found that the miR-199a and miR-214 genes are located 6 kb apart within the intron of the dynamin-3 gene and that Twist-1 had previously been reported to drive the expression of a single noncoding transcript from this region (6). Twist is a nuclear basic helix–loop–helix (bHLH) transcription factor known to be crucial for mesoderm formation in Drosophila (7). In mammals, Twist-1 is expressed in cranial neural crest derivatives and in mesenchymal structures (8–11). Mutations in the Twist-1 gene cause the Saethre- Chotzen syndrome, an autosomal dominant craniosynos- tosis characterized by abnormal fusion of the cranial sutures (12,13). Studies have also indicated that Twist family members bind preferentially to E-box DNA promoter sequences as homodimers (14) or heterodimers (15). We present data to show that during embryonic development Twist-1 binds an E-box region within the DNM-3 gene intron to drive the expression of a single transcript that is processed to miR-199a and miR-214. MATERIALS AND METHODS Generation of recombinant adenovirus constructs Mouse Twist-1 cDNA was cloned into the PxCx-CMV shuttle vector and recombinant E1-deleted adenoviral constructs produced as described previously (16). The pre- dicted DNM3os promoter was recovered from mouse genomic DNA by PCR. The primer sequences were: pro- moter (–640:0) forward (F), AAA GGG GGG AGC CCC AAC TTA TCT G; reverse (R), TTC CTG CAC CAG GGG CTT GT; E-box deleted (–640:-357) forward, AGG GGG GAG CCC CAA CTT ATC TGA; reverse, TGG *To whom correspondence should be addressed. Tel: +117 3313088; Fax: +117 3313086; Email: james.uney@bristol.ac.uk 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.