The Igf2/H19 muscle enhancer is an active transcriptional complex

Nucleic Acids Research, Sep 2013

In eukaryotic cells, gene expression is mediated by enhancer activation of RNA polymerase at distant promoters. Recently, distinctions between enhancers and promoters have been blurred by the discovery that enhancers are associated with RNA polymerase and are sites of RNA synthesis. Here, we present an analysis of the insulin-like growth factor 2/H19 muscle enhancer. This enhancer includes a short conserved core element that is organized into chromatin typical of mammalian enhancers, binds tissue-specific transcription factors and functions on its own in vitro to activate promoter transcription. However, in a chromosomal context, this element is not sufficient to activate distant promoters. Instead, enhancer function also requires transcription in cis of a long non-coding RNA, Nctc1. Thus, the insulin-like growth factor 2/H19 enhancer is an active transcriptional complex whose own transcription is essential to its function.

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The Igf2/H19 muscle enhancer is an active transcriptional complex

Bokkee Eun 0 1 Megan L. Sampley 1 Matthew T. Van Winkle 1 Austin L. Good 1 Marika M. Kachman 1 Karl Pfeifer 1 0 Core-Laboratory, College of Medicine, Korea University , Seoul 136-701, Republic of Korea 1 Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health , Bethesda, MD 20892, USA In eukaryotic cells, gene expression is mediated by enhancer activation of RNA polymerase at distant promoters. Recently, distinctions between enhancers and promoters have been blurred by the discovery that enhancers are associated with RNA polymerase and are sites of RNA synthesis. Here, we present an analysis of the insulin-like growth factor 2/H19 muscle enhancer. This enhancer includes a short conserved core element that is organized into chromatin typical of mammalian enhancers, binds tissue-specific transcription factors and functions on its own in vitro to activate promoter transcription. However, in a chromosomal context, this element is not sufficient to activate distant promoters. Instead, enhancer function also requires transcription in cis of a long noncoding RNA, Nctc1. Thus, the insulin-like growth factor 2/H19 enhancer is an active transcriptional complex whose own transcription is essential to its function. - Promoters and enhancers are generally thought of as two distinct regulatory elements. Functionally, promoters have been defined as the regions where RNA transcription initiates, whereas enhancers are DNA elements that work over distance to activate transcription at promoter elements (1). Furthermore, genomic analyses have defined and distinguished promoters and enhancers by their distinctive epigenetic marks, specifically their unique patterns of histone methylation (24). More recently, these functional and structural distinctions between enhancers and promoters have become somewhat blurred with the identification of enhancers with promoter-like chromatin features (2,5,6) and also with the realization that enhancer regions are frequently enriched for RNA Polymerase II (RNAP) and are sites for transcription of all kinds of RNAs including bidirectional transcripts (eRNAs) and multi-exonic polyadenylated RNAs (713). However, the functional significance of enhancer associated RNAs remains unclear (14). Insulin-like growth factor 2 (Igf2) and H19 are linked coregulated genes on the distal end of mouse chromosome 7. In humans, mis-expression of these genes on chromosome 11p15.5 is associated with developmental disorders and with several types of cancer including rhabdosarcoma (15,16). Igf2 and H19 are co-ordinately regulated in that they share tissue and developmental specificities that are dependent on a series of shared tissue-specific enhancer elements. The enhancer required for in vivo expression of Igf2 and H19 in muscle has been defined by mouse knockout studies (17). The ME mutation, a 20 kb deletion, centred 25 kb downstream of H19 (or 105 kb downstream of Igf2) (Figure 1A) that reduces Igf2 and H19 expression in myocytes to essentially undetectable levels (17,18). Recently, transient transfection analyses identified a 294 bp myocyte-specific core enhancer region [here called the core muscle enhancer (CME)] within the sequences defined by the ME deletion (19). In addition to carrying the CME, the minimal enhancer region, as defined by the mouse knockout and also by transgene analyses, completely coincides with the gene, Nctc1 (Figure 1A and B). The Nctc1 promoter lies 7 kb upstream of the CME and generates a spliced long noncoding RNA (lncRNA) expressed only in myocytes (18,20). In this study, we sought to identify a role for the Nctc1 gene and/or RNA in muscle enhancer function, and therefore we performed detailed molecular and genetic analyses of the enhancer. We show that the Igf2/H19 enhancer is bipartite. Enhancer activity requires the CME element that binds transcription factors, is organized into chromatin typical of an enhancer and Published by Oxford University Press 2013. This work is written by US Government employees and is in the public domain in the US. functions in classical in vitro reporter assays to activate promoter transcription. However, in a chromosomal context, enhancer function also requires the Nctc1 promoter and its transcription in cis. Altogether, our results demonstrate that this enhancer is an active transcriptional complex and that enhancer transcription is integral to enhancer function. MATERIALS AND METHODS Animal work was done according to NIH policy and approved by the Institutional Animal Care and Use Committee. Primary myoblast culture Primary myoblasts were isolated from neonatal pups (21) and differentiated into myotubes by growth in limiting horse serum (5%) for 2448 h. RNA isolation and analysis RNAs were extracted from snap-frozen muscle tissue using TriPure Extraction Reagent (Roche) or from cultured cells by the QiaShredder column (Qiagen) and then purifie (...truncated)


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Bokkee Eun, Megan L. Sampley, Matthew T. Van Winkle, Austin L. Good, Marika M. Kachman, Karl Pfeifer. The Igf2/H19 muscle enhancer is an active transcriptional complex, Nucleic Acids Research, 2013, pp. 8126-8134, 41/17, DOI: 10.1093/nar/gkt597