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.
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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)