MOLECULAR GENETICS OF AN IMPRINTED GENE CLUSTER
ON MOUSE DISTAL CHROMOSOME 7
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Karl
Pfeifer, PhD, Head,
Section on Genomic Imprinting Mathew
Casimiro, PhD, Visiting Fellow Sangkyun
Jeong, PhD, Visiting Fellow Kurt
Inman, BS, Student David
Kunkel, BS, Student Young Soo Yoon, BS, Student |
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Genomic
imprinting is an unusual form of gene regulation in which expression of an
allele is restricted in accordance with its parental origin. Imprinted genes
are not randomly scattered throughout the chromosome but rather are localized
in discrete clusters. One cluster of imprinted genes is located on the distal
end of mouse chromosome 7. The syntenic region in humans (11p15.5) is highly
conserved in both gene organization and expression patterns. Mutations
disrupting the normal patterns of imprinting at the human locus are
associated with Beckwith Wiedemann syndrome, a developmental disorder, and
with many types of tumors. In addition, inherited cardiac arrhythmia is
associated with mutations in the maternal-specific Kcnq1 gene. We use
mouse models to address the molecular basis for allele-specific expression in
this region and hope to use imprinting as a tool with which to understand
fundamental features of epigenetic regulation of gene expression. We are also
generating mouse models of the numerous inherited disorders associated with
this region. We have generated models to study defects in cardiac
repolarization associated with loss-of-function mutations at Kcnq1 and
specifically to understand the effect of beta-adrenergic–mediated
stress on the cardiac phenotype. Molecular
basis for allele-specific expression of the mouse H19 and Igf2
genes Jeong, Kunkel, Rong,
Yoon, Pfeifer Our
studies on the mechanisms of genomic imprinting focus on the H19 and
the Igf2 genes, which lie at one end of the distal 7 imprinted cluster.
Paternally expressed Igf2 lies about 70 kb upstream of the
maternal-specific H19 gene. Using cell culture systems as well as
transgene and knockout experiments in vivo, we have identified the
enhancer elements responsible for activation of the two genes. The elements
are largely shared and located downstream of the H19 gene.
Parent-of-origin–specific expression of both genes is dependent on a
shared element (called the H19DMR) located just upstream of the H19
promoter and thus juxtaposed between the Igf2 gene and the shared
enhancers. The CpG sequences within the shared element are methylated
specifically on the paternally inherited chromosome. Our conditional ablation
of the element in vivo demonstrates that the nonmethyated H19DMR
(i.e., the copy on the maternal chromosome) is continually required for
silencing of the maternal Igf2 allele. Knockin experiments demonstrate
that the H19DMR contains a methylation-sensitive transcriptional
insulator. Thus, on the nonmethylated maternal chromosome, the active
insulator within the H19DMR prevents activation of Igf2 by the
downstream enhancers. Methylation of the paternal chromosome inactivates the
insulator and permits Igf2 expression. Unexplained by this model is
the effect of several small DMRs proximal to the Igf2
transcription unit. We are currently investigating the mechanistic
significance of these elements and have so far shown that expression of Igf2
does not correlate with methylation of the sequences. Imprinting of H19
occurs via a distinct genetic mechanism. The conditional ablation of the H19DMR
indicates that it is not continuously required for silencing the paternal
allele. Rather, the H19DMR is required early in development to
establish an epigenetic state at the H19 promoter that itself prevents
transcription. Current studies indicate that the epigenetic program includes
but is not solely the hypermethylation of the H19 promoter. To
determine which elements are necessary and sufficient for imprinting at the
locus, we have moved the H19DMR and its mutated derivatives to
heterologous loci. Our results demonstrate that the DMR alone is
sufficient to imprint a normally nonimprinted chromosome. Moreover, such
activity is not dependent on germline differences in DMR methylation.
Thus, the DMR likely marks its parental origin by a mechanism
independent of DNA methylation. Using genetic and molecular analyses of
embryonic stem cells derived from mutant mice, we are now determining the
epigenetic signals that constitute the genomic imprint. We
are continuing a series of experiments to understand the molecular mechanisms
by which the H19DMR can act as a transcriptional insulator. Several
groups have demonstrated the presence of four CTCF binding sites within the H19DMR.
CTCF is a DNA binding protein previously demonstrated to interact with the
chicken beta-globin insulator. The ability of CTCF to recognize DNA is
sensitive to methylation, i.e., CTCF cannot bind to the methylated paternally
inherited DMR, thus explaining the activation of the paternal Igf2
allele. To understand the molecular basis for insulator function, we have
begun a series of experiments to characterize the three-dimensional
organization of the Igf2/H19 locus, comparing maternal with paternal
and wild-type with mutant chromosomes. Specifically, we are examining the
long-range interactions between the Igf2 and H19 promoters and
the shared enhancer elements and the effect of a working insulator on the
interactions. We are also identifying interactions between the H19DMR and
the promoter and enhancer sequences. Srivastava M, Frolova E, Rottinghaus B, Boe SP, Grinberg A, Lee
E, Love PE, Pfeifer K. Imprint control
element-mediated secondary methylation imprints at the Igf2/H19 locus.
J Biol Chem 2003;278:5977-5983. Mouse
models for inherited long QT syndrome Casimiro, Inman, Rong,
Pfeifer; in collaboration with Ebert, Knollmann Inherited
long QT syndrome (LQTS) is characterized by an abnormal electrocardiogram
indicative of repolarization defects and can result in syncope or sudden
death. Romano-Ward syndrome (RWS) patients inherit the LQTS disorder
generally as a dominant phenotype and show no other traits. Jervell and
Lange-Nielsen syndrome (JLNS) patients display profound congenital deafness
in addition to LQTS, with both phenotypes recessive. We have generated
several mutations in the mouse Kcnq1 gene to model the human diseases.
Ablation of the gene results in vestibular and auditory defects. Histological
analyses suggest that the defects are attributable to a deficiency in the K+
recycling pathway, which is crucial for generating endolymph, the specialized
fluid bathing the inner hair cells. ECG tracings of mutant mice indicate
profound defects in cardiac repolarization when measured in vivo.
However, the defects are not in evidence in isolated hearts ex vivo,
indicating that the Kcnq1 protein plays a key role in mediating critical
extracardiac signals. Further analyses demonstrate that Kcnq1 is specifically
required to modulate cardiac function in the presence of beta-adrenergic
stimulation. We
have also generated three point mutations to model RWS. We have analyzed
mutations in the central pore region and in the sixth membrane-spanning
domain. The phenotypes of the mutations are each a distinct subset of those
seen in the null mutation and thus demonstrate that the Kcnq1 protein plays
distinct roles in the heart versus the inner ear and in various aspects of
cardiac function. While inherited LQTS is relatively rare, our genetic models
represent excellent paradigms for addressing mechanisms for acquired LQTS,
the single largest cause of death in Western societies. Biochemical
and pharmacological studies both predicted that the key biological role of
the Kcnq1 protein was its association with the helper protein Kcne1 to form
the IKS potassium channel. One of the most novel results of our
studies is the discovery that ablation of the Kcnq1 gene leads to
cardiac defects in addition to those noted in Kcne1-deficient mice.
The results suggest a novel role for Kcnq1 in heart development and/or
function. We have used our mutant mice as tools to detect a previously
unappreciated potassium channel that was dependent on Kcnq1 but not Kcne1.
The role of the channel in mouse and human hearts is now under investigation.
Casimiro MC, Knollmann BC, Yamoah EN, Nie L, Vary JC, Sirenko
SG, Greene AE, Grinberg A, Huang SP, Ebert SN, Pfeifer K. Targeted point
mutagenesis of mouse Kcnq1: phenotypic analysis of mice with point
mutations that cause Romano-Ward syndrome in humans. Genomics 2004;84:555-564. Tosaka T, Casimiro MC, Rong Q, Tella S, Oh M, Katchman AN,
Pezzullo JC, Pfeifer K, Ebert SN. Nicotine induces a long QT phenotype in Kcnq1-deficient
mouse hearts. J Pharmacol Exp Ther 2003;306:980-987. Role
of cells synthesizing beta-adrenergic hormones in the development of the
cardiac conduction system Rong, Pfeifer; in
collaboration with Ebert During early development, the
heart is the primary (and probably only) site of synthesis of the
beta-adrenergic hormones norepinephrine and epinephrine. This
cardiac-specific synthesis is transient and disappears by late gestation.
Intriguingly, the cells synthesizing the beta-adrenergic hormones are located
in positions that predict the location of the developing cardiac conduction
network. To understand the fate of cells that synthesize these hormones, we
generated a mouse with a mutated Pnmt locus (which encodes
phenylethanolamine N-methyltransferase, the enzyme that converts
norepinephrine to epinephrine) such that the cre recombinase enzyme is synthesized
in any cell normally making epinephrine. When crossed with appropriate tester
strains, Pnmt-expressing cells and their descendants become
beta-galactosidase–positive and thus can be readily identified and
isolated. Our analyses indicate that epinephrine is synthesized by cells that
give rise to cardiac conduction cells. However, the analyses suggest that the
intrinsic cardiac adrenergic cells represent a stem cell population that
contributes extensively to fetal cardiac development. We have recently
generated a new mouse model in which the green fluorescent protein is
expressed specifically in intrinsic adrenergic cells in the heart. We are
purifying these cells from fetal mouse hearts and characterizing their
ability to act as cardiac stem cells in vitro and in vivo. Ebert SN, Rong Q, Boe S, Thompson RP, Grinberg A, Pfeifer K.
PNMT as a novel marker for cardiomyocyte progenitor cells. Dev Dyn 2004;231:849-858. Pfeifer K, Boe SP, Rong Q, Ebert SN. Generating a mouse model
for studying the function and fate of intrinsic cardiac adrenergic cells. Annals
NY Acad Sci 2004;1018:418-423. COLLABORATORS Steven Ebert, PhD, Bjorn Knollmann, MD, PhD,
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