[Cangen-L) The Regulation of Genomic Imprinting and X Inactivation in Mice

  • From: Rievaulx Canines <rievaulx@xxxxxxxxxxxxx>
  • To: cangen-l@xxxxxxxxxxxxx
  • Date: Fri, 19 Jul 2002 08:09:44 -0700

The Regulation of Genomic Imprinting and X Inactivation in Mice



Marisa S. Bartolomei, Ph.D.
Assistant Investigator,
University of Pennsylvania School of Medicine

Summary: Marisa Bartolomei is interested in the processes that control genomic
imprinting and X inactivation in mice.


Genomic imprinting results in the differential expression of the parental 
alleles of a
gene. This parent-of-origin-specific effect is observed for a small number of 
genes in
mammals and is likely to involve novel regulatory events. The mechanism by which
parental origin affects this subset of genes remains largely unknown and is the 
focus
of our research. To this end, we are using the maternally expressed mouse H19 
gene as
a model imprinted gene.

The mouse H19 gene was one of the first imprinted genes to be identified. 
Although the
H19 transcript is typical of protein-coding RNAs, it does not encode a protein, 
and
the active gene product may be the RNA itself. The function of the H19 gene, 
which is
predominantly expressed in mesodermal and endodermal derivatives in embryonic 
and
neonatal tissues, is not, however, entirely clear. Interestingly, H19 is closely
linked to the oppositely imprinted insulin-like growth factor II gene (Igf2). 
Evidence
suggests that these genes are part of an imprinting domain in which a common 
set of
enhancers drives expression of H19 on the maternal chromosome and Igf2 on the 
paternal
chromosome.

Our initial studies investigated how the two parental alleles of imprinted 
genes are
distinguished so that the transcriptional machinery knows which allele to 
express. A
2-kb region located from ?2 kb to ?4 kb relative to the start of transcription
(designated the imprinting control region [ICR] or differentially methylated 
domain
[DMD]) is preferentially methylated on the repressed paternal allele throughout
development. While hypermethylation of this region represents the best 
candidate for
conferring the allele-specific imprinting mark, this region also exhibits
maternal-specific DNase hypersensitivity in somatic cells. We have deleted the
differentially methylated sequence at the endogenous locus and found that the
imprinting and methylation of both the H19 and the Igf2 genes are disrupted,
indicating that this region is required for the reciprocal imprinting of H19 
and Igf2.
We have also used conditional mutants to demonstrate that the ICR is required 
in the
germline for the establishment of the imprinting of these two genes; in 
differentiated
cells, however, the ICR is required for Igf2 imprinting but is no longer 
required for
H19 imprinting.

More recently, it has been shown in vitro that the ICR displays insulator 
activity
that is sensitive to methylation. Insulator elements can isolate a gene and its
regulatory elements from position effects and block enhancer-mediated 
transcription
when placed between a gene and its enhancer. At the H19 locus, it is postulated 
that
the ICR insulates H19 and its enhancers from Igf2 on the maternal allele, 
thereby
causing the exclusive expression of H19. On the paternal allele, the ICR is 
methylated
and inactivated, and Igf2 has access to the 3' enhancers. (This work has been
supported by the National Institutes of Health.)

To determine which part of the 2-kb ICR is essential to imprinting, we cloned 
and
characterized the corresponding sequence from human and rat. Sequence comparison
revealed a conserved 21-bp CpG-rich element that is repeated four times in 
mouse and
rat and six times in human. The ubiquitous transcriptional regulator CTCF binds 
this
sequence in a methylation-sensitive manner in vitro. CTCF also binds to the 5' 
chicken
ß-globin insulator via a sequence that is similar to the H19 repeats. While in 
vitro
experiments point to the repeat sequence as mediating the ICR insulator 
activity, the
insulating capacity of these sequences has not been demonstrated in vivo. To
understand the nature of the insulator, we have mutated the CpG dinucleotides 
within
the CTCF sites at the endogenous locus. Although the mutant ICR retains 
insulating
capacity, preliminary analysis indicates that the ICR does not become 
methylated in
the male germline, resulting in mice harboring hypomethylated parental alleles. 
These
results suggest that the H19 locus possesses distinct 5' sequences that permit
insulator activity on the maternal allele and attract methylation that leads to
repression on the paternal allele.

While deletion of the ICR disrupts imprinted expression and methylation at the 
H19
locus, there is still a small amount of parental-specific methylation that 
remains on
the mutant alleles. These results suggest that other cis-acting sequences may be
crucial to imprinted expression. Such elements are likely located 3' to the
transcription unit, since additional conserved sequences have been described in 
this
region and deletion of a large segment of DNA harboring these sequences perturbs
imprinting of H19 transgenes. We are performing gene targeting in embryonic 
stem cells
and transgenic analysis to investigate which sequence is required for H19 and 
Igf2
imprinting.

It is also essential to determine at what point during gametogenesis the
parental-specific epigenetic mark is conferred. Since mature oocytes and sperm 
are
differentially methylated, we have analyzed H19 methylation in earlier 
populations of
gametes. Determination of the time and tissue in which the methylation pattern 
is
established will ultimately allow us to dissect the mechanism by which the 
parental
alleles are identified and subsequently marked. We have found that primordial 
germ
cells in 13.5-day male embryos lack methylation on both parental alleles, 
indicating
that during male germ cell development, the paternal-specific methylation 
inherited
from sperm is erased. By embryonic day 15.5, the paternal H19 allele is 
methylated in
prospermatogonia. In contrast, the maternal H19 allele acquires significant
methylation after birth. These experiments indicate that the parental alleles 
of H19
remain distinct in the male germline, despite the fact that methylation 
differences
are transiently erased. Thus an epigenetic modification such as differential 
chromatin
structure may act in the absence of methylation to maintain parental identity. 
Once
the embryo recovers its ability to methylate genomic DNA, allelic methylation
differences may either resume a primary role in allelic identity or act with 
another
epigenetic modification to confer parental identity. Experiments are in 
progress to
assess the nature of such an allele-distinguishing epigenetic modification.

We are also interested in the processes that govern X inactivation in mice. X
inactivation is the dosage compensation mechanism that female mammals use to 
silence
one X chromosome and to achieve X-linked expression equivalent to that of males.
Certain aspects of this complex multistep process have been well established, 
but the
molecular and genetic mechanisms controlling this process remain poorly 
characterized.
While all factors known to be involved in X inactivation map to the X 
chromosome, it
is probable that unidentified autosomal factors are essential to the process. To
isolate such factors, we used N-ethyl-N-nitrosourea (ENU) mutagenesis in the 
mouse to
select for mutations that affect X inactivation. In collaboration with 
Huntington
Willard (Case Western Reserve University School of Medicine), we have recovered 
two
independent autosomal-dominant mutations that perturb X-inactivation patterns.
Affected heterozygous females exhibit alterations in the proportion of cells
expressing a given X chromosome. The observation that 6.5-day embryos are 
affected by
the mutations suggests that we have disrupted autosomal factors that act early 
in the
X-inactivation pathway. Such factors may regulate the choice process of X
inactivation. These results are the first evidence of an autosomal mutation 
affecting
any component of the X-inactivation pathway. We have mapped one mutation to 
chromosome
15 and are refining its location, mapping the second mutation, and 
characterizing the
phenotype of the mutant animals.







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