Download Methylation of an upstream Alu sequence on the Imprinted H19

Methylation of an upstream Alu sequence on the Imprinted H19
gene during spermatogenesis in rhesus monkeys
Amanda Stafford
Figure 1. DNA methyltransferase aiding in attaching a methyl
group to a 5’-cytosine of a CpG island.
Project Summary
Alu sequences are repetitive 300 base pair, site specific elements
interspersed in primate genomes. They contain numerous CpG
islands that are sometimes methylated. Alu methylation differs
between somatic and germ cell DNA, suggesting a possible role
for Alu sequences in genomic imprinting. The objective of this
proposed research will be to examine Alu methylation on the
imprinted H19 gene throughout spermatogenesis in the rhesus
monkey. Genomic DNA will be isolated from spermatogenic cells,
digested with RSAI and electrophoresed. Southern blot
hybridization and identification of the H19 Alu sequence will
follow. The isolated Alu sequence will be re-digested with
methylation-specific restriction enzymes. Results from this
proposed experiment should reveal a better understanding of
H19 Alu methylation and further our knowledge of genomic
imprinting.
Introduction
Genomic imprinting involves “marking” parental alleles
as either maternal or paternal. Such imprints are
established during gamete production and involve
differential DNA methylation. Unfortunately, however,
the exact imprinting mechanism is unknown. DNA
methylation is aided by the enzyme DNA methyltransferase, which adds methyl groups to 5’-cytosine
residues of CpG islands; this methylation results in gene
silencing.
Previous studies have shown that differential DNA
methylation patterns exist in the genome of sperm and
eggs (Kierszenbaum 2002). One highly studied
methylated/imprinted gene is H19. Research shows that
H19 is methylated in sperm but not eggs. The paternal
H19 allele is silenced due to hypermethylation. In
contrast, the maternal allele is hypomethylated and,
thus, expressed. How the paternal H19 imprint is made,
however, is not fully understood.
The upstream region of the H19 gene in primates
contains an Alu sequence (Figure 2). The Alu sequence
is a repeating element containing ~300 base pairs and
several CpG islands. Could the CpG islands in the H19
Alu sequence be involved in imprinting this gene?
Objectives
1. To isolate the upstream Alu sequence
on the imprinted H19 gene and
observe Alu sequence methylation
during spermatogenesis in the rhesus
monkey.
2. To determine if there is any correlation between Alu sequence methylation and the imprinted H19 gene
methylation.
Department of Biological Sciences, York College of Pennsylvania
Review of Literature
Expected Results
Research Design and Methods
Kierszenbaum 2002
Figure 4. Expected Results of methylated and unmethylated
upstream H19 Alu sequence.
• Methylation occurs on cytosine residues that are present
in dinucleotide motifs that consist of a 5’-cytosine followed
by a guanine (CpG) and requires the activity of DNA
methyltransferase (Figure 1).
Testes will be surgically
removed from a rhesus
monkey, placed in
medium RPMI-H
containing collagenase
IA and shaken until
seminiferous tubules
become separated from
each other.
• Methylation of paternal genes occurs during
spermatogenesis as cells undergo mitotic and meiotic
division.
Davis et al. 1999
• A 2kb region of paternal-allele specific DNA methylation
located from –2 to –4 kb relative to the H19 transcription
start site is present throughout development and has been
proposed as the allelic imprinting mark in mice.
Elongated
Spermatids
Round
Spermatids
Secondary
Spermatocyte
• Methylation of the imprinted region of H19 is acquired
during spermatogenesis.
Primary
Spermatocyte
Spermatogonia
Cells will be centrifuged
and separated using the
Staput Sedimentation
apparatus. Spermatogenic
cells will be identified
microscopically (fig. 3).
Rubin et al. 1994
• Southern blot hybridization combined with methylation
sensitive restriction digests reveal that Alu sequences are
undermethylated in mature sperm.
M
Hp
M
Hh
Hp
Hh
728
553
536
536
275
124
124
68
68
Table 1. Expected results based on previous experiments
• Methylation of this region was shown to be different for
maternal and paternal alleles.
• Alu methylation studies of sex cells and somatic cells
reveal unuique patterns between these two groups.
Unmethylated
Methylated
Figure 3. Cross section of
a testis indicating the
various sperm cell types
during spermatogenesis.
Genomic DNA will be isolated
using Qiagen DNA isolation
kit, follwed by digestion with
RsaI (GT/AC).
• The methylation of Alu sequences has not been studied
in developing sperm.
Upstream Alu
H19a
Alub
Spermatogonia
hypomethylated
hypomethylated
Primary
Spermatocyte
increased
methylation
increased
methylation
--
Secondary
Spermatocyte
methylation
increase
methylation
increase
--
Round
Spermatids
methylation
increase
methylation
increase
--
hypermethylation
Hyper-methylation Hypomethylation
Elongated
Spermatids
aDavis
et al. (1999)
bRubin
et al. (1994)
--
Sanford et al. 1987
Literature Cited
5’ CTGGGTGCGG TGGCTCACGA CCAACCTGGC
Davis, T.L., Trasler, J.M., Moss, S.B., Yang, G.J. and Bartolomei, M.S.
1999. Acquisition of the H19 methylation imprint occurs differentially on
the paternal and maternal alleles during spermatogenesis. Genomics
58:18-28.
• It is possible to isolate spermatogenic cells using tissue
digestion followed by differential centrifugation.
TAACACGGTG
AAACCCCATC
ATACAAAAAA
TTAGCCGGGT GAGGTGGTGG
GCGCCTGTAG TCCCACCTAC
TCTACTAAAA
TTGGGAGGCT
GAGGCAGGAG AATGGTGTGA ACCCGGGAGG
CGGAGCCTGC
AGTGAGCCGA GATCATGCCA
CTGCACTCCA
GCCTAGGGGA CAGAGCGAGA
CTCAAAAAAC
Southern Blot
AAAAAACAAA CAAAAA 3’
Figure 2. Upstream Alu sequence of the H19 gene. Highlighted areas indicate
methylation sites. Pink is MspI/HpaII and blue is HhaI.
Fragment
containing
H19 upstream
Alu sequence
(~728 bp)
+
Digested DNA will be
electrophoresed and
subjected to Southern Blot
hybridization to identify the
H19 gene.
Kierszenbaum, A.L. 2002. Genomic imprinting and epigenetic
reprogramming:unearthing the garden of forking paths. Molecular
Reproduction and Development 63:269-272.
Rubin, C.M., Vande, C.A., Teplitz, R.L. and Schmid, C.W. 1994. Alu
repeated DNA are differentially methylated in primate germ cells. Nucleic
Acids Research 22:5121-5127.
Sanford, J.P., Clark, H.J., Chapman, V.M. and Rossant, J. 1987.
Differences in DNA methylation during oogenesis and spermatogenesis
and their persistence during early embryogenesis in the mouse. Genes
and Development 1:1039-1046.
MspI C/CGG H19 will be cut from
the blot and digested
HpaII C/CGG with methylationHhaI GC/GC specific enzymes.
Acknowledgements
Deborah Ricker, Ph.D, YCP Research Mentor