1. Methylation of an upstream Alu sequence on the Imprinted H19 gene during spermatogenesis in rhesus monkeys Amanda Stafford Department of Biological Sciences, York College of Pennsylvania 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 methyl- transferase, 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? Review of Literature Kierszenbaum 2002 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). 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. Methylation of this region was shown to be different for maternal and paternal alleles. Methylation of the imprinted region of H19 is acquired during spermatogenesis. Rubin et al. 1994 Alu methylation studies of sex cells and somatic cells reveal unuique patterns between these two groups. Southern blot hybridization combined with methylation sensitive restriction digests reveal that Alu sequences are undermethylated in mature sperm. The methylation of Alu sequences has not been studied in developing sperm. Sanford et al. 1987 It is possible to isolate spermatogenic cells using tissue digestion followed by differential centrifugation. 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 corre- lation between Alu sequence meth- ylation and the imprinted H19 gene methylation. Research Design and Methods Genomic DNA will be isolated using Qiagen DNA isolation kit, follwed by digestion with RsaI (GT/AC). Southern Blot H19 will be cut from the blot and digested with methylation- specific enzymes. + MspI C/CGG HpaII C/CGG HhaI GC/GC M Hp Hh Expected Results Literature Cited 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. 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. Acknowledgements Deborah Ricker, Ph.D, YCP Research Mentor 728 536 124 68 553 536 275 124 68 Table 1. Expected results based on previous experiments Upstream Alu H19 a Alu b Spermatogonia Primary Spermatocyte Secondary Spermatocyte Round Spermatids Elongated Spermatids hypomethylated -- increased methylation -- methylation increase -- methylation increase -- hypermethylation Hyper-methylationHypo- methylation a Davis et al. (1999) b Rubin et al. (1994) Elongated Spermatids Secondary Spermatocyte Primary Spermatocyte Round Spermatids Spermatogonia Figure 4. Expected Results of methylated and unmethylated upstream H19 Alu sequence. 5’ CTGGGTGCGG TGGCTCACGA CCAACCTGGC TAACACGGTG AAACCCCATC TCTACTAAAA ATACAAAAAA TTAGCCGGGT GAGGTGGTGG GCGCCTGTAG TCCCACCTAC TTGGGAGGCT GAGGCAGGAG AATGGTGTGA ACCCGGGAGG CGGAGCCTGC AGTGAGCCGA GATCATGCCA CTGCACTCCA GCCTAGGGGA CAGAGCGAGA CTCAAAAAAC AAAAAACAAA CAAAAA 3’ Figure 3. Cross section of a testis indicating the various sperm cell types during spermatogenesis. 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. Cells will be centrifuged and separated using the Staput Sedimentation apparatus. Spermatogenic cells will be identified microscopically (fig. 3). Fragment containing H19 upstream Alu sequence (~728 bp) Digested DNA will be electrophoresed and subjected to Southern Blot hybridization to identify the H19 gene. Figure 1. DNA methyltransferase aiding in attaching a methyl group to a 5’-cytosine of a CpG island. Figure 2. Upstream Alu sequence of the H19 gene. Highlighted areas indicate methylation sites. Pink is MspI/HpaII and blue is HhaI. Methylated Unmethylated