BACKGROUND: Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. RESULTS: Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis. CONCLUSIONS: These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation.

Bioinformatics Group Babraham Institute Cambridge CB22 3AT UK

Department of Epigenetics and Molecular Carcinogenesis The University of Texas M D Anderson Cancer Center Smithville TX 77030 USA

Department of Histology and Cell Biology School of Medicine Yokohama City University Yokohama 236 0004 Japan

Epigenetics Programme Babraham Institute Cambridge CB22 3AT UK Biotech Research and Innovation Centre University of Copenhagen 2200 Copenhagen Denmark

Epigenetics Programme Babraham Institute Cambridge CB22 3AT UK Centre for Trophoblast Research University of Cambridge Cambridge CB2 3EG UK

Epigenetics Programme Babraham Institute Cambridge CB22 3AT UK Computer Science Department KASIT University of Jordan Amman Jordan

Epigenetics Programme Babraham Institute Cambridge CB22 3AT UK Friedrich Miescher Institute for Biomedical Research 4058 Basel Switzerland

Epigenetics Programme Babraham Institute Cambridge CB22 3AT UK Laboratory of Developmental Biology and Genetics Department of Molecular Biology University of South Bohemia 37005 Ceske Budejovice Czech Republic

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$a Gahurova, Lenka $u Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT UK. Laboratory of Developmental Biology and Genetics, Department of Molecular Biology, University of South Bohemia, 37005 Ceske Budejovice, Czech Republic.

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$a Transcription and chromatin determinants of de novo DNA methylation timing in oocytes / $c L. Gahurova, SI. Tomizawa, SA. Smallwood, KR. Stewart-Morgan, H. Saadeh, J. Kim, SR. Andrews, T. Chen, G. Kelsey,

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$a BACKGROUND: Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. RESULTS: Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis. CONCLUSIONS: These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation.

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$a Tomizawa, Shin-Ichi $u Department of Histology and Cell Biology, School of Medicine, Yokohama City University, Yokohama, 236-0004 Japan.

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$a Smallwood, Sébastien A $u Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT UK. Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland.

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$a Stewart-Morgan, Kathleen R $u Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT UK. Biotech Research and Innovation Centre (BRIC), University of Copenhagen, 2200 Copenhagen, Denmark.

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$a Saadeh, Heba $u Epigenetics Programme, Babraham Institute, Cambridge, CB22 3AT UK. Computer Science Department, KASIT, University of Jordan, Amman, Jordan.

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