title: Methylation analysis of histone H4K12ac-associated promoters in sperm of healthy donors and subfertile patients.
abstract: BACKGROUND
  Histone to protamine exchange and the hyperacetylation of the remaining histones are hallmarks of spermiogenesis. Acetylation of histone H4 at lysine 12 (H4K12ac) was observed prior to full decondensation of sperm chromatin after fertilization suggesting an important role for the regulation of gene expression in early embryogenesis. Similarly, DNA methylation may contribute to gene silencing of several developmentally important genes. Following the identification of H4K12ac-binding promoters in sperm of fertile and subfertile patients, we aimed to investigate whether the depletion of histone-binding is associated with aberrant DNA methylation in sperm of subfertile men. Furthermore, we monitored the transmission of H4K12ac, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) from the paternal chromatin to the embryo applying mouse in vitro fertilization and immunofluorescence.
  
  
  RESULTS
  Chromatin immunoprecipitation (ChIP) with anti-H4K12ac antibody was performed with chromatin isolated from spermatozoa of subfertile patients with impaired sperm chromatin condensation assessed by aniline blue staining. Fertile donors were used as control. DNA methylation analysis of selected H4K12ac-interacting promoters in spermatozoa was performed by pyrosequencing. Depletion of binding sites for H4K12ac was observed within the following developmentally important promoters: AFF4, EP300, LRP5, RUVBL1, USP9X, NCOA6, NSD1, and POU2F1. We found 5% to 10% hypomethylation within CpG islands of selected promoters in the sperm of fertile donors, and it was not significantly altered in the subfertile group. Our results demonstrate that the H4K12ac depletion in selected developmentally important promoters of subfertile patients was not accompanied by a change of DNA methylation. Using a murine model, immunofluorescence revealed that H4K12ac co-localize with 5mC in the sperm nucleus. During fertilization, when the pronuclei are formed, the paternal pronucleus exhibits a strong acetylation signal on H4K12, while in the maternal pronucleus, there is a permanent increase of H4K12ac until pronuclei fusion. Simultaneously, there is an increase of the 5hmC signal and a decrease of the 5mC signal.
  
  
  CONCLUSIONS
  We suggest that aberrant histone acetylation within developmentally important gene promoters in subfertile men, but not DNA methylation, may reflect insufficient sperm chromatin compaction affecting the transfer of epigenetic marks to the oocyte.
authors: Section Molecular Andrology, Biomedical Research Center Seltersberg, Justus Liebig University of Giessen, 35392 Giessen, Germany.
authors: Markus
authors: M
authors: Vieweg
authors: Laboratory of Reproductive Biology, Institute of Biotechnology AS CR, v.v.i., Videnska 1083, 14220 Prague 4, Czech Republic.|Biocev Group, Department of Zoology, Faculty of Science, Charles University in Prague, 12844 Prague, Czech Republic.
authors: Katerina
authors: K
authors: Dvorakova-Hortova
authors: Biocev Group, Department of Zoology, Faculty of Science, Charles University in Prague, 12844 Prague, Czech Republic.
authors: Barbora
authors: B
authors: Dudkova
authors: Department of Urology, Pediatric Urology and Andrology, Justus Liebieg University of Giessen, 35392 Giessen, Germany.
authors: Przemyslaw
authors: P
authors: Waliszewski
authors: Fertility Center, 35578 Wetzlar, Germany.
authors: Marie
authors: M
authors: Otte
authors: Fertility Center, 35578 Wetzlar, Germany.
authors: Berthold
authors: B
authors: Oels
authors: Fertility Center, 35578 Wetzlar, Germany.
authors: Amir
authors: A
authors: Hajimohammad
authors: Fertility Center, 65189 Wiesbaden, Germany.
authors: Heiko
authors: H
authors: Turley
authors: Fertility Center, 65189 Wiesbaden, Germany.
authors: Martin
authors: M
authors: Schorsch
authors: Department of Urology, Pediatric Urology and Andrology, Justus Liebieg University of Giessen, 35392 Giessen, Germany.
authors: Hans-Christian
authors: HC
authors: Schuppe
authors: Department of Urology, Pediatric Urology and Andrology, Justus Liebieg University of Giessen, 35392 Giessen, Germany.
authors: Wolfgang
authors: W
authors: Weidner
authors: Section Molecular Andrology, Biomedical Research Center Seltersberg, Justus Liebig University of Giessen, 35392 Giessen, Germany.
authors: Klaus
authors: K
authors: Steger
authors: Section Molecular Andrology, Biomedical Research Center Seltersberg, Justus Liebig University of Giessen, 35392 Giessen, Germany.
authors: Agnieszka
authors: A
authors: Paradowska-Dogan
keywords: H4K12ac in spermatozoa; Promoter methylation; Pyrosequencing; Subfertility; μChIP