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Molecular genetic analysis of functional FMR1/FMRP expression on human folliculogenesis and ovarian reserve

Youness, Berthe

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Abstract

Aim of this thesis was to investigate various possible mechanisms involved in FMR1 gene epigenetic expression control in human granulosa cells. Systematic CpG site methylation analysis within the extended FMR1 promoter domain in COV434 and in primary human granulosa cells by subsequent sequence analysis revealed four distinct regions with a unique CpG site methylation pattern: “FMR1-UMR, FMR1-DMR1;-DMR2 and –DMR3”, of which two (FMR1-DMR1 and –DMR2) with distinct patterns were found earlier in leukocytes. Most interesting, FMR1-DMR3 contained a conserved double binding site for E2F1, binding only to unmethylated CpG sites. I experimentally confirmed that E2F1 binds to its predicted consensus sequence within FMR1-DMR3 but only when containing an unmethylated CpG 94 site. In human primary granulosa cells, the CpG 94 site methylation rate within FMR1-DMR3 was found to be dependent on patient ovarian reserve; being lower when the amount of matured follicles seems low (i.e., in POR patients). Increased binding of E2F1 to CpG 94 is the consequence activating FMR1 transcript expression. This, however, results in reduction of the cellular FMRP protein level due to the well-known gene ́s negative feedback loop mechanism. My experimental data therefore indicate that there is an epigenetic control mechanism of FMR1/FMRP expression in human granulosa cells that is functionally associated with the rate of E2F1 binding to CpG94 located in FMR1-DMR3 and that its impairment probably interferes with women ovarian reserve. FMR1-UMR a completely CpG unmethylated region, covered the FMR1 minimal promoter and the CGG repeat block in exon1. CpG sites located within FMR1-UMR were demethylated on both gene alleles but only in granulosa cells indicating that FMR1 transcription is activated on both alleles in these cells. Escape from X inactivation seemed to be focused on FMR1-UMR, since outside of this region, the CpG sites methylation pattern reflected the presence of one CpG methylated and one unmethylated allele, respectively three differentially methylated regions coined DMR1, DMR2 and DMR3, respectively. Analysis of the complex splicing pattern of the Anstisense FMR1 gene (ASFMR1), a long non coding RNA expressed in antisense direction, revealed three novel and probably granulosa cell specific initiation sites characterized by at least 8 different ASFMR1 transcript splice forms in human granulosa cells, not previously described in the literature. Similar to FMR1, ASFMR1 expression varied between patients with different ovarian reserve. Taken together, these observations suggest that in addition to the CpG methylation control of FMR1 expression, ASFMR1 expression may also contribute to the variable ovarian reserve observed in patients entering the in vitro fertilization (IVF) clinic. Analysis of putative signal pathways involved in the regulation of FMR1-gene expression in human granulosa cells, by microarray assays after FMR1 gene knock down revealed 748 genes with significant differential expression. They could be grouped into 7 main signal pathways covering a wide range of regulatory networks, including cell cycle regulation, apoptosis, mRNA decay and vesicle transport. Among the most important signal pathways is the PI3K/AKT/mTOR pathway already known to be involved in primordial follicle activation and follicular pool maintenance. Inhibition of FMR1 gene expression induced an increase of mTOR, AKT, S6K at both transcripts and protein levels. Most interesting S6K expression reached a statistical significant difference at both mRNA (p=0.05) and protein level (p=0.03). Another identified signal pathway was the methionine salvage pathway (MTA). This pathway is highly conserved from yeast to human and was reported to be involved in Drosophila fecundity. These results provide new starting points to study functional FMR1/FMRP expression in human folliculogenesis and ovarian reserve.

Item Type: Dissertation
Supervisor: Vogt, Prof. Dr. Hans-Peter
Place of Publication: Heidelberg
Date of thesis defense: 10 January 2018
Date Deposited: 19 Jan 2018 08:23
Date: 2019
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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