Long non coding RNAs and sex determining gene regulation in ruminants
Résumé
While microRNAs represent a well studied part of the non-coding genome, long non-coding transcripts are much more intricate, and are likely to contain as yet unidentified classes of molecules forming transcriptional regulatory networks. Long non-coding RNAs (ncRNAs) have long been considered as negative regulators, promoting chromatin silencing, but recently, several studies pointed out a role for a class of long ncRNAs in positive regulation of protein coding genes [1]. In order to identify long ncRNAs that could be implicated in testis or ovary differentiation, a high throughput RNA sequencing project was initiated in the bovine species. Strand-orientated libraries were prepared from transcripts of early testes and ovaries and next generation sequencing technologies (NGS) were used to identify all RNAs (coding and non coding) expressed at this early stage in gonads (RNA-sequencing). Our attention will be focused on loci known to enclosed sex determining genes, to highlight putative regulatory function of long ncRNAs, as it looks like to exist for one of the most important genes for the female gonad, the transcription factor FOXL2. Indeed, to date, very few numbers of long ncRNAs are known to be expressed during sex determination or gonadic differentiation. Interestingly in the goat, FOXL2 gene is lying inside a complex locus containing various long ncRNAs. This locus was characterized following studies on the mutation responsible for the Polled Intersex Syndrome (PIS) in goats. This mutation induces hornlessness (as soon as heterozygous) and XX sex-reversal (when homozygous) [2]. The PIS mutation consists in an 11.7kb deletion of putative regulatory regions (called the PIS element) located at 300kb upstream of FOXL2[3]. Other genes of this locus, coding for long ncRNAs are also misregulated in PIS+/- or PIS-/- goats [3,4].The PIS mutation affects the expression of FOXL2 and the long ncRNAs, inducing an ectopic expression of these genes in hornbuds (resulting in hornless phenotype), while they are repressed in XX gonads, leading to testes differentiation (female to male sex-reversal - XX males). Among the PIS long ncRNAs, two are well characterized PISRT1 (PIS regulated Transcript 1) [5] that is close to the PIS element and PFOXic (Promoter FOX inverse complementary) expressed from FOXL2 bidirectional promoter [4]; but recent data demonstrated the existence of transcriptional activities in a 100kb area encompassing the PIS element. Interestingly depending on FOXL2-expressing tissues examined (mutant hornbuds or ovaries), two distinct regions showed expression of long ncRNAs. These ncRNAs were expressed from one or both strands, suggesting multiple transcription start sites. It appears that their transcription could be a prerequisite to FOXL2 expression. Chromatin structure of this ncRNAs containing area was evaluated, comparing different tissues. Differential epigenetic marks have been observed and will be presented in regards to the transcriptional activity of this region. The existence of a direct link between ncRNAs expression, chromatin conformation and long-range regulation of FOXL2 gene is hypothesized and should be determined. In addition, specific-strand RNA-sequencing studies performed on early fetal bovine gonads, may point out new long ncRNAs co-expressed together with other sex determining genes. These NGS data may participate to the characterization of crucial regulatory regions and will be presented depending on the progress of bioinformatic analysis.