Study Of Genetic And Epigenetic Determinants Of Gene Expression Regulation For Both Sl Genes In Response To Estrogen In Common Carp (Cyprinus Carpio)

Abstract

Somatolactin (SL), a fish hypophyseal hormone involved in background adaptation, reproduction and fatty acid metabolism might be affected by estrogenic endocrine disruptor compounds. Two sl transcripts were detected in pituitary of Cyprinus carpio, slα and slβ, with only about 47,6% identity of coding sequences, but conserved key features in the derived amino acid sequences such as cysteine number and position. At basal conditions slα showed higher mRNA levels than slβ and SLβ was immunodetected in different subregions of pars intermedia indicating spatiotemporal divergent expression patterns. When exposed to estrogen only slβ but not slα responded with increased expression in pituitary of male adult carp to 17β-estrogen treatment respect to control as shown by RT-qPCR analyses. The in depth comparative analyses of regulatory elements of slα and slβ revealed highly divergent, promoter regions besides the presence of Pit-1 binding sites in both, specially the one located in the neighborhood of TATA box was conserved. Indeed, in slβ but not in slα promoter a cognate Estrogen Response Element (ERE) half site was found. A functional assay on GH3/BH6 cells demonstrated that this ERE half site was directly related with differential response of sl genes to 17β-estrogen. No mayor differences were detected on CpG methylation of promoter and coding sequences of both sl genes in response to the estrogen treatment. However, slα showed a decreased methylation level respect to slβ promoter under basal conditions, suggesting a role of methylation on regulation of sl expression. slβ promoter showed a decrease in inactive chromatin marks in response to estrogen that correlated with increased transcript levels. Taken together these data suggest that sl paralogs diverged at genetic and epigenetic levels since last duplication event.