mRNA expression of Hoxa10 gene and epigenetic marks on its regulatory regions in the uterus of non-exposed and glyphosate or a glyphosate-based herbicide exposed Wistar rats
; Doná, Florencia ; Milesi, Maria Mercedes ; Varayoud, Jorgelina Guadalupe Otros responsables
Fecha
Publicador:
Consejo Nacional de Investigaciones Científicas y Técnicas
Fecha de depósito:
6/09/2024
Fecha de creación:
2022-2022
Clasificación temática
Palabras clave
Resumen
In this work, we investigated the effect of perinatal exposure to a glyphosate-based herbicide (GBH) or glyphosate (Gly), the active ingredient, on the transcriptional regulation of Hoxa10 gene and epigenetic marks such as DNA methylation status and histone post-translational modifications (hPTMs) on Hoxa10 regulatory regions (RRs) and major epigenetic enzymes during the receptive stage in Wistar rats. GBH and Gly down-regulated Hoxa10 mRNA. GBH and Gly increased DNA methylation levels and Gly also induced higher levels than GBH in all the RRs analyzed. Both GBH and Gly enriched histone 3 (H3) and H4 acetylation in most of the RRs. While GBH caused higher H3 acetylation, Gly caused higher H4 acetylation in all RRs. Finally, GBH and Gly enhanced histone H3 lysine 27 trimethylation (H3K27me3) marker at 3 out of 4 RRs studied which was correlated with increased levels of the histone methyltransferase EZH2. In conclusion, exposure to GBH or Gly during perinatal period induces epigenetic modifications in RRs of uterine Hoxa10 gene.
Métodos
F0 dams were treated with a glyphosate-based herbicide (GBH) or glyphosate (Gly), the active ingredient, through a food dose of 2 mg Gly/kg bw/day from gestational day (GD) 9 up to lactational day 21. A F1 female offspring was randomly selected from each litter in the control, GBH- and Gly-exposed group. Then, F1 female rats reached sexual maturity and on postnatal day 90 they became pregnant with males of proven fertility. Finally, on the morning of GD5 (corresponding to the preimplantation period) F1 female rats were sacrificed and uterine samples were collected from control (n= 9), GBH (n= 8) and Gly (n= 8) rats from a different F0 dam. Transcripts levels of Hoxa10, DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b), histone deacetylases (Hdac-1 and Hdac-3) and histone methyltransferase (EZH2) were assessed by quantitative polymerase chain reaction (qPCR). Four CpG islands containing sites targeted by BstUI methylation-sensitive restriction enzyme and predicted transcription factors were identified in Hoxa10 gene. qPCR-based methods were used to evaluate DNA methylation and histone post-translational modifications status in four regulatory regions along the gene by performing methylation-sensitive restriction enzymes and chromatin immunoprecipitation assays, respectively. Glossary: Dnmt: DNA methyltransferase; EZH2: enhancer of zeste homolog 2, GBH: glyphosate based-herbicide; GLY: glyphosate (active ingredient); Hdac: histone deacetylase; H3: histone 3; H3K27me3, histone 3 lysine 27 trimethylation; H4: histone 4; Hoxa10, Homeobox A10; hPTMs, histone post-translational modifications; RR: regulatory region.
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