Differences in the pattern of X-linked gene expression between fetal bovine muscle and fibroblast cultures derived from the same muscle biopsies

Abstract

The sex determination system in mammals creates an imbalance between males and females in the number of X chromosomes. This imbalance is compensated through transcriptional silencing of one of the two X chromosomes in female diploid cells by epigenetic modifications. Although common for mammals, X inactivation shows marked species-specific differences in mechanisms and end results, and provides a unique opportunity to study epigenetic regulation of gene expression. The aim of the present study was to establish the expression pattern of selected X-linked genes in bovine fetal muscle tissue and muscle fibroblast cultures in order to follow possible modifications at the transcriptional level attributable to in vitro culture. We used heterologous cDNA microarray hybridization and quantitative real-time PCR to study the pattern of expression of X-linked genes including SLC25A6, GAB3, MECP2, RPS4X, JARID1C, UBE1, BIRC4 and SLC16A2. Quantitative real-time PCR analysis in fetal bovine muscle showed higher transcript levels in females for all X-linked genes tested with the exception of SLC25A6, with differences being significant for RPS4X, JARID1C and UBE1. The expression in fibroblast cultures derived from the same samples differed, with significantly higher levels for UBE1, GAB3 and BIRC4, while the rest of the panel of X-linked genes remained unchanged. The changed expression pattern in vitro, probably reflecting modifications in the epigenetic mechanisms that regulate transcriptional activity and gene silencing in X inactivation, has important implications for the advancement of new biotechnologies such as somatic cell nuclear transfer and stem cell therapy.

Spatial Distribution of Histone Isoforms on the Bovine Active and Inactive X Chromosomes

“…Previous studies from this laboratory had shown that the expression status of genes on the bovine X chromosome is more similar to that of the human X than the murine X and that the levels of expression of many of these genes are highly variable and under epigenetic control [Nino-Soto et al, 2005. Since histone modifications and histone variants are intimately involved in epigenetic regulation of gene expression we undertook a study to examine the spatial relationship of different histone variants on the bovine X chromosome using an immunostaining approach (with 4 different antibodies) in the context of DNA replication, heterochromatinization and gene silencing.…”

Impact of in vitro production techniques on the expression of X‐linked genes in bovine (bos taurus) oocytes and pre‐attachment embryos

“…Histone 2A (H2A) was chosen as autosomal control based on previous reports (Robert et al, 2002;Lonergan et al, 2003a). Design of specific PCR primers and quantification of transcripts using quantitative realtime PCR was performed as previously described (Nino-Soto et al, 2005). Briefly, PCR products for each gene tested were used to build specific standard curves to perform absolute quantification (Bustin, 2000) in a Light Cycler instrument with the Light Cycler FastStart DNA Master Mix SYBR Green I kit.…”

Rare Missense and Synonymous Variants in UBE1 Are Associated with X-Linked Infantile Spinal Muscular Atrophy

“…However, as has been reported, UBE1 displays significant differences in gene expression depending on whether it is analyzed in native tissue or in cell cultures derived from the same biopsies. 13 Therefore, we performed real-time PCR in white blood cells of the still living index patient of family #15. PCR amplification and detection was performed on a Sequence Detection System (ABI PRISM 7000, Applied Biosystems) applying the standard two-step protocol (45 cycles, annealing temperature 61 C) recommended by ABI.…”