109 Dosage Compensation of the X Chromosome in Ovine Embryos, Late Gestation, and Adult Somatic Tissues

Abstract

Deviations from proper gene dosage of the autosome range from severe to lethal consequences in mammals. Eutherian males (XY), however, have reduced gene dosage compared with females (XX) due to a single X and deteriorating Y chromosome. This dosage imbalance is resolved through X chromosome dosage compensation, according to Ohno’s hypothesis: X-linked gene expression is doubled in both males and females to balance expression of the X chromosome and autosomes. To compensate for doubled X chromosome expression in females, X chromosome inactivation (XCI) inactivates a single X chromosome in each cell. Although these mechanisms have been well studied in mice and humans, controversies exist due to the analysis and interpretation of RNA sequencing data. Here we described X chromosome dosage compensation in the sheep. Twelve ewes were fed 100% (control), 60% (restricted), or 140% (overfed) of the National Research Council requirements for a ewe pregnant with twins (NRC, 1985; Nutrient Requirements of Sheep, 6th ed.). Day 135 brain, lung, and kidney tissues were collected from fetuses of the control, restricted, and overfed groups (n = 7, 4, and 4; respectively). RNA seq libraries were prepared using the Illumina TruSeq stranded mRNA kit and sequenced on the NextSEqn 500 (Illumina Inc., San Diego, CA, USA). Two additional RNA-seq datasets were downloaded from Sequence Read Archive (SRA), including Day 14 embryos (PRJNA254105), and adult and juvenile heart, brain, liver, muscle, rumen, and female- and male-specific tissues (PRJEB6169). The RNA-seq data were trimmed and mapped to the ovine reference genome assembly Oar_v4.0 using Hisat2 (version 2.0.5; https://ccb.jhu.edu/software/hisat2/index.shtml) aligner. The mRNA level of each gene was estimated by transformed transcripts per kilobase million (TPM) and was quantified using IsoEM (version 1.1.4; http://dna.engr.uconn.edu/). The relative expression of X to autosomal (A)(RXE) was calculated using RXE = log₂(X expression) – log₂(A expression) with an average of 486 X-linked genes and 13,001 autosomal genes after TPM >1 filtering. RXE ≥0 (or X:A ratio ≥ 1); <0, = –1 indicate complete, incomplete, or no dosage compensation; respectively. Control, restricted, and overfed ovine fetal somatic tissues displayed incomplete dosage compensation. Incomplete dosage compensation was also observed in juvenile and adult somatic major organs and female specific tissues. Brain tissues, apart from the cerebellum, displayed complete dosage compensation with an RXE range of 0 to 0.16. An interesting pattern was observed in the male specific tissues with complete dosage compensation in the epididymis (RXE = 0.32) and incomplete dosage compensation in the testes (RXE = –0.84). No significant RXE differences were observed between ovine female and male somatic tissues, supporting Ohno’s hypothesis of balanced expression of X-linked genes to autosomal genes. Our results indicate that a mechanism for dosage compensation exists in the sheep, although it is largely incomplete.