Dosage Compensation and Gene Expression of the X Chromosome in Sheep

Abstract

Ohno's hypothesis predicts that the expression of the single X chromosome in males needs compensatory upregulation to balance its dosage with that of the diploid autosomes. Additionally, X chromosome inactivation ensures that quadruple expression of the two X chromosomes is avoided in females. These mechanisms have been actively studied in mice and humans but lag behind in domestic species. Using RNA sequencing data, we analyzed the X chromosome upregulation in sheep fetal tissues from day 135 of gestation under control, over or restricted maternal diets (100%, 140% and 60% of National Research Council Total Digestible Nutrients), and in conceptuses, juvenile, and adult somatic tissues. By computing the mean expression ratio of all X-linked genes to all autosomal genes (X:A), we found that all samples displayed some levels of X chromosome upregulation. The degrees of X upregulation were not significant (P-value = 0.74) between ovine females and males in the same somatic tissues. Brain, however, displayed complete X upregulation. Interestingly, the male and female reproduction-related tissues exhibited divergent X dosage upregulation. Moreover, expression upregulation of the X chromosome in fetal tissues was not affected by maternal diets. Maternal nutrition, however, did change expression levels of several X-linked genes, such as sex determination genes SOX3 and NR0B1 In summary, our results showed that X chromosome upregulation occurred in nearly all sheep somatic tissues analyzed, thus support Ohno's hypothesis in a new species. However, the levels of upregulation differed by different subgroups of genes such as those that are house-keeping and "dosage-sensitive".

Keywords: Maternal nutrition; Ohno’s hypothesis; Ovine; X chromosome upregulation.

Figure 1

Boxplots of log₂-transformed relative expression of the X chromosome (RXE) and each autosome pair (RGE) in major tissues (A) and brain (B) of juvenile and adult sheep. Red dots: mean RXEs for all replicates within a tissue type. Black dots: mean RGEs for each autosome pair. Numbers by black dots: autosomes whose RGEs fell outside of the expression quartiles for the tissue. Red dotted line: the border for complete (above line) and incomplete (below line) dosage compensation. The X:A ratio was calculated as the Relative X expression, RXE = log₂ (X) − log₂ (A), the difference between the log₂-transformed mean TPM values of X and A. An RXE value of 0 means the expression of X and autosome is equal, suggesting X dosage compensation. Positive and negative RXE values indicate complete and incomplete dosage compensation, respectively. An RXE of -1, however, represents the lack of X dosage compensation. RGE of each autosome pair over all other chromosomes was used to evaluate the deviation of X expression to autosomes.

Figure 2

Boxplots of log₂-transformed relative expression of the X chromosome (RXE) and each autosome pair (RGE) in female- and male-specific tissues. Red dots: RXEs for all replicates within a tissue type. Black dots: RGEs for each autosome pair. Numbers by black dots: autosomes whose RGEs fell outside of the expression quartiles for the tissue. Red dotted line: the border for complete (above line) and incomplete (below line) dosage compensation. The X:A ratio was calculated as the Relative X expression, RXE = log₂ (X) − log₂ (A), the difference between the log₂-transformed mean TPM values of X and A. An RXE value of 0 means the expression of X and autosome is equal, suggesting X dosage compensation. Positive and negative RXE values indicate complete and incomplete dosage compensation, respectively. An RXE of -1, however, represents the lack of X dosage compensation. RGE of each autosome pair over all other chromosomes was used to evaluate the deviation of X expression to autosomes.

Figure 3

Boxplots of log₂-transformed expression of the X chromosome (RXE) and each autosome pair by fetal tissues from mothers under different nutritional treatments: Control (A), Overfed (B) and Restricted (C). Red dots: RXEs for all replicates within a treatment group. Black dots: RGEs for each autosome pair. Numbers by black dots: autosomes whose RGEs fell outside of the expression quartiles for the tissue. Red dotted line: the border for complete (above line) and incomplete (below line) dosage compensation. The X:A ratio was calculated as the Relative X expression, RXE = log₂ (X) − log₂ (A), the difference between the log₂-transformed mean TPM values of X and A. An RXE value of 0 means the expression of X and autosome is equal, suggesting X dosage compensation. Positive and negative RXE values indicate complete and incomplete dosage compensation, respectively. An RXE of -1, however, represents the lack of X dosage compensation. RGE of each autosome pair over all other chromosomes was used to evaluate the deviation of X expression to autosomes.

Figure 4

Boxplot of RXE values in the categories of “All genes”, “Expressed genes”, “Genes subject to XCI (removal of genes in PAR)” and “Dosage sensitive genes”. Red dotted line: the border for complete (above line) and incomplete (below line) dosage compensation.