Spike-in normalized analyses of gene expression in the set-4(n4600) null mutant suggest that autosomal repression results in a net effect of X derepression.
(A) Schematic representation of the spike-in mRNA-seq experiment. Here we counted and mixed in C. briggsae L1s with C. elegans L1s prior to total mRNA isolation. Since the spiked in C. briggsae worms are the same and mixed-in at the same proportion, we normalized expression from C. elegans to that of C. briggsae. Normalization was done by determining the coefficients of a linear regression that equalized C. briggsae expression ratio between mutant and wild type, and applying these parameters to the C. elegans expression ratios (S8B Fig). (B) Boxplot shows distribution of corrected log2 fold-changes in mRNA level between set-4(n4600) and wild type L1 larvae. (C) Comparison of differential expression values determined by spike-in mRNA-seq and RT-qPCR for a number of genes on the X and autosomes. Since RT-qPCR also relies on the assumption that the total RNA does not change between wild type and mutant, we used amplification of two C. briggsae genes to correct expression difference between wild type and mutant (see Materials and Methods). Darker bars show RT-qPCR relative expression (mutant/wild type) values and lighter bars show spike in corrected values. Circles show corresponding mRNA-seq differential expression ratios with (red) and without (pink) spike in correction. Error bars represent relative the standard error of the mean from 3 biological replicates. (D) Interpretation of the spike-in mRNA-seq and RT-qPCR data suggests that stronger repression from the autosomes in the set-4(n4600) mutant results in a relative increase from the X. (E) Current model for the temporal dynamics and the role of DCC and H4K20me1 in X chromosome repression. Solid arrows and their thickness indicate the level of repression. Dashed arrow represents a possible role for H4K20me1 in strengthening DCC mediated repression. Within the developmental timeline, early stage embryos before and after DCC activation, comma stage embryos, and larva are depicted. Ovals represent nuclei with DCC (top) and H4K20me1 (bottom) signals in pink and yellow. For simplicity, X chromosome localization is represented as darker colors in a single subnuclear domain. In early embryogenesis, as the DCC starts loading onto the X chromosomes, it starts repression. DCC binding leads to H4K20me1 increase on the X chromosome, which contributes slightly to repression in later development.