Investigating Regulatory Networks Through Transcription Factor Knockdown

Population genetics theory predicts that the unique inheritance pattern of the X chromosome, where it is heterogametic in one sex, causes DNA sequences on the X to exhibit a faster rate of evolution than similar sequences on autosomes. While work on this effect, termed "faster-X," has focused largely on sequence divergence, recent evidence suggests that a faster-X pattern of gene expression divergence also exists and that this pattern is resulting from both cis- and trans-regulatory changes. Preliminary analysis of a computationally predicted Drosophila transcription factor network has shown that X-linked transcription factors preferentially regulate targets on the X chromosome and that the relationship is reciprocal. It has been previously hypothesized that the observed trans-regulatory faster-X effect is due to protein sequence evolution of X-linked transcription factors. Here, we attempt to explore the relationship between X-linked transcription factors and their targets by using RNA interference to knockdown ten X-linked transcription factors with high predicted proportions of X-linked target genes and confirming or identifying their targets based on correlated regulation analysis from RNA-seq data. We do not find that this is a feasible method for large scale analysis of the transcription factor network because a singular RNAi methodology is not equally effective for all RNAi constructs. We also find potential targets of the transcription factor Htk, however these do not overlap with the computationally predicted targets.