Shared Transcriptional Machinery at Homologous Alleles Leads to Reduced Transcription in Early Drosophila Embryos

Abstract

The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. We explored the possibility that interallelic interactions between two homologous alleles might affect gene regulation. Using an MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcripts of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the corresponding hemizygous allele, suggesting the possibility of allelic competition in homozygotes. However, the competition was not observed when the enhancer-promoter interaction was weakened by placing the reporter construct in a different chromosome location or by moving the enhancer further away from the promoter. Moreover, the reporter gene showed reduced transcriptional activity when a partial transcription unit (either an enhancer or reporter gene only) was in the homologous position. We propose that the transcriptional machinery that binds both the enhancer and promoter regions, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. We showed that the degree of allelic interference increased over developmental time as more Pol II was needed to activate zygotic genes. Such allelic competition was observed for an endogenous gene as well. Our study provides new insights into the role of 3D interallelic interactions in gene regulation.

Synergistic insulation of regulatory domains by developmental genes and clusters of CTCF sites

“…Instead, promoter competition might involve non-structural mechanisms whereby multiple promoters compete for rate-limiting factors (e.g. TFs, GTFs, RNA Pol2) present within shared transcriptional hubs 42,43,73 .…”

“…Instead, promoter competition might involve non-structural mechanisms whereby multiple promoters compete for rate-limiting factors (e.g. TFs, GTFs, RNA Pol2) present within shared transcriptional hubs 42,43,73 . Although these models and the putative rate-limiting factors await experimental validation, we speculate that nearby promoters with strong enhancer responsiveness and similar architecture (e.g.…”

“…However, more recent observations support non-structural mechanisms whereby promoters and enhancers share transcriptional hubs/condensates 39,40 , within which promoters might compete for rate-limiting factors (e.g. TFs, GTFs, RNA Pol2) required for gene transcription 42,43 . In contrast, promoter-driven enhancer blocking occurs when the preferred gene prevents the expression of its neighbours only if placed between the shared enhancer/s and the other gene/s, thus resembling how insulators work 21,36 .…”

Mitfover-expression leads to microphthalmia and coloboma inMitf-cremice

“…However, work by Pal-Bhadra et al 34 has suggested that high copy number transgenes can instigate homology mediated co-suppression of its unlinked endogenous counterpart. In addition, other studies have proposed that transgenes can affect neighbouring genes in cis through a distance dependent manner due to competition for transcriptional machinery and transcription factors, as well as disruption of topologically associated domains (TADs) 31,33,[35][36][37] .…”