Interchromosomal communication coordinates intrinsically stochastic expression between alleles

Abstract

Sensory systems use stochastic mechanisms to diversify neuronal subtypes. In the Drosophila eye, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) determines a random binary subtype choice in R7 photoreceptors. Here, we show that a stochastic, cell-autonomous decision to express ss is made intrinsically by each ss locus. Stochastic on or off expression of each ss allele is determined by combinatorial inputs from one enhancer and two silencers acting at long range. However, the two ss alleles also average their frequency of expression through up-regulatory and down-regulatory interallelic cross-talk. This inter- or intrachromosomal long-range regulation does not require endogenous ss chromosomal positioning or pairing. Therefore, although individual ss alleles make independent stochastic choices, interchromosomal communication coordinates expression state between alleles, ensuring that they are both expressed in the same random subset of R7s.

Figure 1. The stochastic decision to express ss is made early and maintained

A. Ss is absent from pR7s allowing for Rh3 expression. Ss is expressed in yR7s activating Rh4 and repressing Rh3. B. Stochastic distribution of Rh3- and Rh4-expressing R7s. C. Ss is expressed in a random subset of R7s throughout development. Pros marks all R7s.

Figure 2. The cis-regulatory logic controlling intrinsically stochastic ss expression

A. ss locus schematic. F = Fragment; red boxes = silencers; blue box = InterCom element; purple box = minimal promoter; green box = R7/R8 enhancer; gray circles = untranslated exons; yellow circles = translated exons; arrows = transcriptional starts. For B–E, white circles indicate expression and gray circle indicate no expression. B. Mini-gene1 is expressed in all R7s and R8s. C. Sal is expressed in all R7s and R8s. D. Ss expression is completely lost in sal mutants. E. Ectopic Sal expression in svp mutants causes Ss expression in a random subset of PRs. F. Mini-gene2 induces expression in a subset of R7s independently of endogenous Ss/Rh4 expression. Mini-gene2 localizes to the nucleus whereas Rh4/Ss localizes to membranous rhabdomere structures. The four possible combinations of expression are observed: 1. white solid ovals = Mini-gene2 and Ss/Rh4; 2. white dashed ovals = Ss/Rh4 only; 3. gray solid ovals = Transgene only; and 4. gray dotted ovals = no expression. G. Four expression combinations in F. H–J. Models for random expression decisions H. The ss locus randomly assumes one of two (i.e. active and repressed) DNA looping configurations. I. One silencer facilitates the nucleation of closed chromatin state spreading from the other silencer. J. One silencer lowers expression in all R7s whereas the other specifically provides the stochastic input (through looping or spreading).

Figure 3. ss regulatory regions upregulate and downregulate expression frequency through interchromosomal communication

A. Wild type ss locus over deficiency1. B. transgene4 upregulates expression frequency from the endogenous ss gene in cis. C. Quantification of A, B, and D D. transgene4 upregulates expression frequency from the endogenous ss gene in trans. E. transgene4 upregulates expression, though less efficiently, from the ss locus on the non-homologous 2^nd chromosome. F. Quantification of E. G. Ss/Rh4 is not expressed in ss^{low freq1} hemizygous mutants. H. The normal regulatory regions of the ss^{prot null1} allele upregulate Ss/Rh4 expression from the ss^{low freq1} allele. I. Wild type ss homozygous loci. J. The regulatory regions of the ss^{low freq1} allele downregulate Ss/Rh4 expression from the wild type ss allele. K. Quantification of G–J.

Figure 4. Interchromosomal communication coordinates expression from ss alleles

A. Rh3 is expressed in pR7s and Rh4 is expressed in yR7s in wild type hemizygous animals. B. Rh3 is expressed in pR7s and Rh4 and Rh3 are expressed in yR7s in ss^trunc hemizygous animals. C. In ss^{wild type(inv2)}/ss^trunc animals, Rh3 and Rh4 are always expressed exclusively. D. In ss^{low freq1}/ss^trunc animals, the normal regulatory regions of the ss^trunc allele upregulate expression from ss^{low freq1} into the same subset of R7s since Rh3 and Rh4 are expressed exclusively. E–G. Models for the coordination of expression state through interchromosomal communication E. A temporally distinct two-step mechanism involving both alleles making independent expression decisions followed by an activating and repressing tug of war. F. A temporally distinct two-step mechanism in which one allele makes the decision and then imposes the decision onto the other naïve allele. G. A mechanism involving contemporaneous decisions that average the activating and repressing inputs from each allele.