trxG regulators associated with highly expressed genes, not genome reorganization Robert Cornman, USGS 27 June 2013 I do not believe these data should be interpreted as evidence that trxG regulation promotes the aggregation of target genes into physical clusters over evolutionary time. I believe the clustering reflects primarily tandem duplication of trxG-responsive genes. While the two mechanisms are not mutually exclusive, segmental duplication is a sufficient explanation founded on well studied phenomena, and can propagate both protein-coding sequences and cis-regulatory elements simultaneously. In contrast, I am unaware of a theoretical or empirical basis for arguing a fitness advantage of a clustered genotype over a dispersed genotype with respect to trxG targets. The authors acknowledge that their clustering analysis did not consider the contribution of tandem duplication of paralogs. They do note, however, that the conserved clustering of trx-responsive genes largely reflects the very strong trend of cuticular proteins to be clustered. (Indeed, ash2-responsive genes were much less clustered, and that gene list includes only a few cuticular proteins.) In Drosophila, physical clustering of cuticular protein genes is strongly associated with phylogenetic clustering, implicating tandem duplication in their origin. Examples include clusters 2, 3, 9, 19, and 21 containing genes of the CPR family of cuticular proteins and cluster 24 containing genes of the Tweedle family (Cornman PloS One e8345, 2009). Tandem duplication is also the likely cause of physical clustering of some of the other trx-responsive genes identified. BLAST searches indicate that the lysozyme genes (cluster 6), the heat-shock protein genes (cluster 11), and the innate immunity genes (cluster 5) in each case include homologs much more similar to each other than to homologs elsewhere in the genome. That some trx-responsive clusters were found to be conserved with Anopheles does not necessarily imply purifying selection against rearrangements: a null model for the rate of decay of synteny would need to be rejected. It is also worth pointing out that the functional annotation of trx-responsive genes and the availability of modENCODE expression data together highlight that genes in these clusters have very high peak expression (maximum expression of genes in trxG-responsive clusters is ~7 times greater than the genome average [unpublished observation]). Responsive genes often exhibit peak expression during narrow windows of development, a profile typical of cuticular protein genes (Togawa et al. Insect Biochem Mol Biol 38:508, 2008; Cornman and Willis Insect Mol. Biol. 18:607, 2009). Indeed, recent annotations reveal that even more trx-responsive genes in clusters are cuticle-related than the authors could have appreciated at the time. Clusters 16 and 17 contain genes of the recently described CPLCA/retinin cuticular protein family (Cornman and Willis Insect Mol. Biol. 18:607, 2009). Clusters 4, 8, and 20 contain genes encoding short motifs (YLP and GYR motifs) that are characteristic of extracellular matrix proteins including cuticular proteins (Cornman PloS One e12536, 2010). Short proteins with signal peptides that are rich in alanine, proline, and valine are also characteristic of cuticle (Willis Insect Biochem Mol Biol 40:189, 2010), found in cluster 10. Other families of highly expressed genes that are trx-responsive include salivary gland proteins, lysozymes, and peritrophic matrix proteins. Since it has been shown that dSET1 (Hallson et al. Genetics 190:91, 2012) is the primary H3K4 methyltransferase in Drosophila, I would suggest that trxG genes specialize in the regulation of chromatin states favoring very rapid transcription. Competing interests No competing interest. Comment reflects a personal opinion and is not endorsed by or intended to represent the views of my employer.