Enhancer RNAs (eRNAs) play an important role in transcriptional regulation and serve as key intermediates linking genomic enhancers to their target genes. Although ongoing efforts aim to annotate enhancers in the chicken genome, the current understanding of avian enhancers remains less developed compared to that of mammals. We utilized CAGE-seq data from chicken tissues obtained through the “Genetic Technologies in Poultry” project to predict enhancers in the chicken genome. Preliminary predictions focused on non-coding regions exhibiting bidirectional transcription, which were subsequently validated using explicit Markov models and refined with hidden Markov models. To assess sequence family homogeneity, we developed a method based on Euclidean distances between explicit Markov model matrices. Our analysis revealed that the proportion of enhancer-associated DNA in chicken is approximately similar to that observed in mammals, encompassing 9.29% of the entire chicken genome, roughly similar to the estimate made by the ChickenGTEx project. A relatively small number of them (12,242 enhancers) were significantly expressed among all tissues. Notably, more than half of the enhancer DNA overlapped intronic regions. Additionally, based on the bimodal distribution of enhancer lengths combined with the homogeneity of their Markov models, we identified a class of long enhancer elements which we hypothesize to be absent in mammals.

The promoter shift is a long known phenomenon which implies changes in the coordinates of a transcription start site (TSS). Analysis of CAGE experiments allows one to assess the impact and statistical significance of the shift. The promoter shift can be associated not only with the change of stages in early development or tissue differentiation, but also with the environmental influence on the cells. Differential promoter usage suggests non-constitutive expression activity of the regulated gene, which, in turn, suggests the usage of few sharp promoters. On the other hand, the housekeeping genes are assumed to demonstrate stable levels of expression while using multiple broad promoters. Additionally, the housekeeping genes are assumed to be normally expressed in most of the tissues. Our results show that many ubiquitously expressed genes use single sharp promoters and are subject to the significant promoter shift, which implies an additional level of complexity to the problem of definition of a housekeeping gene. The reaction of genes in cells and tissues to external stimuli is often studied in the context of differential gene expression. Our study employs a different approach of differential promoter usage, which takes the genes whose promoters undergo significant promoter shifting as the signatures of tissue response and phenotypic effect. Our results suggest that the difference in growth rate of chickens is controlled by the rate of metabolism of nutrients via the differential usage of promoters of the relevant genes.

Gene expression from promoters is influenced by interactions with genomic enhancers located within the same topologically associating domain (TAD). Enhancer activity can be evaluated by measuring the transcriptional output of enhancer RNAs, and the CAGE methodology enables the simultaneous assessment of enhancer and promoter activities within a single experiment. In this study, we examined the correlation between gene and enhancer activities within individual TADs across multiple tissues in slow- and fast-growing chickens, and we assessed the biological significance of genes with promoters that are regulated by enhancers. Our analysis revealed a statistically significant association between gene expression levels and enhancer activity in all tissues examined. Notably, enhancer-mediated regulation appears to activate key pathways involved in transcriptional control and nucleic acid biosynthesis.