Description
BACKGROUND: Macroautophagy (autophagy) is one of the primary homeostatic mechanisms that the cells of organisms use to maintain energy, nutrient, and metabolic balance. The pathway shows dynamic regulation and can be rapidly enhanced under conditions of starvation, infection or stress. In addition, it has been shown that nutritional excess and enhanced insulin/TOR signaling serves as a potent suppressor of autophagy. Autophagic defects have been shown to have progressive consequences within cells. Depending on the tissue-specific requirements of the pathway, autophagy has been linked to disorders in the immune, cardiac and neural systems. It is hypothesized that the position of individual transcription factor binding sites and regulatory cassettes within the promoter will be conserved between subsets of autophagy genes that are interacting partners within the pathway. These conserved sequences may be predictive of transcriptional co-regulation between autophagy components. The research outlined in this proposal uses several computational matrix-based approaches to compare upstream promoter sequences of individual human and macaque autophagy genes. Both pattern matching and detection algorithms were used to identify, map, and characterize several putative cis-regulatory elements in the 5' promoter sequences of multiple autophagy genes.RESULTS: Pairwise comparisons of individual autophagy genes were done between human and macaque promoter sequences. A substantial degree of conservation was found in the 5' sequence within 4.0kb of the gene's transcriptional initiation (start) sight. On average 70-92% identity was found between the species within this non-coding sequence, and a significant degree of spatial conservation was observed for most binding sites. In addition, a highly conserved cassette sequence was identified that was initially identified from BLAST-z alignments of the human Map1LC3b and Atg4A 5' promoter regions. We found that cassette number and spatial organization show considerable overlap among those pathway components that closely interact or function in concert for autophagosome biogenesis, further suggesting potential co-regulation of these genes. Conversely, cassette positions and distributions varied greatly between the promoter regions of duplicated autophagy gene paralogs such as Atg4A and Atg4D. The rVista software was used for a detailed analysis of the regulatory cassette. We found multiple highly conserved transcription factor binding sites within the cassette, which included NRF2/MAF/TAXCREB, PITX2/CRX, PITX2/LUN1, HNF3/FOXD3 clusters. Functional analysis of cassette clusters was further broken down into five major physiological responses; (1) early development and pattern formation, (2) early immune and the inflammatory responses, (3) metabolic development and insulin and glucose metabolism, (4) visual development and circadian cycles, (5) stress responses. CONCLUSIONS: By comparing the 5' promoter sequences of human and macaque autophagy genes we identified multiple conserved cis-regulatory elements. In addition to transcription factor binding sites, we identified and characterized a unique cassette sequence that was conserved in nearly all autophagy genes but was absent in a select set of control promoters. The number and position of these autophagy gene cassettes suggested that pathway components that interact or function together at a particular point during autophagosome formation had conserved promoters. This work may also serve as a foundation for the further analysis of autophagy gene evolution. The identification of these cis-element sub-groupings will facilitate the design of experiments that examine the regulation of autophagy genes under a variety of physiological conditions, including metabolic syndrome, aging, immune dysfunction, cardiac disease, cancer, and neurodegenerative disorders.
