Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii

Abstract

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences.Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

Molecular characterization and genetic authentication assay for Anopheles ‘hemocyte-like’ cell lines 4a-3A and 4a-3B

“…Relative to 4a-3A cells, 4a-3B cells contain 21 noncoding indels (20 intronic, 1 in the 3’–UTR), 134 noncoding SNPs (2 in the 5’-UTR, 122 intronic and 10 in the 3’-UTR) and 13 synonymous coding SNPs (4 in Exon 2, 4 in Exon 3, 2 in Exon 4, 3 in Exon 5) within the PPO1 gene region. Using a genome-wide catalog of transcriptional enhances [ 9 ], variation was also examined in two predicted enhancer regions, 2R:27996659-27997160 and 2R:28016526-28017040, close to PPO1. Examining sequence difference across these two candidate enhancers, 4a-3B cells had 0 indels and 2 SNPs relative to 4a-3A cells for the enhancer at 2R:27996659-27997160 and 3 indels and 13 SNPs in the enhancer located at 2R:28016526-28017040.…”

“…Current work indicates that only one of these genes, PPO1, is genetically variable between 4a-3A cells and 4a-3B cells, and the coding variation segregating between the cell lines is all synonymous. Using our recently reported genome-wide catalog of transcriptional enhancers [ 9 ], genetic variations in candidate enhancers near all six PPO genes were examined. Most non-coding variation lies in enhancers near the PPO1 gene, with most occurring in a predicted enhancer located at 2R:28016526-28017040, just 1665 bp upstream of the PP01 gene.…”

“…There are over 500 insect cell lines originating from different tissues from species within Diptera, Lepidoptera and Hemiptera insect orders. Anopheline cell lines have been used to examine a wide variety of traits, including to determine if the Minos transposable element integrates in a transposase-dependent manner [ 2 ], to characterize the cellular response to Bin toxin, a candidate vector control tool [ 3 , 4 ], to study hemocyte properties and prophenoloxidase (PPO) expression [ 5 ], for studies of phagocytosis and hemocyte gene expression [ 6 ], to characterize the effect of genetic variation enhancer activity [ 7 ], to characterize interaction of proteins with a role in malaria infection [ 8 ] and to perform STARR-seq and establish a genome-wide catalog of transcriptional enhancers [ 9 ]. Establishment of a male-specific anopheline cell line suitable for the study of processes unique to male mosquitoes was also recently reported [ 10 ].…”

Use of Insect Promoters in Genetic Engineering to Control Mosquito-Borne Diseases

“…Computational predictions and comparative genomics tools have assisted in the identification of cis-acting regulatory regions and transcriptional enhancers in mosquitoes [8,9].…”