Enhancers are non-coding DNA fragments that increase the concentration of active transcriptional machinery at promoter sites, therefore to positively drive gene expression. Furthermore, enhancers are known to reside in regions of open chromatin, range from 50 bases to 20 kilobases long, and contain sequences necessary for transcription factor and/or coactivator binding (Li et al., 2016). Beyond these characteristics and a handful of previously identified enhancers, there is much more to learn. A good way to start is by identifying enhancers sequences at a genome-wide level.
We aim to identify enhancers that regulate meiotic genes, specifically genes involved in spermatogenesis. Spermatogenesis is the male version of meiosis, where primordial germ cells undergo two rounds of cellular division to produce four haploid spermatozoa. It is critical that all genes necessary for spermatogenesis are transcribed at the correct time and place, otherwise viable sperm cannot be produced and the organism is infertile. To identify enhancer sequences involved in spermatogenesis, we will use two genome-wide sequencing techniques, ATAC-seq and PRO-seq, on mouse spermatocyte DNA. Both techniques allow us to map the chromatin state, nucleosome patterning, and RNA polymerase positioning across the entire spermatocyte genome. Along with the fact that enhancers are situated within 50KB of a gene’s transcription start site (TSS), we can compare our list of spermatogenesis genes and their known TSS loci to the genomic profile and predict enhancer sequences.
Loss-of-function mutants are usually associated with having point mutations or indels in the gene’s coding sequence. Therefore, translation goes awry and growing peptide chains have incorrectly incorporated amino acids or truncation due to early stop codons. However, an alternative hypothesis is that disrupted gene expression is due to mutations in the gene’s non-coding sequence, like an enhancer. A future aim is to incorporate previously discovered non-synonymous, single nucleotide polymorphisms (SNPs) into identified enhancer sequences and look at the effects on gene transcription.