At one point within our educational careers, we have all sat in a classroom and learned about the notion of the central dogma of biology. This concept describes how the genetic information of an organism is transcribed then subsequently translated into proteins. The transcription and translation of a specific section of our genetic information is referred to as “gene expression.” DNA serves as the initial component of this process and plays a pivotal role in gene expression. Experimental methods to understand the relationship between DNA accessibility, exposed sites of DNA capable of binding transcription machinery, and gene expression have been progressively developed within recent decades. In this article, we describe an unbiased emerging method that improves current sequencing technology to study DNA organization and transcriptional regulation by taking a different experimental approach.
Let us first differentiate terms – DNA and genes – often used interchangeably to refer to the genetic information within organisms. DNA is a molecule composed of two antiparallel polynucleotide chains that twist around each other to create a three-dimensional double helix. An individual polynucleotide chain, or strand of DNA, is a polymer of nucleotide subunits composed of a phosphate group, sugar, and nitrogenous base connected by a phosphodiester bond. Phosphodiester bonds are strong chemical bonds that maintain the sequential integrity of DNA by creating a covalently linked sugar-phosphate backbone. The two polynucleotide strands that form the double helix are paired up by hydrogen bonding between complementary nucleotides. Hydrogen bonds are comparatively weak bonds that allow for the array of transient interactions that occur within living organisms.
The human genome spans over three billion nucleotides, and if you were to unwind DNA in a single diploid cell and stretch out the individual polynucleotide strand then it would span over six feet in length! For this reason, our cells have developed mechanisms to organize DNA within the confines of their nucleus, which is only 10 micrometers in diameter on average1. The structural organization of DNA begins with the formation of a nucleosomal complex, or nucleosome. The formation of nucleosomal complexes within eukaryotic systems are driven by dynamic processes that are heavily modulated and change over time. Histones, proteins responsible for the dynamic structural organization of DNA, interact with DNA through electrostatic interactions and assist in the wrapping around the histone core by the direction of histone tails. The formation of the nucleosomal complex occurs when DNA wraps around a collection of eight histone cores that are separated by a stretch of linker, or free, DNA. Further compaction of DNA, by interactions with other DNA-binding proteins, lead to the recruitment of transcription factors and development of higher-ordered chromosome states such as: chromosome fibers, chromosome loops, and, ultimately, the full-length chromosome.
Genes are a subset, or segment, of DNA that contain essential information for the production of a specific protein. Of the six billion nucleotides contained within the diploid human genome, only 3% encode for functional proteins. For genes to be encoded into proteins, these segments of genic DNA must be accessible by the transcribing machinery of the cell. The relationship between DNA packing and function is not yet known. However, a recent development in Peter B. Becker’s lab at the Ludwig Maximilian University of Munich in Germany provides new insights of nucleosomal positioning and its relation to gene expression through nanopore sequencing of Drosophila melanogaster2.
In the context of the human genome, understanding the role of DNA packaging and gene expression is a daunting task. For this reason, like many geneticists, the Dr. Becker investigates nucleosomal positioning in Drosophila melanogaster, a relatively small and well-understood genome consisting of only 180 million base pairs. Current experimental methods to study nucleosomal positioning are limited to around a few million base pairs, subjecting datasets to bias if repeated. The Becker lab has successfully developed an interdisciplinary approach that accurately and effectively broadens the resolution of a genome-wide search.
The Becker Lab’s novel approach exploits current Illumina-based sequencing to obtain improved experimental data. Illumina-based sequencing is a technique developed by Shankar Balasubramanian and David Klenerman at Cambridge University that, in principal, cuts a long strand of DNA into small segments, loads the small segments onto a flow cell, tags the small segments with a certain marker, and amplifies the small segments to determine the sequence of the original strand3. The Becker lab has discovered that utilizing longer sequences leads to clustering of strands at specific locations, which all get read simultaneously together in the same sequencing cycle. In creating this approach, the Becker lab has identified that nucleosomal positioning is dependent on the state of DNA packing and similarly positioned near areas of the chromosome where genes are not generally expressed. This novel nanopore sequencing technology provides to a new approach to understanding the positioning of nucleosomes and gene expression.
Sequencing technology explores new areas of research and approaches to a number of problems faced by the life sciences community. Current sequencing technology extends to the study of gene expression through later stages of the central dogma. RNA sequencing, the study of the transcriptome of an organism, has enabled us to quantify relative gene expression by measuring the production of RNA4. As technological advancements occur and become more readily available, the ability for researchers to answer the complex questions that occur at the molecular level improve as a function of the tools they are able to utilize.
- Thakur, V. (2018, September 13). How a 2 meters long DNA is fitted into a 2 micrometers Nucleus? Retrieved from https://sciencesamhita.com/packaging-of-dna-inside-nucleus/
- Baldi, S., Krebs, S., Blum, H., & Becker, P. B. (2018, August). Genome-wide measurement of local nucleosome array regularity and spacing by nanopore sequencing. Retrieved from https://www.nature.com/articles/s41594-018-0110-0/
- Shen, R., Fan, J. B., Campbell, D., Chang, W., Chen, J., Doucet, D., . . . Oliphant, A. (2005, June). High-throughput SNP genotyping on universal bead arrays. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/15829238/
- Wang, Z., Gerstein, M., & Snyder, M. (2009, January). RNA-Seq: A revolutionary tool for transcriptomics. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2949280/