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Investigating the impact of the mechanical code of the genome on protein:DNA interactions

   Department of Biosciences

   Applications accepted all year round  Funded PhD Project (UK Students Only)

About the Project

Training opportunity: This project will appeal to a motivated student who wants training in the cutting-edge technologies surrounding genomic research. The student will explore the use of experimental genomic and high-throughput DNA and RNA sequencing to decipher how mechanical information encoded in DNA impacts critical processes involving DNA:protein interactions. In addition to mastering genomic techniques, the student will gain significant computational experience in analyzing large experimental genomic datasets, and molecular biology experience pertaining to studying protein:DNA interactions. The student will also have the potential to complement high-throughput genomic investigations with high-resolution single-molecule fluorescence imaging. This topic offers an exciting and innovative program of work at the cutting edge of genomic, molecular biology, and biophysical research.

Background: The transcription, storage, copying, and repair of genetic information stored in DNA is critical to life, and requires a host of specialized proteins to interact with DNA at various genomic loci. Almost all such critical protein:DNA interactions require some form of mechanical and structural deformation of DNA, such as stretching, bending, or twisting. We recently revealed that the mechanical properties of DNA, which facilitate or hinder such deformations, are encoded in sequence via a “mechanical code” (Basu et al, Nature, 589, 462 – 467, 2021, https://www.nature.com/articles/s41586-020-03052-3). This permits evolution to encode in sequence not just genetic information, but also mechanical information that regulates the interaction of important proteins at various genomic loci. We achieved this deciphering of the mechanical code by developing novel genomic methods that permit investigation of the structural properties of DNA via next-generation high-throughput sequencing.

In this project, we will investigate the impact of the genetically-encoded physical properties of DNA on fundamental protein:DNA interactions, with a particular focus on transcription. Transcription requires RNA polymerase to displace highly bent DNA:protein complexes called nucleosomes in its path. The stability of these complexes, and therefore the efficiency of transcription, depends in part on the local mechanical pliability of DNA, which in turn is sequence-dependent. We will design high-throughput in vitro biochemical experiments to understand how the mechanical profile of DNA along nucleosomes impact the pausing and translocation of polymerases. We will complement these with experiments involving generic alterations of the physical properties of DNA at functionally important loci in vivo, and map the impact of altered DNA mechanics to downstream readouts like nucleosome positioning or gene expression. Finally, we will understand how chemical modifications of DNA and nucleosomes, such as methylation, that occur routinely as part of development or disease like various cancers, can alter the manner in which genetics encodes mechanics, and therefore provide a novel regulatory pathway to control critical protein:DNA interactions.

Aims: The student will (1) engineer a large array of transcribable nucleosomes formed on more than 100,000 pre-specified mechanically diverse DNA sequences taken from various genomic loci in yeast, (2) map the pausing and translocation extent of RNA polymerase transcribing through these nucleosomes via high-throughput next-generation (Illumina) sequencing of the produced nascent mRNA, (3) measure how the mechanical properties of DNA vary long the lengths of these DNA sequences via the novel technique of loop-seq, (4) analyze the data obtained from aims (2) and (3) to build an understanding of how mechanical information encoded in DNA sequence impacts the transcription of genetic information, (5) test the ideas developed in aim 4 via genome-editing in yeast, and/ or via high-resolution single-molecule fluorescence microscopy measurements.

Broad Picture: Great strides in molecular biology have constantly been ushered by our discoveries of novel ways in which DNA encodes information, such as in sequence, in epigenetic modifications, and in the 3D structure of chromatin. We have recently developed tools to explore how DNA encodes information in yet another mode - in the sequence-dependence of its local physical and mechanical properties. This presents, therefore, a very exciting platform to explore how nature and evolution have taken advantage of this to encode powerful regulatory information. Parallel developments in the field of gene-editing editing will certainly benefit from our exploration of how the altered mechanical properties of edited loci can impact critical DNA:protein interactions. Finally, we would be in a position to explore how diseases like cancers and programs like development, which induce chemical alterations to DNA, could be achieving downstream effects by altering the mechanical code. 

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