Communication between chromosomes: understanding regulation of gene expression in trans

The single cell of a fertilized egg contains all of the information necessary for making a complete animal, stored in the form of DNA, inside the cell. One of the most ambitious goals of modern biology is to decode this information and to predict the complete course of development, from single cell to adult. Within the last two decades, researchers have determined the entire DNA sequence (genome) of most model organisms. It is possible to 'read' these genomes, and identify within them 'genes', sections of DNA that contain the code to make proteins, the key molecules that perform the majority of biological functions. Interestingly, the majority of DNA sequence does not encode protein. For example, in humans the 20-25,000 protein-coding genes account for only around 1.5% of the full genome sequence. The remaining sequence is not all 'junk', however. It contains regulatory information that dictates when and where (which tissues and organs) particular genes will be switched on, making proteins, or off, which ultimately shapes the development and function of a particular tissue or organ. To understand the differences between individuals or species, or understand how certain genetic diseases arise, we must look beyond just the genes themselves, to this regulatory information.
For many years, understanding the mechanisms of gene regulation has formed a major focus of biological research. Different types of 'regulatory DNA elements', that are specific sequences or sections of DNA, have been identified. DNA sequences that interact with specific target genes work, sometimes over long stretches of intervening DNA, to switch on or off target genes. Often this occurs via physical interaction between proteins and specific DNA sequences. In the last two decades it has become apparent that much of the DNA sequence between genes that does not encode protein is 'read', in a process called 'transcription', to produce RNA molecules that do not make proteins but that may function somehow differently. This transcription process itself can regulate genes, as can RNA molecules that physically interact with proteins, guiding them to specific target genes.
In the work proposed here, we will use new techniques to study a poorly understood type of gene regulation called 'transvection'. In each cell the entire DNA genome is compartmentalized in discrete 'packages' of DNA called chromosomes. In most animals, chromosomes exist as pairs, one coming from each parent, and each containing equivalent genes at equivalent positions. Most known gene regulation processes involve interactions between regions on the same chromosome. Transvection refers to the special case when a regulatory sequence on one chromosome targets a gene on the other chromosome. This has been observed at several different genes and in a variety of types of animal from flies to humans. However, it is unknown what types of molecules are involved, how it occurs, and how widespread the process is. We have devised a set of experiments in the fruit fly that will allow us to separately measure transcription of genes from each chromosome in a pair. By making specific alterations to DNA sequences, we can then assess the effect this has on genes on the same chromosome vs those on the partner chromosome. This will allow us to identify the regulatory sequences that are involved in transvection. For example, we will test if RNAs produced from one chromosome affect transcription on the other. We will also test different types of proteins that bind to RNAs to determine whether they play a role in transvection. We envisage that our work will reveal components and processes of a new important type of gene regulation relevant to all animals, moving us closer towards the ultimate goal of fully decoding the information hidden within the genome.

Understanding gene regulatory mechanisms is essential to understanding biological diversity, development and disease, and has been a major focus of biological research for many years. Hox gene complexes integrate multiple layers of regulation to control precise spatial and temporal patterns of expression during development, providing an excellent model for studying complex gene regulation. Early studies in the Drosophila Hox complex revealed a 'transvection' phenomenon, where regulatory sequences on one chromosome influenced gene expression on its homolog. Though genetically well-described, it is not mechanistically understood. Our lab has used a nascent transcript fluorescent in situ hybridisation (FISH) approach to directly visualise transvection at the molecular level at two distinct Drosophila Hox loci, and uncovered trans-regulation at long noncoding RNAs (lncRNA) in the cis regulatory regions. This suggests a possible functional link between lncRNAs and trans-regulatory phenomena. In the proposed study we aim to utilise recent advances in single-molecule FISH and precise genome editing, combining them in a multi-pronged approach to quantitatively and mechanistically dissect trans-chromosome regulation at the Sex combs reduced and Abdominal-B Hox loci. The proposed experiments will enable allele-specific quantification of Hox and lncRNA transcription with single molecule and single cell resolution in order to assess both cis and trans chromosome organisation and transcriptional output. We will also use a catalytically inactive CRISPR-Cas9 system to directly regulate allele specific lncRNA expression, in order to test the effect on Hox expression in cis and trans. Finally, we will identify and test candidate proteins for involvement in trans-chromosome regulation and lncRNA binding. Together, our results will provide unprecedented insight into this little understood but potentially widespread mode of transcriptional regulation.

The most identifiable and immediate impact of this work will be on the larger biological research community. However insight into gene regulation will have long term impact on fields such as synthetic biology and genetic reprogramming. Thus, the beneficiaries of this work will be primarily academic researchers that study transcriptional regulation and RNA biology, particularly those with interest in lncRNA regulation of transcription and chromatin biology. Advances in understanding regulation of genes in cis and trans will ultimately have broad relevance for virtually every field of biology as the regulation of gene exprssion plays a role in most biological process. Therefore this proposal has a potentially large impact on the greater biological community.

A second group of beneficiaries will be non-academic individuals in the commercial and private sector that exploit gene regulation for genetic research and those that are investigating protocols to direct the development of stem cells or other multi-potent cells towards specific differentiated states (e.g. pancreatic beta cells...) for therapeutic use. This work will provide a mechanistic insight into DNA elements controlling transcriptional regulation in trans relevant to basic and clinical research studying mutations associated with perturbed development and disease, as this mode of regulation currently is poorly understood but potentially of critical importance. The methods developed in this proposal for quantitative imaging of transcription in Drosophila have significant potential for application with limited modification to most other animal model systems. Specifically, the outcomes of Objective 1 in this proposal will include data regarding regulated gene expression in Drosophila but also a generally applicable protocol for multiplex imaging and quantitation with single cell resolution in an intact embryo. We have previously had great success in disseminating and helping adapt our multiplex nascent transcript RNA FISH protocols to Xenopus and mouse labs. Another pool of beneficiaries may be groups and companies that use whole genome association studies to identify functional elements associated with phenotypes and disease. Understanding the mechanisms of regulatory control in trans may identify conserved candidate sequence or chromatin signatures that provide a better interpretation for existing datasets.

The two primary methods for ensuring impact will be communication of results through publication in international journals and presentations at national and international meetings. The PI will perform these activities and training will be provided to develop the skills of the PDRA to assist in publicizing results. The research Co-I has significant experience and he will therefore be able to contribute to this effort at a high level from early on in the project. Novel methods for disseminating large imaging data sets as a web resource will also be explored. Currently the Faculty of Life Sciences at UoM is actively supporting and facilitating efforts to develop novel means to publicize and disseminate research data.

The timescale for realizing the impact on academic research will be short, with publications and presentations providing rapid dissemination of results and methods. On a slightly longer scale the publication of this work will further this impact. The relevance and influence of this work for synthetic or diagnostic use is anticipated to be more long range (~8-10 years).

Beyond the academic and commercial impact of this project there will be a social impact as well. The Drosophila Core Facility at The University of Manchester has an extensive and involved outreach and community engagement program that is supported by all participating researchers. Presentations and activities utilizing Drosophila Hox mutations have been very successful and we will develop resources to explain the importance of transcriptional regulation to the public.