Focusing precision medicine through unravelling genetic variation

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  • Except for identical twins, each individual has a unique genetic makeup encoded within their DNA. Genetic variation in combination with environmental factors influences a person’s susceptibility to certain diseases, as well as the outcome of the disease. It also contributes to how successful specific medical treatments will be for each individual. However, our understanding of the precise mechanisms behind why these differences occur remains far from complete. Dr Frances Sladek from the University of California, Riverside is using state-of-the-art protein binding microarray technology to change this though, aiming to deepen our understanding of how single nucleotide polymorphisms in DNA sequences alter gene expression. Her research is helping to lay the foundations for the future of precision medicine, providing data that can be applied clinically to help predict, prevent and treat disease.

    Dr Frances Sladek runs a lab at the University of California, Riverside, USA, where her team is working to characterise the DNA binding specificity of proteins that regulate gene expression. They are also investigating differences between human genetic sequences, linking these to individual variations in physiology, disease susceptibility and drug response.

    The majority of these differences are in the form of single nucleotide polymorphisms (SNPs) – single base changes within DNA. This means that some individual nucleotides within the sequence differ between individuals. Since the sequencing of the first human genome in 2001, more than 12,000 individual human genomes have been sequenced. Within these, over 150 million SNPs have been identified. It has been predicted that the majority of these do not result in any functional differences. However, many SNPs have been found to play a major role in not only variation in characteristics such as hair and eye colour, but also susceptibility to diseases including cancer, heart disease, diabetes and some mental health problems. They also account for the great variability that exists between individuals’ drug response, explaining why a drug can be life-saving for one person and cause serious side effects in another.

    Uncovering regulators of gene expression
    Due to these medical implications, much research is focused on identifying SNPs that may be of clinical importance. SNPs occur throughout the genetic sequence and can be found in the gene regions that encode proteins, thus altering the amino acid sequence encoded and its function. However, most SNPs are outside the protein-coding regions of DNA, where they can influence the expression of specific genes and therefore protein levels. In this way, SNPs that lie in non-protein-coding regions can have as dramatic an impact on phenotype and protein function as those that are housed in protein-coding regions. SNPs that affect the ability of proteins to control gene expression and bind the regulatory regions of genes are known as affinity-altering SNPs (aaSNPs).

    Dr Sladek and her team have set out to exhaustively
    determine the DNA binding specificity of a group of gene
    expression regulators, known as nuclear receptorsQuote_brain

    Dr Sladek and her team have set out to exhaustively determine the DNA-binding specificity of a group of these regulators of gene expression, known as nuclear receptors. Nuclear receptors are essential proteins found in all animal cells and are involved in nearly every aspect of human physiology and disease. Humans possess 48 nuclear receptor genes, including those encoding for vitamin A and D receptors, thyroid hormone receptors and sex hormone and other steroid receptors. Loss of regulation of nuclear receptor signalling pathways contributes to the development of a wide range of diseases, including breast cancer, prostate cancer, ovarian cancer, diabetes and obesity.

    Enhancing treatment specificity
    In addition to playing a key regulatory role in physiology and development, due to their implication in a wide range of diseases, nuclear receptors are common drug targets. For example, cortisol cream for rashes works by acting on the glucocorticoid receptor that is involved in the stress response. Common cancer drugs, such as tamoxifen, also target nuclear receptors, as well as drugs used to treat inflammation, osteoporosis and diabetes. Although research over the past few decades has led to a basic understanding of nuclear receptors’ DNA binding, there is still a vast amount of detail left to uncover regarding the intricacies of how nuclear receptors interact with DNA and which factors are involved.

    An aaSNP in a TFBS that significantly alters the affinity of a given TF for its response element in the promoter/enhancer region of a target gene

    In addition to determining nuclear receptor-binding specificity, Dr Sladek’s research group is investigating aaSNPs in the genomic sequences to which they bind. As many of these regions are associated with variations in drug metabolism and susceptibility to disease, furthering our knowledge of these sites offers great potential for medical progress.

    Incredibly powerful microarrays
    To do this, Dr Sladek and her team are utilising a cutting-edge approach that employs protein binding microarrays (PBMs), a very powerful new tool for high throughput analysis of protein binding. They are pushing the boundaries of what can be achieved with PBMs to further enhance the efficiency of this novel methodology. Usually, PBMs have the capacity to carry out around 10,000–100,000 reactions per experiment. The researchers have succeeded in enhancing this, enabling them to run one million reactions per PBM on a single microscope slide.

    Dr Sladek hopes to connect the nuclear receptor and genetic
    variation research communities, bringing the fields one step
    closer towards facilitating precise, personalised medicineQuote_brain

    Once Dr Sladek and her team have gathered their PBM data, they will use the results to search human genome data to identify potential nuclear receptor target genes. Once identified, they can then cross reference these with genetic sequence data to uncover details of associated binding locations and gene expression. One of their key objectives is to create a complete database of aaSNPs for nuclear receptors, particularly those that have an impact on gene expression. Such a database could then be used for informing precision medicine. So far, they have identified over 20,000 aaSNPs for seven different nuclear receptors.

    Combining data to catalyse progress
    All of Dr Sladek and her team’s findings are or will be made available on public databases, as well as their own website dedicated to the project. In doing this, they help other researchers to use the data to further progress this venture. This information-sharing approach is invaluable for researchers and is set to fast track work linking nuclear receptors to drug metabolism and disease. Importantly, public availability of this knowledge is another step towards finding the most effective and precise ways of using drugs that target nuclear receptors.

    One of these public databases, funded by the National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) at the National Institutes of Health (NIH), is called Transcriptomine, which has made big leaps towards gathering current data on nuclear receptors, their ligands and the genes they regulate, in one easily accessible place. Now, Dr Sladek’s lab will use PBMs to combine that data with human genomic variation, such as that in GTEx (Genotype-Tissue Expression), an NIH-funded consortium correlating tissue-specific gene expression with genetic variation, all with the ultimate goal of being able to predict and therefore prevent or better treat disease. Bridging this knowledge gap is essential for the development of personalised treatment for patients.

    Dr Sladek’s team is also working towards integrating their existing online databases into Transcriptomine by developing data integration and visualisation tools within the Transcriptomine platform. Dr Sladek hopes to enhance the existing tools and connect the nuclear receptor and genetic variation research communities, and in doing so bring the fields one step closer towards facilitating precise, personalised medicine.

  • What led you to focus your work on nuclear receptors specifically?
    As a postdoc at Rockefeller University I purified and cloned a protein that bound a DNA response element that was known to be important in liver-specific gene expression. That protein, called hepatocyte nuclear factor 4 (HNF4), turned out to be a member of the nuclear receptor superfamily. Since I purified HNF4 based on its ability to bind DNA, I have always been interested in the DNA binding properties of nuclear receptors. Shortly after publishing the first HNF4 paper in 1990 (1), I helped a group in the Netherlands show that Haemophilia B Leyden was due to a mutation in an HNF4 binding site in the regulatory region of factor IX, a blood coagulation gene (2). I have been fascinated by the notion that changes in nucleotide sequences can alter the ability of transcriptional regulators to bind DNA ever since.

    Could you please give us an insight into how it is possible to carry out up to one million individual reactions in a single protein binding microarray experiment?
    Agilent uses inkjet technology to print very small spots of DNA on a microscope slide that can be read at two micron resolution. We provide Agilent with the sequences that we want printed as single-stranded DNA, which we then make double-stranded using a universal linker. We apply crude nuclear extracts containing the nuclear receptor of choice to the slide, wash off non-specifically bound proteins and visualise the bound nuclear receptor using immunoblot technology. Considering that nuclear receptors can search through three billion base pairs of DNA in a single nucleus the size of about six microns, there is plenty of space on the slide for a nuclear receptor to bind a million spots of DNA.

    Is a collaborative, information sharing approach between research groups a relatively new development in genetic research?
    There certainly has been a big push in recent years for more public sharing of data. The first human genome that was sequenced by Craig Venture’s group in 2001 was considered private property and you had to pay a subscription to view it. Fortunately, at the same time the international consortium of the Human Genome Project released their sequence for free. Now, you cannot publish any sort of ‘omics’ paper without making all the primary data publically accessible so that others can analyse it on their own. This is a wonderful development as most genomic research done in the US is funded by taxpayer money via the NIH, so in essence it belongs to the public.

    What do you think are the most exciting achievements of your research thus far?
    Just getting the protein binding microarrays to work for the first time was very exciting. Originally we used an array with 48 spots that we printed ourselves. Once the printing technology became more affordable, we ordered slides with eight grids of 15,000 spots each and soon found that even 5,000 unique sequences in triplicate were not sufficient to cover the range of DNA sequences that even a single nuclear receptor could bind. As we have scaled up to a million spots of DNA, I am continually impressed at how sensitive and reproducible the arrays are. We can even apply crude nuclear extracts from mouse liver and detect endogenous nuclear receptors in the PBMs. We can also detect co-regulators binding to those nuclear receptors.

    What are your hopes for the future of your work into aaSNPs and nuclear receptors?
    We are currently designing PBMs based on eQTL data from the Genotype-Tissue Expression (GTEx) database from the NIH Common Fund. I want to get all the data that we have so far out into the public domain so that others can start using it in their studies, especially genome wide association studies (GWAS). I would also like to secure additional funding so that we can analyse the other 40+ nuclear receptors in a similar fashion.

    (1) Sladek et al. (1990) Genes and Development 4: 2353-65 (PMID: 2279702)
    (2) Reijnen et al. (1992) PNAS 89: 6300-3 (PMID: 1631121)

  • Research Objectives
    Dr Sladek’s research focuses on characterising the impact of single nucleotide changes in the human genome on the ability of nuclear receptors to bind DNA. This, in turn, could enable predictions to be made in terms of a patient’s susceptibility to a certain disease or response to a particular medical treatment.

    Funding

    • National Institute of Mental Health (NIMH)
    • National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
    • Nuclear Receptor Signalling Atlas Data Source Project (NURSA NDSP)
    • University of California Riverside Seed Grant

    Collaborators

    • Dr Eugene Bolotin, PhD
    • Dr Bin Fang, PhD
    • Dr Daniel Mañé Padrós, PhD
    • Dr Nina Titova, PhD
    • Jonathan Deans
    • Prof Tao Jiang, PhD
    • Prof Thomas Girke, PhD
    • Dr Neil McKenna, PhD

    Bio
    Professor Sladek received her PhD from Yale University in 1988 and cloned the nuclear receptor HNF4a as a postdoctoral fellow at Rockefeller University. She started her own lab at the University of California, Riverside in 1992, where she continues to investigate the role of HNF4a and other nuclear receptors.

    Contact
    Frances M Sladek, PhD
    Professor of Cell Biology and Toxicologist
    Department of Cell Biology and Neuroscience
    Associate Director, UCR Stem Cell Center
    2115 Biological Sciences Building
    University of California
    Riverside, CA 92521-0314
    USA

    E: frances.sladek@ucr.edu
    T: +1 (951) 827 2264
    W: http://www.sladeklab.ucr.edu/
    W: http://nrdbs.ucr.edu

  • Focusing precision medicine through unravelling genetic variation
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