Andrew's research programme focuses on the regulation of the oestrogen receptor (ER) in breast cancer. In 70% of all breast cancer cases, this receptor drives the growth and proliferation of the tumour, and is a key target for the drug Tamoxifen. The ER is known to associate with pioneer factors and numerous co-factors, including many with enzymatic activity that can modify ER and other co-factors.
Andrew aims to investigate the role of these co-factors and how they interact with the ER by using state-of-the-art computational methods to combine proteomic and genomic data. In the long term he aims to produce a model could be used to predict the effects of perturbing components of the pathway with drugs and potentially lead to improved combinatorial treatment regimes.
Proteomics uses mass spectrometry to detect the levels of proteins within the cell. Building on the technologies Andrew developed at the MRC Laboratory of Molecular Biology, his research programme makes use of state-of-the-art instrumentation to investigate how the Oestrogen Receptor activates genes while it is bound to the DNA in the cell's nucleus. This allows for the identification of novel protein interactions. If these interactions are involved in the regulation of the gene activation they could be of therapeutic interest.
By combining high-throughput sequence and computational methods we are able to analyse the activation of specific genes by the Oestrogen receptor. This technology is used in tandem with proteomics to provide a complete understanding of both which genes are activated and how they are activated in response to a specific stimulus.
Marisa Di Monaco
XL-MS allows for the study of protein-protein binding by way of using small isotopically-labelled linker molecules. These linker molecules bind between residues that are within range of each other and then the resultant cross-linked protein complex is digested and analysed by mass spectrometry. The software developed by Andrew Holding to interrogate the mass spectrometry data was named Hekate and is freely available.
More details are available on the Hekate page of this site and the results of this work were published as:
Figure 1. Cartoon image outlining the method of protein cross-linking. A protein sample is cross-linked with a homobifunctional reagent that links residues within a certain distance of each other. The protein sample is then digested into small peptides and the cross-linked fragments are detected by mass spectrometry. These peptides are then fragmented to provide an amino acid sequence detailing where in the protein the cross-linkers occur, and thereby we can work out the structure of the protein and how multiple proteins interact.
Brundle is an R package that provides a series of functions for the normalisation of ChIP-Seq data to internal or external controls. It was developed to resolve challenges in estimating the precise fold-change between conditions.
Figure 2. MA plots showing Estrogen Receptor-alpha (ER) ChIP-seq data before and after the treatment of MCF7 cells with fulvestrant. This example shows the difference between the raw data (left) and normalised data (right). Without normalisation biological signal can be supressed. In this example normalisation was achieved using a second control antibody against CTCF. The CTCF binding sites were then used to normalise the ER binding data.
1These authors contributed equally to this work.
* Corresponding author.
PhD in Chemical Biology
Studies on the biosynthesis of non-ribosomal peptides
University of Cambridge, UK
Masters in Chemistry
University of Oxford, UK
Senior Research Associate
Cancer Research UK Cambridge Research Institute
2013 - 2014
Cancer Research UK Cambridge Research Institute
2009 - 2013
Career Development Fellow
MRC Laboratory of Molecular Biology
2005 - 2009
Deparment of Chemistry, University of Cambridge, UK.
Advisors: Dr. J. Spencer and Dr. F. Leeper
2017 Thomas Jefferson Visiting Fellow at University of Virginia
2017 CRUK Research Travel Award
2015 CRUK Rising Star in Research Engagement Prize
2015 Fellow Downing College
2014 Unltd Try-It Award
2013 College Lecturer and Bye-Fellow Downing College
2012 Wellcome Trust People Award
2012 British Mass Spectrometry Society Travel Award
2012 MRC Award via the Special Awards Scheme for contributions to the MRC
2012 British Science Association Media Fellowship
2011 UnLtd Catalyst Award for Social Entrepreneurship
2009 MRC Career Development Fellowship
2005 BBSRC PhD Studentship
2015 Fellow of the Higher Education Academy
2014 Member Biochemical Society
2009 Member Royal Society of Chemistry
Abstract: The problem of bacterial resistance is of growing concern within the medical community. Eventually, even with responsible use of antibiotics, new compounds will be required to bypass the resistance that bacteria have acquired. Thus the expansion of knowledge and understanding of antibiotics is key in the development of new compounds in the fight against infection. One attractive starting point for the development of new compounds are those natural products generated by non-ribosomal peptide synthetases (NRPS), which include a range of clinically relevant glycopeptide antibiotics.
Several aspects of the biosynthesis of glycopeptide antibiotics were examined: first, the investigation to identify, by the use of directed evolution if 4-hydroxymandelic acid synthase (HmaS) from the gene cluster of the antibiotic chloroeremomycin may have evolved from its homologue 4-hydroxyphenylpyruvate dioxygenase (HppD). The summation of this work is published in FEBS Lett. 2006; 580:3445. Following on from this work was an investigation into the hypothesis that HmaS catalyses the turnover of the non-natural phenylpyruvic acid to produce a product with an inverted chiral centre compared to that of the natural substrate due to differences in substrate binding. Results showed that, while not the major product, the inverted product was detected via chiral GCMS. Secondly, it is shown that all three cytochrome P450 enzymes (OxyA-C) that catalyse the sequential formation of three essential oxidative cross-links within the chloroeremomycin molecule do so with the retention of the oxygen atom on the peptide backbone and without the incorporation of oxygen in the air. This portion of the work is published in ChemBioChem 2008; 9:2209. The final part of the study was the development of a high-throughput screening method for NRPS A-domains, with the aim of both rapid characterisation and directed evolution of novel substrate specificity. This led to the identification that the amino acid loaded by the first A-domain of the teicoplanin NRPS was shown to load the d-amino acid in preference to the l-amino acid. This is in contrast to the equivalent domain in the chloroeremomycin gene cluster that loads the l-amino acid.