Current research

Andrew is currently based at Cancer Research UK's Cambridge Institute where he leads a research team.

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

Proteomics

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.


Genomics

Genomics

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.

Team

Current Members

Amanda Donnelly
PhD student
MSc Mol. Medicine, Imperial College London, UK, 2013
Funding: Cancer Research UK

Amy Cullen
Research Assistant
BSc (Hons), Imperial College London, UK, 2016
Funding: Cancer Research UK

Anze Godicelj
Research Assistant
MSci BA (Hons), Univesrity of Cambridge, UK, 2017
Previously Part III Student in lab, 2016, University of Cambridge: Biochemistry awarded overall First Class.

Jade Talbot
MPhil Project Placement
University of Essex
Funding: University of Essex

Previous Members

Gabriella Gellen
Visiting Student
University of Debrecen, Hungary
Funding: Erasmus

Simon Wengert
Visiting Student
Molecular Biotechnologist (M. Sc.), University of Heidelberg, Germany, 2017
Funding: Cancer Research UK/Erasmus+

Ryan Blake
MPhil Project Placement
University of Essex
Funding: University of Essex

Jannik Lauenstein
Work Experiance Research Assistant
Apprenticeship in biology laboraty technician, Lise-Meitner-Schule, Berlin, Germany
Funding: Lise-Meitner-Schule, Berlin

George Wood
Visiting Student
Natural Sciences Tripos Undergraduate, University of Cambridge
Funding: Downing College — Bill Willetts and Mays Wild funds

Marisa Di Monaco
MPhil Project Placement
University of Essex
Funding: University of Essex

Software Packages

xlinkHekate

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:

  1. Holding et. al (2013) J. Proteome Res. [PubMed]

  2. Liu et. al (2013) Genes Dev. [PubMed]

  3. Rego et. al (2013) EMBO J. 32. 1334-1343. [Pubmed] [F1000Prime]

Figure showing XCMS workflow


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.

More...

Download Hekate

TeleportBrundle

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 showing Brundle workflow


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.

Brundle on Github

Published Research

Journal Articles

  1. Andrew N. Holding*, Amy E. Cullen and Florian Markowetz (2018)
    Genome-wide Estrogen Receptor-alpha activation is sustained, not cyclical
    Preprint. [bioRxiv]

  2. Andrew N. Holding*, Federico M. Giorgi, Amanda Donnelly, Amy E. Cullen, Luke A. Selth and Florian Markowetz (2018)
    Network Dynamics of ER-α activation reveals reprogramming of GRHL2
    Preprint. [bioRxiv]

  3. Euan S. Polson, Verena B. Kuchler, Christopher Abbosh, Edith M. Ross, Ryan K. Mathew, Hester A. Beard, Bárbara da Silva, Andrew N. Holding, Stephane Ballereau, Eulashini Chuntharpursat-Bon, Jennifer Williams, Hollie B. S. Griffiths, Hao Shao, Anjana Patel, Adam J. Davies, Alastair Droop, Paul Chumas, Susan C. Short, Mihaela Lorger, Jason E. Gestwicki, Lee D. Roberts, Robin. S Bon, Simon J. Allison, Shoutian Zhu, Florian Markowetz, Heiko Wurdak (2018)
    KHS101 disrupts energy metabolism in human glioblastoma cells and reduces tumor growth in mice
    Science translational medicine, 10, 454. [PubMed]

  4. Evangelia K. Papachristou, Kamal Kishore1, Andrew N. Holding1, … , Rasmus Siersbaek, Clive S. D’Santos and Jason S. Carroll (2018)
    A quantitative mass spectrometry-based approach to monitor the dynamics of endogenous chromatin-associated protein complexes
    Nature Communications, 9, 2311. [PubMed]

  5. Michael J. Guertin, Amy E. Cullen, Florian Markowetz and Andrew N. Holding* (2018)
    Parallel Factor ChIP Provides Essential Internal Control for Quantitative Differential ChIP-Seq
    Nucleic Acids Research. 46, 12. [PubMed]

  6. Andrew N. Holding* (2015)
    XL–MS: Protein cross-linking coupled with mass spectrometry
    Methods 89. 54-63. [Science Direct]

  7. Jennifer Lee, ShuJing Ding, Thomas B. Walpole, Andrew N. Holding, Martin G. Montgomery, Ian M. Fearnley and John E. Walker (2015)
    Organisation of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.
    J. Biol. Chem. 290. 13308-20. [PubMed]

  8. Andrew N Holding,* Meindert H Lamers, Elaine Stephens and J Mark Skehel (2013)
    Hekate: software suite for the mass spectrometric analysis and three-dimensional visualization of cross-linked protein samples.
    J Proteome Res. 12. 5923-33. [PubMed]

  9. Yang Liu,1 Hannah K Salter, 1 Andrew N Holding, Christopher M Johnson, Elaine Stephens, Peter J Lukavsky, John Wlashaw and Simon L Bullock (2013)
    Bicaudal-D uses a parallel, homodimeric coiled coil with heterotypic registry to coordinate recruitment of cargos to dynein.
    Genes Dev. 27. 1233-46. [PubMed]

  10. Andrew N Holding,1 Ana Toste Rego,1 Helen Kent and Meindert H Lamers (2013)
    Architecture of the Pol III—clamp—exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair.
    EMBO J. 32. 1334-1343. [Pubmed] [F1000Prime] - recommended as being of special significance.

  11. Andrew N Holding* and Jonathan B Spencer. (2008)
    Investigation into the mechanism of phenolic couplings during the biosynthesis of glycopeptide antibiotics.
    ChemBioChem. 9. 2209-14. [Pubmed]

  12. Helen M O’Hare, Fanglu Huang, Andrew Holding, Oliver W Choroba and Jonathan B Spencer. (2006)
    Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution.
    FEBS Lett. 580. 3445-50. [Pubmed]

1These authors contributed equally to this work.
* Corresponding author.

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