Dr Zac Chatterton

Dr Zac Chatterton, Lecturer, The University of Sydney.

Zac graduated with a BMSc from Deakin University in 2008, a BSc (Hons) from The University of Melbourne in 2009, a PhD from The University of Melbourne in 2014, and is now a postdoctoral Research Fellow/ Adjunct Assistant Professor Ichan School of Medicine at Mt Sinai since 2014. He is also a lecturer at The University of Sydney since 2017.

Zac and his lab are interested in epigenetics, the genetic basis of disease and the development of in vitro and in silico methods for cell-free DNA diagnostics. With the advent of high-density microarrays and high throughput sequencing an unbiased and detailed picture of the human genome and its cell-specific regulation is emerging.

To add to this knowledge, Zac is characterising the epigenomes of single-cell types within disease susceptible regions of the human brain during normal aging. This new information will provide insights into aging and cell-specific regulation of neurogenetics whilst also defining biomarkers with potential utility for neurological disease diagnosis and monitoring using cell-free DNA.

Forefront Group:

  • BMC Neurogenetics and Epigenetics Research Group

Affiliate Organisations

Icahn School of Medicine at Mt Sinai (USA)

Neurodegeneration of interest:

AD, FTD, MND, Ageing

Expertise:

  • Epigenetics
  • Biomarkers
  • Bioinformatics

Specific Skills:

  • Molecular and Computational Biology

Project - Epigenetic profiling of brain cell-types

Research Project Abstract

DNA methylation is an epigenetic modification that is found covalently bound to cytosines typically within a CpG context. DNA methylation regulates gene expression. During early development, the gene promoters of pluripotency genes are expressed which is accompanied by an absence of DNA methylation within their gene promoters. As cells commit to lineage, pluripotency genes are repressed and their gene promoters become methylated (Feldman et al, Nat Cell Biol, 2006). Simultaneously, DNA methylation patterning occurs (losses and gains) across the entire genome, laying the foundations for cell-specific gene regulation of brain cells (Lister et al, Science, 2013).

DNA methylation is highly specific to cell-types. Only in the past few years have technology developments in single-cell sequencing now enabling characterisation of the genome-wide DNA methylation. This project intends to characterise single-base resolution maps of DNA methylation off all cell populations within brain regions susceptible to neurodegeneration in Alzheimer’s Disease, Frontotemporal Dementia and Motor Neurone Disease.

To achieve this we have established new implementations of the recently described single-nuclei combinatorial indexing method (Mulqueen et al, Nat Biotech, 2017) and novel bioinformatic procedures which have been generously supported by the 2018 Professor Tony Basten Fellowship for Medical Research, The University of Sydney Postdoctoral Fellowship scheme (2018-2021) and CCS Collaborative Research Infrastructure Awards.

Challenges within the field

Single-cell sequencing are relatively new technologies that are rapidly evolving and can be split into plate-based, droplet-based, and combinatorial indexing methods. Single-cell combinatorial indexing is the most recent of the technologies. Batches of cells or nuclei are pooled, fixed and DNA indexes are ligated through the use of engineered transposome enzymes followed by subsequent pooling, redistribution and indexing (Mulqueen et al, Nat Biotech, 2017). This technique has the advantage of the lowest cost and highest throughput of all methods.

We are working to improve the cost-per-cell and throughput by improving inefficient library preparation steps (amplification and DNA fragmentation) and bioinformatic processes (demultiplexing and alignment).

Research Project Description

The project is broken up into 3 distinct phases with the overarching aim to catalogue DNA methylation of human brain cells to be used for diagnosis of neurodegenerative disease (classifying brain-derived cell-free DNA in blood) and further understanding of mechanisms of disease onset (cell-specific gene regulation during aging).

Phase I - Optimising single-cell combinatorial indexing and DNA methylation sequencing
In the wet-lab, we are creating engineered Tn5 enzymes with molecular indexes (transposomes) that can be used for Single-cell Combinatorial Indexing (SCI). We are also optimising oligonucleotides used for linear amplification of SCI DNA fragments, investigating 3-tier systems for higher multiplexing of cells/nuclei in addition to non-bisulfite based enzymatic methods for SCI DNA methylation sequencing. In the dry-lab, we are developing new bioinformatic approaches to improve demultiplexing of next-generation sequencing reads using Levenshtein distances and base quality scores.

Phase II – Characterising single-cell DNA methylation of human brain cells
In the wet-lab, we have begun optimizing nuclei isolation methods followed by Fluorescent Activated Nuclei Sorting (FANS) from post-mortem tissues. Our aim is to characterise DNA methylation of all brain-cell types within brain regions that are susceptible to neurodegeneration within AD (Hippocampus), FTD (inferior / parietal regions), MND (Primary Motor Cortex) and SCA (Cerebellum).

Phase III - Characterising single-cell DNA methylation of the aging human brain.
The incidence of neurodegenerative diseases such as AD, FTD and MND increase across the decades 40-80 yrs. Due to the relatively young nature, post-mortem brain specimens without evidence of neurological disease are quite rare. Following a global search we have obtained a unique collection of 156 human post-mortem brain tissue specimens (gender balanced) generously donated by the National Institute of Health (USA). Our aim is to extend the characterization of DNA methylation in brain-cell types to additional brain-regions (eg. Insula, Amygdala) of the aging human brain.

Research Objectives

  • Optimising single-cell combinatorial indexing and DNA methylation sequencing
  • Characterising single-cell DNA methylation of human brain cells
  • Characterising single-cell DNA methylation of the aging human brain.

Project - Epigenetic k-mers

Research Project Abstract

Epigenetics is a field of research that aims to explain how DNA is regulated, why, how and when some genes are turned “off” or “on”. With the advent of massively parallel Next Generation Sequencing (NGS) enormous amounts of epigenetic data have been produced, however to capitalise on the potential of epigenetic information for disease diagnosis and monitoring new bioinformatic tools are required for data processing, statistical analysis and interpretation. Within our lab we are developing k-mer based search strategies for epigenetic information. These represent ultra-fast methods that can be applied directly to raw NGS data. This project aims to curate epigenetic k-mer databases and improve search strategies to improve assignment of DNA fragments to their cell-of-origin.

Research Project Description

https://www.sydney.edu.au/medicine-health/study-medicine-and-health/undergraduate-courses/honours/project-detail-876.html

Project - Brain-derived cell-free DNA

Research Project Abstract

DNA can be released into the blood stream following cell-death and is referred to as “cell-free” DNA. Cell-free DNA taken by simple blood test from an expecting mother is frequently used to diagnose fetal genetic abnormalities. Recently, cell-free DNA has been used to diagnose and monitor cancer. The brain is notoriously hard to monitor due to its location within the skull and the blood-brain-barrier. This project aims to investigate brain-derived cell-free DNA to diagnose and monitor neurodegenerative disease.

We have developed new next generation sequencing technology in order to detect brain-derived cell-free DNA, generously funded by a Brain Foundation Research Gift in 2017 and the 2018 Professor Tony Basten Fellowship for Medical Research. We are now leading a multinational initiative to evaluate the method for precision medicine in behavioural variant frontotemporal dementia generously funded by the NHMRC- European Union Joint Program on Neurodegenerative Disease Research (JPND)(2020-2025)

Challenges within the field

It is hypothesised that therapies for neurodegenerative diseases such as dementia may need to be initiated several years prior to the emergence of clinical symptoms to be maximally effective. However, even in the presence of clinical symptoms diagnoses of neurodegenerative diseases are often delayed or even missed.

Brain-derived cell-free DNA holds promise as a cheap blood test to identify neurodegeneration with the potential to be applied early, identifying patients for preventative therapies.

In clinical trials assessing new therapies in dementia the therapeutic efficacy is determined by cognitive and functional primary outcomes that are typically measured over the 18 month trial period. These tests, however, have significant measurement error that impedes the identification of even highly efficacious therapies (80%) in trials lasting < 5-years (Anderson et al, Lancet, 2017).

Such issues are not unique to clinical trials in dementia. Clinical trials for Spinocerebellar Ataxia (SCA) also employ functional testing to assess therapeutic efficacy which, due to considerable measurement error, requires large multinational cohorts (>250, eg. Transatlantic SCA consortia) in order to assess small to medium effect sizes (Schmitz-Hubsch et al, Neurology, 2010).

Brain-derived cell-free DNA holds promise to measure neurodegeneration for the purpose of evaluating therapeutic efficacy in neurodegenerative disease clinical trials over shorter periods of time and within smaller trial cohorts.

Research Project Description

https://www.youtube.com/watch?v=VerOJuwX3Rw