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Song Group Research Overview

The goal of research in our group is to develop and apply novel tools to probe epigenetic and epi-transcriptomic modifications, thereby understanding their functions in human health and disease. To do so, we combine various chemical biology and genome approaches to develop novel tools to analyse the epigenome. We then apply our tools to detect the epigenetic information in body fluids for non-invasive disease diagnostics, including early detection of cancer. In addition, we are investigating the epigenetic heterogeneity of tumours to understand the contribution of epigenetics to cancer.

1. Novel technologies to sequence the epigenome

Bisulphite sequencing is the current gold standard for sequencing DNA epigenetic modification including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). However, it has always been an unsatisfactory method due to its indirect detection and harsh chemical treatment, which damages most of the DNA in the samples. Recently, we developed TAPS (TET-assisted pyridine borane sequencing), a mild bisulphite-free and base-resolution sequencing method that detects 5mC and 5hmC directly without affecting unmodified cytosines. TAPS could replace bisulphite sequencing as the new standard in DNA epigenetic analysis and could have wide applications in academic research and clinical diagnostics, especially in sensitive low-input samples, such as circulating cell-free DNA (cfDNA), single-cell epigenetics, and long-read epigenetic sequencing.

 

A schematic of the TAPS (TET-assisted pyridine borane sequencing), TAPSbeta (TAPS with beta-glucosyltransferae (betaGT) protection) and CAPS (chemical-assisted pyridine borane sequencing) reactions. In TAPS, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are converted using TET enzyme to 5-carboxylcytosine (5caC). Pyridine borane converts 5caC to dihydouracil (DHU), which is converted to thymine (T) during PCR. Cytosine (C) to T transitions during TAPS therefore read out cytosines that were originally modified with either 5mC or 5hmC. In TAPSbeta, betaGT converts 5hmC (but not 5mC) to 5gmC. TET enzyme converts 5mC (but not 5gmC) to 5caC, which is then converted using pyridine borane to DHU (leaving 5gmC untouched). In the PCR step, DHU is converted to T and 5gmC is converted to C. C to T transitions during TAPSbeta therefore read out cytosines that were originally modified with 5mC but not those originally modified with 5hmC. In CAPS, potassium perruthenate is used to convert 5hmC (but not 5mC) to 5-formlycytosine (5fC). Pyridine borane converts 5fC to DHU, which is then converted to T during PCR. C to T transitions during CAPS therefore read out cytosines that were originally modified with 5hmC but not those originally modified with 5mC.A schematic of the TAPS (TET-assisted pyridine borane sequencing), TAPSbeta (TAPS with beta-glucosyltransferae (betaGT) protection) and CAPS (chemical-assisted pyridine borane sequencing) reactions. In TAPS, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are converted using TET enzyme to 5-carboxylcytosine (5caC). Pyridine borane converts 5caC to dihydouracil (DHU), which is converted to thymine (T) during PCR. Cytosine (C) to T transitions during TAPS therefore read out cytosines that were originally modified with either 5mC or 5hmC. In TAPSbeta, betaGT converts 5hmC (but not 5mC) to 5gmC. TET enzyme converts 5mC (but not 5gmC) to 5caC, which is then converted using pyridine borane to DHU (leaving 5gmC untouched). In the PCR step, DHU is converted to T and 5gmC is converted to C. C to T transitions during TAPSbeta therefore read out cytosines that were originally modified with 5mC but not those originally modified with 5hmC. In CAPS, potassium perruthenate is used to convert 5hmC (but not 5mC) to 5-formlycytosine (5fC). Pyridine borane converts 5fC to DHU, which is then converted to T during PCR. C to T transitions during CAPS therefore read out cytosines that were originally modified with 5hmC but not those originally modified with 5mC.

2. Epigenetic-based diagnostics

Aberrant patterns of DNA epigenetic modifications are hallmarks of cancer development. Detecting epigenetic alterations from circulating cfDNA hold promise as sensitive and specific detection strategies for disease diagnostics and prognostics. In particular, the tissue and cancer-specific epigenetic information could provide important tissue-of-origin information crucial for early cancer detection. One of our long-term goals is to develop comprehensive cell-free DNA epigenetic sequencing using TAPS and related methods to achieve the full potential of liquid biopsy for early cancer detection.

3. Epigenetic heterogeneity of tumours

Epigenetic factors are known to have pivotal roles in tumour development and maintenance. It is also becoming clear that tumour tissue is highly heterogeneous in terms of the genome, transcriptome and epigenome. Defining cell lineages within a tumour based on epigenetic information may provide insights to identity crucial events in tumour development and to guide cancer prevention and therapeutics. We are applying TAPS and other methods to develop novel single-cell epigenetic sequencing to study the epigenetic heterogeneity of tumours.

 

Chunxiao Song graphical abstract