Single-molecule long-read sequencing reveals chromatin basis of gene expression

Audrey opened her talk by stating that there is great complexity in the organisation of the genome, with different levels of folding required for packaging the genome into chromosomes. It is known that there is a relationship between these degrees of folding and the control of gene expression, and the research of Audrey and her team involves determining this relationship.

One technique that can be used to investigate this is NOME-seq (nucleosome occupancy and methylome sequencing). This method involves treating the target sample with methyltransferase to investigate endogenous CpG-specific vs. exogenous GpC-specific 5mC methylation, to reveal chromatin state. The nucleosome protects nucleosomal DNA from being methylated by the exogenous methyltransferase, but cytosines which are exposed in the linker sequences between the nucleosomes are preferentially methylated to 5mC.

Audrey described how their method, MeSMLR-seq - methyltransferase treatment followed by nanopore single-molecule long read sequencing - can be used to map nucleosome occupancy at the single-molecule level, to differentiate open/accessible from closed/inaccessible regions of the genome. Furthermore, Audrey stated that, because they used yeast cells in their experiments, single molecules represented single cells in these instances.

Similar to NOME-seq, MeSMLR-seq profiles 5mC modifications at GpC sites; these modifications are inserted preferentially at regions of open chromatin. However, by employing long-read nanopore sequencing, methylation could be directly measured via changes in the current, and long reads enabled multiple genes and nucleosomes to be spanned in single reads.

Presenting data from two genomic loci, Audrey described how there was high heterogeneity in nucleosomal positioning around the AUA1 gene, which is transcriptionally silent, at the transcriptional start site, but high uniformity of nucleosome spacing. The EMW1 gene, which is actively transcribed, shows the converse - low heterogeneity in nucleosome positioning but low uniformity in nucleosome spacing. This shows how MeSMLR-seq can relate nucleosome positioning and chromatin accessibility to transcriptional activity of genes.

Next, Audrey showed an example demonstrating a relationship between accessibility and coexpression of the glucose transport genes HXT3 and HXT6 ; as glucose concentration increases, HXT3 expression reduces, whereas the expression of HXT6 increases. Her team found that changes in the chromatin status followed these gene expression changes.

In conclusion, Audrey stated that MeSMLR-seq enables the long-range measurement of chromatin accessibility, and phasing of nucleosomes at the single-molecule/cell level, to demonstrate the link between chromatin accessibility and gene expression.

Together with single-cell RNA sequencing, MeSMLR-seq can be used to reveal the quantitative link between chromatin accessibility and gene transcription.

For more information about MesSMLR-seq, check out the publication from Audrey's team, in the Resource Centre section of our website:

Authors: Audrey Bollas