Novel approaches for epigenomic profiling of repetitive elements
Lead Research Organisation:
Queen Mary University of London
Department Name: Blizard Institute of Cell and Molecular
Abstract
Transposable elements (TEs) are mobile genetic elements that are highly abundant in virtually all
eukaryotic genomes. Host defense strategies against TE expansion involve epigenetic mechanisms, such as DNA methylation, that ensure their silencing. This can in turn have an impact on local chromatin environment and host gene expression. In plants, targeting of DNA methylation to TEs can create epialleles that drive phenotypic changes and that are transgenerationally inherited (Iwasaki & Paszkowski 2014 EMBO J). Similar TE-driven epialleles have also been described in mice (Jirtle & Skinner 2007 Nat Rev Gen). Despite the potential of TEs for dramatically affecting phenotype, surprisingly little is known about their epigenetics at the single-copy level, which in turn deeply impairs a deeper and wider understanding of their impact on the host. This shortcoming stems solely from the repetitive nature of TEs, which makes it notoriously difficult to assign short reads from high-throughput sequencing (HTS) to unique locations in the genome.
We propose to develop new sequencing strategies that allow for profiling of DNA modifications at repetitive loci, especially at evolutionarily young TE copies. Cambridge Epigenetix (CEGX) have developed technologies for detecting modified DNA bases (5-methylcytosine, 5-hydroxymethylcytosine), which we will combine with genome phasing solutions that allow for the assembly of short sequencing reads into long, multi-kb contigs. Genome phasing involves partitioning of high molecular weight DNA into hundreds of sub- genomic fractions, followed by fragmentation and sequencing. This partitioning facilitates the assembly of reads from repetitive elements. There are several well-developed genome phasing solutions that have been highly successful in resolving human haplotypes, including techniques based on multi-well partitioning, microdroplet partitioning, and transposase contiguity, which we will explore for profiling DNA modifications. Notably, such a technique would be considerably closer at delivering truly whole-genome epigenetic profiles than the current state of the art. Long read sequencing platforms could also be considered, but the bisulphite treatment underlying DNA methylation detection leads to DNA fragmentation, counteracting the benefits of long reads. Whilst some of these platforms also have the capability to detect DNA modifications without bisulphite treatment, they currently underperform with respect to sensitivity and resolution. The experimental design will be developed through a constant dialogue between the partners. The student will develop a first version of the technique in the Branco lab and then optimize and streamline the protocol with CEGX, coupling it to their DNA modification detection products. We will then implement this technique to profile unique copies of young mouse TEs (e.g., LINE1s, IAPs) in embryonic stem cells, in order to evaluate the epigenetic variability of TEs and how this correlates with the local chromatin environment, as well as with TE and gene transcription. We have previously shown that DNA modifications at LINE1s change
2
dynamically throughout cell differentiation (Ficz, Branco et al 2011 Nature), but the impact on gene expression has remained unclear.
As an alternative, backup solution, we will in parallel explore a PCR-based technique that profiles the junctions between TEs and single-copy regions and for which the Branco lab has promising preliminary data.
eukaryotic genomes. Host defense strategies against TE expansion involve epigenetic mechanisms, such as DNA methylation, that ensure their silencing. This can in turn have an impact on local chromatin environment and host gene expression. In plants, targeting of DNA methylation to TEs can create epialleles that drive phenotypic changes and that are transgenerationally inherited (Iwasaki & Paszkowski 2014 EMBO J). Similar TE-driven epialleles have also been described in mice (Jirtle & Skinner 2007 Nat Rev Gen). Despite the potential of TEs for dramatically affecting phenotype, surprisingly little is known about their epigenetics at the single-copy level, which in turn deeply impairs a deeper and wider understanding of their impact on the host. This shortcoming stems solely from the repetitive nature of TEs, which makes it notoriously difficult to assign short reads from high-throughput sequencing (HTS) to unique locations in the genome.
We propose to develop new sequencing strategies that allow for profiling of DNA modifications at repetitive loci, especially at evolutionarily young TE copies. Cambridge Epigenetix (CEGX) have developed technologies for detecting modified DNA bases (5-methylcytosine, 5-hydroxymethylcytosine), which we will combine with genome phasing solutions that allow for the assembly of short sequencing reads into long, multi-kb contigs. Genome phasing involves partitioning of high molecular weight DNA into hundreds of sub- genomic fractions, followed by fragmentation and sequencing. This partitioning facilitates the assembly of reads from repetitive elements. There are several well-developed genome phasing solutions that have been highly successful in resolving human haplotypes, including techniques based on multi-well partitioning, microdroplet partitioning, and transposase contiguity, which we will explore for profiling DNA modifications. Notably, such a technique would be considerably closer at delivering truly whole-genome epigenetic profiles than the current state of the art. Long read sequencing platforms could also be considered, but the bisulphite treatment underlying DNA methylation detection leads to DNA fragmentation, counteracting the benefits of long reads. Whilst some of these platforms also have the capability to detect DNA modifications without bisulphite treatment, they currently underperform with respect to sensitivity and resolution. The experimental design will be developed through a constant dialogue between the partners. The student will develop a first version of the technique in the Branco lab and then optimize and streamline the protocol with CEGX, coupling it to their DNA modification detection products. We will then implement this technique to profile unique copies of young mouse TEs (e.g., LINE1s, IAPs) in embryonic stem cells, in order to evaluate the epigenetic variability of TEs and how this correlates with the local chromatin environment, as well as with TE and gene transcription. We have previously shown that DNA modifications at LINE1s change
2
dynamically throughout cell differentiation (Ficz, Branco et al 2011 Nature), but the impact on gene expression has remained unclear.
As an alternative, backup solution, we will in parallel explore a PCR-based technique that profiles the junctions between TEs and single-copy regions and for which the Branco lab has promising preliminary data.
People |
ORCID iD |
| Miguel Branco (Primary Supervisor) | |
| Darren Taylor (Student) |
| Description | Transposable elements are repetitive DNA sequences that can copy and paste themselves in the genome, hence often being referred to as jumping genes. In humans, transposons make up between ~45-66% of the human genome and they are implicated in numerous diseases; predominantly cancer, autoimmunity and psychiatric disorders. It is notoriously difficult to assign ChIP-seq reads to individual transposon copies, particularly those which are evolutionarily young. This is due to their repetitive nature, meaning ChIP-seq data can map to multiple positions in the genome. Following this, reads are often discarded leaving so called "dead-zones" or reads are often mapped randomly to one of the many locations. This therefore masks the true regulation of these elements. We have overcome this obstacle and have developed and validated a tool to allow mapping of ChIP-seq data to individual transposon copies for the first time. |
| Exploitation Route | The outcomes once published will allow the transposable element field to accurately assign ChIP-seq data to transposable element copies, thereby allowing better understanding of their regulation. |
| Sectors | Pharmaceuticals and Medical Biotechnology |
| Description | Babrham Institute - Stefan Schoenfelder |
| Organisation | Babraham Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | N/A |
| Collaborator Contribution | Dr Stefan Shoenfelder provided assistance in initially setting up Hi-C in our lab. Dr Schoenfelder and Dr Steven Wingett are currently providing assistance in developing capture probes. |
| Impact | We have successfully implemented Hi-C in our lab and are currently working to design appropriate probes. |
| Start Year | 2017 |
| Description | Dr Gael Cristofari |
| Organisation | University of Côte d'Azur |
| Country | France |
| Sector | Academic/University |
| PI Contribution | We are currently working with Dr Cristofari to validate our tool. We hope the collaboration will be more mutually beneficial following this. |
| Collaborator Contribution | Dr Cristofari provided sequencing data and two cell lines which we have used to help validate our novel approach. |
| Impact | The collaboration has aided in the validation of our approach. |
| Start Year | 2019 |
| Description | Blizard STARS |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | Approximately 10 students from underprivileged backgrounds from schools across London attended the Blizard institute for the week to obtain some practical lab experience with different postgraduate students, during which we also highlighted STEM career paths. |
| Year(s) Of Engagement Activity | 2018 |
| Description | London Interdisciplinary Doctoral Training Programme Retreat |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | ~180 students from across 6 London based universities attended a three day residential retreat in which I was on the committee in 2018 and the committee lead in 2019. At the event we organised career panels with attendees from the following sectors: innovation centres/startups, policy makers/advisors, scientific academics, patent attorney firms, and consultants. We also organised workshops from external organisations in public engagement, communicating your science effectively to the press, presentation skills and entrepreneurship. Additionally, we organised renowned keynote speakers to give an overview of their careers and research alongside team building events |
| Year(s) Of Engagement Activity | 2018,2019 |
| Description | Postgraduate Student Educational Events |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Educational events are organised monthly by the Postgraduate Committee which I am a member of. We provide a variety of monthly career talks and panels for postgraduate students of post-docs from across Queen Mary, University of London. |
| Year(s) Of Engagement Activity | 2018,2019,2020 |