Epigenetic mechanisms underlying neurodegeneration
ABSTRACT
Rationale and significance
To investigate this, conformational changes in the mutant RFTS domain will first be verified, as this domain is thought to regulate DNMT1 autoinhibition and cofactor recruitment. Replication foci will be visualised via nascent DNA labeling, allowing DNMT1 localisation to be directly assessed within replication-associated chromatin environments. The organisation of these foci will be quantified using immunostaining. Also, high-resolution microscopy, including expansion microscopy (ExM) and cryo-electron tomography (cryo-ET) will be used to visualise chromatin domain ultrastructure at nanoscale resolution. Multi-omic profiling methods will complement this by identifying altered epigenetic interactors and native DNA contacts, exploring candidate factors and RNA-modification pathways without presupposing a single mechanism.
To demonstrate that epigenetic defects are a cause rather than a consequence of neurodegeneration, mechanistic discovery in mESCs will be validated in human ESC-derived cells, cerebral organoids, and mice.
Objectives and research questions
The central hypothesis posits that DNMT1 RFTS mutations, specifically DNMT1 A570V mutation, alters cofactor association and recruitment, hence, disrupting replication-focus organisation and RNA-modification pathways. Additionally, this mutation is hypothesised to increased neuronal vulnerability.
The project asks:
1. Does A570V alter DNMT1-associated replication-focus architecture and chromatin structure after point mutation?
2. Which altered DNMT1-associated factors and RNA-modification pathways occupy nascent-DNA environments?
3. Can restoring appropriate recruitment improve cellular or tissue phenotypes in human models and mice?
Preliminary work
Previous work established mESC and hESC culture systems and generated resources, including DNMT1-C-HA mESCs and H9-GFP A570V cells. S-phase enrichment and flow-cytometric validation were performed, and preliminary ExM achieved approximately tenfold nuclear expansion of nuclear punctate DNMT1-associated signals. Reduced growth and DNMT1 abundance in A570V hESCs are preliminary observations rather than proof of a specific mechanism.
Expected outcomes
A reproducible experimental workflow should be documented with quantitative results addressing the selected research questions. Students will develop practical skills in epigenetics, cell culture, high-resolution imaging (ExM, cryo-ET), multi-omic profiling and the interpretation of molecular and cellular data. Their work will contribute to the laboratory’s capacity to define how DNMT1 mutations disrupt chromatin organization and RNA-modification pathways, and to establish causal links between epigenetic dysfunction and neurodegeneration across mESC, human ESC-derived and in vivo models.
SKILLS & EXPERIENCE REQUIRED
1. Interest in epigenetics, neurodegeneration, molecular mechanisms of disease, or developmental biology.
2. Basic laboratory experience in cell culture (mESC/hESC maintenance) or molecular biology techniques (PCR, cloning, immunostaining) is desirable.
3. Willingness to learn advanced imaging techniques (expansion microscopy, cryo-ET) or multi-omic profiling methods.
4. Attention to detail in experimental record-keeping and data documentation.
5. Ability to work independently on defined lab tasks and seek guidance when needed.
6. Good communication and teamwork skills.
7. Commitment to regular project work and supervisory meetings throughout the research period.