Hia5 Chromatin Methylation at the Istituto Italiano di Tecnologia
From December 2, 2024 to February 22, 2025, Tianyuan Liu, LongTREC Doctoral Candidate at the Institute for Integrative Systems Biology (CSIC-UV) in Valencia, had the opportunity to carry out a research secondment at the Istituto Italiano di Tecnologia, supervised by Francesco Nicassio. This secondment offered valuable growth, both in scientific expertise and personal development.
My goal? Mastering and implementing the Hia5 chromatin methylation protocol in two human cell lines: H1 embryonic stem cells and HEK293 embryonic kidney cells. Hia5 is a DNA adenine methyltransferase that methylates accessible DNA in chromatin. The methylation pattern can then be read by long-read sequencing, giving single-molecule maps of chromatin accessibility.
Tianyuan reflects on his experience:
What I Learned ๐
I learned to culture both cell lines, isolate intact nuclei, prepare the required buffers, handle the Hia5 enzyme and its methyl donor, and extract high-molecular-weight DNA for sequencing. The protocol was run on H1 at test scale and on HEK293 at full scale, and I delivered a draft Standard Operating Procedure (Hia5-v1.0) to the host lab.
Takeaways ๐ฑ
Only the HEK293 experiment was completed. H1 nuclei yields were lower than anticipated, and extending the digitonin permeabilisation from 5 to 8 minutes to deal with residual cell clumps fragmented the genomic DNA. The lessons: disperse colonies thoroughly before digitonin treatment, and adhere strictly to protocol timing and concentrations during permeabilisation.
Impact ๐
The HEK293 line yielded intact, sequencing-ready high-molecular-weight DNA. For future H1 experiments, reducing digitonin concentration rather than extending incubation time should avoid the same DNA degradation.
Overall, this secondment established a technical foundation in an advanced epigenetics protocol for long-read chromatin profiling.
