5hmC is restored on newly replicated DNA with a substantial time delay, and therefore in fast proliferating cells where a lot of new DNA is being synthesised, the global levels of 5hmC are diluted down.
Treatment with such high levels of Vitamin C (1 mM) will most certainly slow down proliferation and therefore partially restore levels of 5hmC. This also means that TET enzymes are still present and active in the cells, and their downregulation cannot be fully responsible for the observed "loss" of 5hmC upon placing primary cells in culture.
Competing interests
None declared
Author reply to comment of Martin Bachman
Colm Nestor, Linköping University
16 February 2015
Dear Martin,
Thank you for your interest in our research.
Indeed, proliferation is likely to be fundamental to the loss of 5hmC observed upon adaptation of primary cells to culture conditions, as we clearly stated in the manuscript. However, proliferation alone does not explain the maintenance of 5hmC at key developmental loci or the widespread remodelling of the transcriptome in culture.
The article referred to by Martin Bachmann, which is Martin’s own article (Bachman et al, Nature Chemistry, 2014), was published while our article was under review. Martin’s article provides several fundamental and valuable insights into the dynamics of 5hmC in mammalian tissues and cultured cells, and is a key reference for anyone in the field. It does not, however, analyse the changes that occur during adaptation of primary cells to culture conditions, which is the sole focus of our paper.
Again, we thank you for your interest and valuable comment.
Global levels of 5hmC vs. proliferation
10 February 2015
The relationship between 5hmC levels and proliferation has already been described and explained in detail here:
http://0-dx-doi-org.brum.beds.ac.uk/10.1038/NCHEM.2064
5hmC is restored on newly replicated DNA with a substantial time delay, and therefore in fast proliferating cells where a lot of new DNA is being synthesised, the global levels of 5hmC are diluted down.
Treatment with such high levels of Vitamin C (1 mM) will most certainly slow down proliferation and therefore partially restore levels of 5hmC. This also means that TET enzymes are still present and active in the cells, and their downregulation cannot be fully responsible for the observed "loss" of 5hmC upon placing primary cells in culture.
Competing interests
None declaredAuthor reply to comment of Martin Bachman
16 February 2015
Dear Martin,
Thank you for your interest in our research.
Indeed, proliferation is likely to be fundamental to the loss of 5hmC observed upon adaptation of primary cells to culture conditions, as we clearly stated in the manuscript. However, proliferation alone does not explain the maintenance of 5hmC at key developmental loci or the widespread remodelling of the transcriptome in culture.
The article referred to by Martin Bachmann, which is Martin’s own article (Bachman et al, Nature Chemistry, 2014), was published while our article was under review. Martin’s article provides several fundamental and valuable insights into the dynamics of 5hmC in mammalian tissues and cultured cells, and is a key reference for anyone in the field. It does not, however, analyse the changes that occur during adaptation of primary cells to culture conditions, which is the sole focus of our paper.
Again, we thank you for your interest and valuable comment.
Colm Nestor
Competing interests
None declared