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Saturday, February 7, 2009

Alex Meissner, Harvard University- “From reference genome to reference epigenome(s)”

Background on Chip-Seq.

High-throughput Bisulfite Sequencing. At 72 bp, you can still map these regions back without much loss of mapping ability. You get 10% loss at 36bp, 4% at 47bp and less at 72bp.

This was done with a m-CpG cutting enzyme, so you know all fragments come with at least a single Methylation. Some update on technology recently, including drops in cost and longer reads, and lower amounts of starting material.

About half of the CpG is found outside of CpG islands.

“Epigenomic space”: look at all marks you can find, and then external differences. Again, many are in gene deserts, but appear to be important in disease association. Also remarkable is the degree of conservation of epigenetic patterns as well as genes.

where are the functional elements?
when are they active?
when are they available

Also interested in Epigenetic Reprogramming (Stem cell to somatic cell).

Recap: Takahashi and Yamanaka: induce pluripotent stemcell with 4 transcription factors: Oct2, Sox2, c-Myc & KLF4[?] General efficiency is VERY low (0.0001% - 5%). Why are not all cells reprogramming?

To address this: ChIP-Seq before and after induction with 4 transcription factor. Strong correlation with chromatin state and iPS. Clearly see that genes in open chromatin are responsive. Chromatin state in MEFs correlates with reactivation.

Is loss of DNA methylation at pluripotency genes the critical step to fully reprogram? Test hypothesis that by demethylation, you could cause more cells to become pluripotent. Loss of DNA methylation does indeed allows transition to pluripotency shown. [lots of figures, which I can't copy down.]

Finally: loss of differentiation potential in culture. Embryonic stem cell to neural progenitor, but eventually can not differentiate to neurons, just astrocytes. (Figure from Jaenisch and Young, Cell 2008)

Human ES cell differentiation: often fine in morphology, correct markers... etc etc, but specific markers are not consistent. Lose methylation and histone marks, which cause significant changes in pluripotency.

Can't yet make predictions, but on the way towards it in the future where you can assess cell type quality using this information.



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