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Oliver Hofmann
Ihor R Lemischka: Systems level approaches to stem cell fate (Keynote)
Deriving genetic, epigenetic and dynamic blueprints of stem cells - Oliver Hofmann
Focus on mouse embryonic stem cells (ES) - Oliver Hofmann
Self-renew, differentiate (in vivo or vitro) into around 120 cell types including the germ line - Oliver Hofmann
Set of known key TFs and pathways (Oct4/Sox2 etc), or inhibition of Erk1/2 and GSK3. Nanog levels may define two different states of pluripotency - Oliver Hofmann
Nanong part of an auto-regulatory circuit maintaining pluri-potency - Oliver Hofmann
Knock down nanog, or sox2 or oct4 one gets blocks of gene expression changes that are superinposable (i.e., same target genes) - Oliver Hofmann
See Lemischka review in Nature Reiews on Mol Cell Biology - Oliver Hofmann
Measure network dynamics during changes in cell fate a requirement. Technical limitations restricted to snapshots at one potential regulatory level - Oliver Hofmann
Recap of the epigenetic molecular and temporal cell landscape - Oliver Hofmann
"Pinball origin of nanog" - Oliver Hofmann
How to convert the snapshots to movies: Lu et al, Nature 463 (2009) - Oliver Hofmann
ES cell lines to tune Nanog expression levels. Vector with Nanog shRNA, inducable promoter. Depend on Doxycycline for Nanog expression, tight control of levels (endogenous nanog knocked down, replaced by new version) - Oliver Hofmann
New publication (last week's nature?): Trace histone marks, Pol II, RNA, 1600 proteins by MassSpec. Day 0, Day 1/3/5 of ES cells - Oliver Hofmann
"Sobering results": if anything an anti-correlation on the expression level; activity of encoded proteins cannot be determined for this sample - Oliver Hofmann
Representation of high dimensional data sets: three dimensional heatmaps, interactive - Oliver Hofmann
Views on all known Nanog, Polycomb targets. Group them by RNA, Protein levels etc. Same option for gene sets (all TFs, drill down to targets of TFs) - Oliver Hofmann
Nanog snapshot (Wang, Nature 2006, Orkin lab) converted to a movie, split up into different time points - Oliver Hofmann
Stem cells: the movie -- coming soon (to the lab website) - Oliver Hofmann
Databases and software to follow (GATE), interfacing prior knowledge. Superimpose existing targets and observe how they relate to the experimental data - Oliver Hofmann
What happens when pulsing Nanog? Turn off, turn on a bit later (the theory of nanog levels and different pluripotency states) -- do you recover the pluripotent state? - Oliver Hofmann
nanog changes not reflected in changes of other pluripotency markers, work in progress at the single cell level - Oliver Hofmann
9 markers in 200 different ES cells monitored by qRT-PCR. Some with very narrow distribution (Oct4, Sox2); Nanog with widely distributed expression levels - Oliver Hofmann
[Love the visualization / interaction system, hopefully available at some point] - Oliver Hofmann
Nanog and Esrrb interacting at different regulatory levels. What happens when Esrrb is taken away (same approach as in Nanog)? - Oliver Hofmann
Strong discordance between mRNA/protein levels - Oliver Hofmann
Monitor landscape after Esrrb down-regulation - Oliver Hofmann
Expand pipeline to additional data types, conditions - Oliver Hofmann
Extended prior knowledge system as a predictive tool used to plot expression level changes after taking away Nanog or Essrb: while closely linked to each other (auto-regulation, PPI) strong changes in target fold changes - Oliver Hofmann
Functional validation by comparing against published RNAi hits - Oliver Hofmann
Transfect Nanog-GFP cells with shRNA, after 24h treat with RA+, follow with FACS - Oliver Hofmann
Knockdown of gene required for neuronal differentiation vs knockdown of gene required for self-renewal observable by reaction to RA stimulus - Oliver Hofmann
shRNA against 350 usual suspects. Hit Oct4, report goes away rapidly; hit components of RAR prolongs time in which Nanog is expressed - Oliver Hofmann
Rank targets by relative promoter construct expression - Oliver Hofmann
Identify SWI/SNF as a potential switch to dismante the pluripotency network - Oliver Hofmann
(a differentiation trigger) - Oliver Hofmann
[And yes, they are hiring -- quantitatively minded students, post docs and faculty] - Oliver Hofmann