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Early embryonic stem cell differentiation includes “exit” from the pluripotent state followed by “commitment” to a specific lineage. During both of these processes there are genome‐wide changes in gene transcription levels that are regulated, at least in part, by epigenetic changes in histone tail modifications. We have demonstrated both global and gene specific changes in histone tail modifications. We focused on the role of these marks as they regulate the earliest stages of differentiation in mouse ES cells.Histone H3 phosphoacetylation (activating mark) is increased within the first 3 days of differentiation. Blocking general phosphorylation pathways markedly slowed exit from the pluripotent state. Blocking MSK1, the specific kinase responsible for phosphorylation on histone H3 still resulted in exit, but with diminished expression of early mesoderm markers. We also demonstrated a gene specific increase in acetylation (activating) of histones H3 and H4 and a global increase in acetylation of H4 as cells began lineage commitment. A pulse of the histone deacetylase inhibitor, TSA, maintained expression of markers of the undifferentiated state while also inducing expression of early mesoderm markers. TSA effects on later lineage commitment are being studied.We propose that epigenetic modifications play an important role in early cell fate decisions.This work was supported by NIH RO1 DK064243 from NIDDK.

RA treatment during mESC differentiation results in predominantly neural specification. A transient pulse of the histone deacetylase inhibitor, Trichostatin A (TSA), during early mESC differentiation via embryoid body (EB) formation results in maintenance of undifferentiated characteristics. Our objective was to determine whether a pulse of TSA followed by RA alters neural lineage specification. Undifferentiated mESCs are exposed to 100 nM of TSA during the first 24h of 3 days of EB formation. EBs are plated and RA added for an additional 4 days. Selected RT‐PCR shows repressed neural marker expression and only a small increase in a few endoderm markers. Gene expression array studies analyzed using a lineage annotated gene database demonstrates a marked increase in expression of numerous placental genes and confirms diminished neural gene expression. Immunofluorescence for the trophoblast marker Plac1, shows increased levels in TSA‐pulsed, RA‐treated cells with minimal expression in RA‐only differentiated cells. Conversely, the neural specific product, Doublecortin, is highly expressed in the RA‐only treated cells and shows diminished protein levels in TSA‐pulsed, RA‐treated cells. We conclude that transient TSA treatment prior to RA induces a dramatic lineage switch characterized by repressed neural differentiation and marked trophoblast induction. Supported by NIH grant RO1DK064243 to MKF.