Evidence That Gene Activation and Silencing during Stem Cell Differentiation Requires a Transcriptionally Paused Intermediate State
Figure 1
Characterization of the transcriptional state of developmentally regulated loci with known functions.
(A) H7 human embryonic stem cells (WiCell) were maintained in MEF conditioned medium [35], replated into a high density monolayer in wells of a 24-well plate and differentiated as previously described in RPMI supplemented with B27 with the sequential addition of Activin A or BMP4. (B) The pluripotent cell population was collected from the undifferentiated human embryonic stem cells in colony growth conditions, the mesodermal population collected 48 hours after the addition of Activin A and the cardiac population was collected from cells 14–21 days after Activin A addition and enrichment via a Percoll density gradient purification [36]. (C) Loci were characterized as being silent if they lacked both tri-methylation on lysine 4 of histone H3 and full-length transcript, paused if they had detectable tri-methylation on lysine 4 of histone H3 only, or active if they had both full-length transcript and tri-methylation of lysine 4 on histone H3. (D) The BRACHYURY T locus, essential for mesoderm formation, is paused (P) in pluripotency and active (A) in mesoderm, is bound by tri-methyl lysine 4 on histone H3 (H3K4me3) in both pluripotency and mesodermal cell populations, with full length transcript above our background threshold (green, non-significant transcript in grey) only in the mesodermal (M) population, not in pluripotency (P) or cardiomyocytes (C). (E) The NKX-2.5 locus, encoding a transcription factor essential for cardiomyocyte differentiation, is paused in both early populations, is modified with H3K4me3 in both pluripotent and mesodermal populations of cells, with transcription above our background threshold only in the cardiac cell population. (F) The NEUROD1 locus, encoding for a transcription factor essential for neuronal ectoderm, is paused in the pluripotent population, becomes silent in the mesodermal population, is associated with H3K4me3 in the pluripotent but not mesodermal population of cells, and does not produce full-length transcript above a background threshold in any observed population of this cardiac mesoderm directed differentiation.