P-TEFb is a protein kinase complex consisting of Cdk9 and Cyclin T. P-TEFb can 1) phosphorylate NELF and promote its dissociation from PolII; 2) phosphorylate DSIF and turn it into a transcriptional activator; and 3) phosphorylate PolII on Ser2 and promote elongation. PTEFb is thought to move quickly into promoter sites, e.g. in response to histone modifications or binding of specific transcription factors such as c-Myc. Recent ChIP-seq studies indicate that c-Myc binding is a key regulator of RNA synthesis by promoting PolII elongation in multiple genes. The widespread use of transcriptional pausing as a means to control mRNA expression DAPT Gamma-secretase inhibitor differs significantly from translational regulation during ESC differentiation. We recently performed translation state array analysis on differentiating murine ESCs, and we found that the majority of mRNA transcripts synthesized are loaded directly onto ribosomes. A minority of transcripts are controlled at the level of translation during differentiation, with mRNAs held in a paused state in pluripotent cells and then loaded with ribosomes. These data indicate that the checkpoint between transcriptional initiation and elongation may represent the major control point for gene expression during ESC differentiation. The induction of mesoderm requires coordinated signaling through members of the TGF-beta superfamily and the Wnt/bcatenin pathways. Both pathways are known to be strong regulators of transcription, but their contributions to the independent regulation of initiation and elongation remains to be addressed. Further ChIP-chip or ChIP-seq studies for the downstream effectors of these pathways would be illustrative. While our study has focused on differentiation of hESCs, we hypothesize that the checkpoint of paused transcription is a more general phenomenon as cells undergo other changes of state. For example, activation of endothelium by cytokines, conversion of blood monocytes to macrophages or differentiation of skeletal myoblasts to myotubes all are accompanied by changes in mRNA transcription. It will be of interest to determine whether paused transcription serves as an obligate checkpoint for transcriptional regulation as these cells change state in response to environmental cues. The capacity of long-term self-renewal and the unique ability to differentiate into all three germ layer cell types define pluripotency. According to the source of cells used for the establishment of pluripotent cells, one can currently distinguish between the following pluripotent cell lines: embryonic stem cells derived from the inner cell mass of mouse blastocysts at embryonic day 3.5, embryonic germ cells cultured from primordial germ cells that colonize the genital ridge at E12.5, embryonal carcinoma cells isolated from germ-cell tumors of either testis or ovary, germ line stem cells isolated from mouse neonatal and adult testis.