Distant upstream regulatory domains direct high levels of beta-myosin heavy chain gene expression in differentiated embryonic stem cells

Eukaryotic gene transcription takes place in the context of chromatin, In o rder to study the expression of the beta-myosin heavy chain (MyHC) gene in its appropriate cardiac environment in vitro, embryonic stem cell lines wer e generated and induced to differentiate into the cardiac lineage. We show that the upstream region of the beta-MyHC gene (-5518 to -2490 relative to the transcriptional start site) directed high levels of transcriptional act ivity only when stably integrated, but not when expressed extrachromosomall y in transient assays. These results are consistent with earlier findings u sing an in vivo transgenic approach. The expression of beta-MyHC reporter g ene constructs was strictly correlated to differentiation status and coinci ded with the expression of endogenous cardiac marker genes and with morphol ogical differentiation of embryoid bodies in vitro. Using populations of st ably transfected cell clones, two domains important for high level expressi on were identified. The analysis of individual cell clones suggested that t he positive regulatory domains act according to the graded model of enhance ment. These results show that chromosomal integration is necessary for the appropriate function of the beta-MyHC gene's upstream regulatory region. (C ) 2000 Academic Press.