Abstract
K. L. Schreiber, A. Calderone and H. Rindt. Distant Upstream Regulatory Domains Direct High Levels ofβ -myosin Heavy Chain Gene Expression in Differentiated Embryonic Stem Cells. Journal of Molecular and Cellular Cardiology (2000) 32, 585–598. Eukaryotic gene transcription takes place in the context of chromatin. In order to study the expression of the β -myosin heavy chain (MyHC) gene in its appropriate cardiac environment in vitro, embryonic stem cell lines were generated and induced to differentiate into the cardiac lineage. We show that the upstream region of the β -MyHC gene (−5518 to −2490 relative to the transcriptional start site) directed high levels of transcriptional activity only when stably integrated, but not when expressed extrachromosomally in transient assays. These results are consistent with earlier findings using an in vivo transgenic approach. The expression of β -MyHC reporter gene constructs was strictly correlated to differentiation status and coincided with the expression of endogenous cardiac marker genes and with morphological differentiation of embryoid bodies in vitro. Using populations of stably transfected cell clones, two domains important for high level expression were identified. The analysis of individual cell clones suggested that the positive regulatory domains act according to the graded model of enhancement. These results show that chromosomal integration is necessary for the appropriate function of the β -MyHC gene's upstream regulatory region.
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