Localization of Distant Urogenital System-, Central Nervous System-, and Endocardium-Specific Transcriptional Regulatory Elements in the GATA-3 Locus

Abstract

We found previously that neither a 6-kbp promoter fragment nor even a 120-kbp yeast artificial chromosome (YAC) containing the whole GATA-3 gene was sufficient to recapitulate its full transcription pattern during embryonic development in transgenic mice. In an attempt to further identify tissue-specific regulatory elements modulating the dynamic embryonic pattern of the GATA-3 gene, we have examined the expression of two much larger (540- and 625-kbp) GATA-3 YACs in transgenic animals. A lacZ reporter gene was first inserted into both large GATA-3 YACs. The transgenic YAC patterns were then compared to those of embryos bearing the identical lacZinsertion in the chromosomal GATA-3 locus (creating GATA-3/lacZ “knock-ins”). We found that most of the YAC expression sites and tissues are directly reflective of the endogenous pattern, and detailed examination of the integrated YAC transgenes allowed the general localization of a number of very distant transcriptional regulatory elements (putative central nervous system-, endocardium-, and urogenital system-specific enhancers). Remarkably, even the 625-kbp GATA-3 YAC, containing approximately 450 kbp and 150 kbp of 5′ and 3′ flanking sequences, respectively, does not contain the full transcriptional regulatory potential of the endogenous locus and is clearly missing regulatory elements that confer tissue-specific expression to GATA-3 in a subset of neural crest-derived cell lineages.

ACKNOWLEDGMENTS

Ganesh Lakshmanan and Ken Lieuw contributed equally to this work, and both should be considered first authors.

We thank Jie Fan for outstanding technical assistance, members of the Engel lab, in particular Jorg Bungert, Ko Onodera, and Yinghui Zhou, for insightful discussions and help, and Gauri Tilak for assistance with transgenic analysis. We also thank Rick Gaber, Rob Nakamura, and Hong Liang for advice about yeast and for reagents.

This work was supported by an MSTP grant to Northwestern University (NIH T32 GM08152; to K.H.L.), a Scanlon fellowship from Evanston Hospital (to G. L.), research grants from the NWO (The Netherlands; to F.G. and A.K.) and the National Institutes of Health (GM28896; to J.D.E.).