ELT-2 is the major regulator of genes involved in differentiation, maintenance and function of C. elegans intestine from the early embryo to mature adult. elt-2 responds to overexpression of the GATA transcription factors END-1 and END-3, which specify the intestine, as well as to overexpression of the two GATA factors that are normally involved in intestinal differentiation, ELT-7 and ELT-2 itself. Little is known about the molecular mechanisms underlying these interactions, how ELT-2 levels are maintained throughout development or how such systems respond to developmental perturbations. Here, we analyse elt-2 gene regulation through transgenic reporter assays, ELT-2 ChIP and characterisation of in vitro DNA-protein interactions. Our results indicate that elt-2 is controlled by three discrete regulatory regions conserved between C. elegans and C. briggsae that span >4 kb of 5′ flanking sequence. These regions are superficially interchangeable but have quantitatively different enhancer properties, and their combined activities indicate inter-region synergies. Their regulatory activity is mediated by a small number of conserved TGATAA sites that are largely interchangeable and interact with different endodermal GATA factors with only modest differences in affinity. The redundant molecular mechanism that forms the elt-2 regulatory network is robust and flexible, as loss of end-3 halves ELT-2 levels in the early embryo but levels fully recover by the time of hatching. When ELT-2 is expressed under the control of end-1 regulatory elements, in addition to its own endogenous promoter, it can replace the complete set of endoderm-specific GATA factors: END-1, END-3, ELT-7 and (the probably non-functional) ELT-4. Thus, in addition to controlling gene expression during differentiation, ELT-2 is capable of specifying the entire C. elegans endoderm.
Footnotes
Author contributions
T.W. and J.Y.B. performed and analyzed the immunohistochemical and transgenic reporter experiments. B.G. performed the EMSA assays for in vitro DNA-protein interactions. E.O.N., A.G.R. and J.D.L. performed and analyzed the ELT-2 ChIP-Seq and RNA-Seq experiments. T.W. performed the mutant rescue experiment. T.W., E.O.N., J.D.L. and J.D.M. wrote and edited the manuscript.
Funding
Work in Calgary was supported by an operating grant from the Canadian Institutes of Health Research to J.D.M.; the ChIP-Seq/RNA-Seq experiments were funded by the National Institutes of Health [grant 5R01GM104050 to J.D.L.]. E.O.N. was supported by a Damon Runyon Cancer Research Foundation Postdoctoral Fellowship Award [2083-11]. J.D.M. gratefully acknowledges salary support from the Alberta Heritage Foundation for Medical Research (now AIHS) and from the Canada Research Chairs Program. Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs [P40 OD010440]. Deposited in PMC for release after 12 months.
