The GATA type transcription factor ELT-2 is proposed to be the major regulator of transcription in the C. elegans intestine after endoderm specification. Ectopic expression of END-1 and END-3, redundant GATA factors necessary for endoderm specification, or ELT-7 (another intestine specific GATA factor) can initiate ectopic expression of ELT-2, suggesting that END-1, END-3 and ELT-7 can activate
elt-2 expression in the earliest endoderm lineage either directly or indirectly. Previous experiments showed that ELT-2 can bind to its own promoter in vivo. To understand the molecular details of how
elt-2 transcription is initiated during embryonic development and is maintained thereafter, we are analyzing the promoter region of
elt-2 in C. elegans. Comparison of upstream sequences of the
elt-2 gene from 4 different Caenorhabditis species revealed three conserved regions (CRI-CRIII). 3' and 5' Deletion series as well as analysis of reporter constructs containing different combinations of the CRs suggested that CRI contains the basal promoter and CRIII contains the main enhancer of
elt-2. There are 3, 3 and 4 conserved GATA sites within CRI, CRII and CRIII respectively. Band shift assays showed that END-1 and ELT-2 can bind to at least one and all four GATA sites within CRIII in vitro respectively. Reporter expression was absent after mutating all GATA sites within a CRIII:CRI:gfp construct, indicating that the GATA sites are necessary for
elt-2 expression and that
elt-2 is mainly regulated by GATA factors. To test if ELT-2 can drive intestinal specification and differentiation in the absence of END-1/-3, we expressed
elt-2 under control of the
end-1 promoter in the
end-1/-3 double mutant. Indeed, the
end-1p::
elt-2 construct is able to rescue the
end-1/-3 double mutant with 50% penetrance, showing that the endoderm differentiation factor ELT-2 can also drive endoderm specification. We engineered an
end-1/end-3/elt-4/elt-7 quadruple mutant and the
end-1p:
elt-2 construct is also able to rescue this mutant showing that ELT-2 is the only GATA type transcription factor needed for intestinal development and further supporting the hypothesis that ELT-2 is involved in the regulation of every gene expressed in the intestine.