We are investigating the relationship between the heterochronic genes
lin-28, which controls larval seam cell fates, and
lin-46, which acts immediately downstream of
lin-28 in an antagonistic manner. Specifically, we want to elucidate the effects of these two genes on the expression of
hbl-1, a gene that is also involved in seam cell fates. Using the integrated
hbl-1::GFP::
hbl-1 3'UTR fusion construct as a reporter, we found that
lin-28 positively regulates
hbl-1 expression. In wildtype animals, this reporter is strongly expressed in the hypodermis from late embryogenesis and decreases until it is undetectable by the early L3. In a strain lacking the three
let-7 sisters (
let-7s),
mir-48,
mir-84 and
mir-124, this
hbl-1 reporter was constitutively expressed (Abbott et al. 2005). However, when
lin-28 was removed in this
let-7s(0) background, the
hbl-1 reporter was downregulated at the normal time. Our lab has also found that in the yeast two-hybrid system LIN-46 interacts with the conserved C-terminal zinc fingers of HBL-1. In fact, when these zinc fingers were expressed in wildtype animals, they displayed a retarded phenotype like
lin-46 null animals. These findings suggest that LIN-46 negatively regulates HBL-1 at the protein level. To further explore the relationships of
lin-28 and
lin-46 with
hbl-1, we constructed a
lin-28;
lin-46;
let-7s mutant strain and found the
hbl-1 reporter was constitutively expressed, consistent with its retarded phenotype (Abbott et al. 2005). Since the
hbl-1 reporter lacks the zinc fingers, it is not clear how LIN-46 effects the reporter's expression. Possibly, this constitutive expression resulted from a feedback loop involving HBL-1 protein. In parallel, we constructed a
lin-28;
lin-46 mutant strain containing the same reporter. Surprisingly, the
hbl-1 reporter was expressed constitutively in this strain, and the GFP fluorescence was significantly brighter than wild type. This is in spite of the fact that
lin-28;
lin-46 double mutants have wildtype development. These results are even more unexpected because we found that
lin-28;
lin-46 mutants have precocious expression of mature
let-7, a miRNA which probably targets the
hbl-1 3'UTR. Furthermore, the
hbl-1 reporter has a dominant
rol-6 marker, whose rolling phenotype was suppressed in this strain. Future experiments aim to determine if the high reporter expression in the
lin-28;
lin-46 strain reflects an increase in endogenous
hbl-1 expression. Additionally, a strain will be constructed of
lin-28;
lin-46 carrying the
hbl-1 reporter as an extrachromosomal array without the
rol-6 marker. This will rule out any effects from the integrated transgene, as well as any from the
rol-6 marker.