The
let-7 microRNA was discovered as a regulator of the L4 larval stage to adult transition.
let-7 transcription oscillates across larval development, with a peak in every stage. But the transcriptional activator of
let-7 and functional significance of its oscillatory expression are not characterized. Here, we report that the transcription factor NHR-23 activates transcription of
let-7, and in turn,
let-7 represses the expression of
nhr-23, thus forming a feedback loop. NHR-23 is the homolog of the Retinoid-related Orphan Receptor (ROR) and is predicted to bind consensus ROR Response Elements (RORE) in the promoter of
let-7. Chromatin immunoprecipitation of NHR-23 followed by qPCR of the
let-7 promoter supports direct binding of NHR-23 to the
let-7 promoter. Scrambling 2 of the
let-7 RORE sites abrogates NHR-23 binding to the
let-7 promoter. In addition, the
let-7 RORE mutants exhibit penetrant defects in vulval development and increased seam cell number, consistent with the partial loss of
let-7 activity.
let-7 targets the 3'UTR of the
nhr-23 transcript through
let-7 complementary sequences (LCSs). Expression of a reporter gene fused to the
nhr-23 3'UTR is enhanced when one of the LCSs is mutated. In the endogenous context,
let-7 dampens oscillations of
nhr-23 as worms go through development. Mutating the
nhr-23 LCSs diminishes dampening of
nhr-23 transcript oscillations and leads to supernumerary molts. Furthermore, overexpressing
nhr-23 also leads to aberrant supernumerary molts in adults, similar to
let-7 hypomorphs. The NHR-23-
let-7 negative feedback loop also affects the pace of development.
let-7 hypomorphs,
let-7 RORE mutants, and
nhr23 overexpressing strains all precociously exit quiescent phases between molts (lethargus). RNAi of
nhr-23 leads to delayed exit from lethargus that is rescued by
let-7 mutations. Taken together, these data suggest that the NHR-23-
let-7 negative feedback loop regulates both the number of molts and the pace of the molting cycle. The negative feedback loop between RORs and
let-7 may be conserved, as evidenced by the presence of homologous cis-regulatory elements in mammalian genes. Transcription of a reporter driven by the mouse
let-7 promoter in C. elegans is responsive to the levels of
nhr-23. LCSs in the 3'UTRs of human RORbeta and mouse RORalpha also regulate reporter gene expression in C. elegans. Therefore, the NHR-23-
let-7 negative feedback loop may represent a conserved genetic oscillator that regulates biological rhythms.