The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. We show that Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (
SummaryThe methylation of CpG dinucleotides located in key protein binding sites within gene regulatory regions often leads to gene silencing. A mechanism of aging is proposed whereby an accumulation of methylation at gene regulatory sites contributes to cellular senescence. DNA methyltransferases (DNMTs) are enzymes that catalyze the transfer of a methyl moiety from S-adenosyl-L-methionine (SAM) to the cytosine of a CpG dinucleotide and are responsible for establishing and maintaining methylation patterns in the genome. It is important to study not only transcription of the DNMTs, but also their protein expression because studies illustrate that it is possible for the enzymes to undergo posttranslational physical changes in response to stimulation even though gene transcription remains unchanged. Here, we discuss an in vitro method to study protein expression of DNMTs in aging systems.
Hedgehog acts as an organizer during development. Its signaling involves the receptor Patched, signal transducer Smoothened and a cytoplasmic complex containing the transcription factor Cubitus interruptus tethered to the Smoothened carboxyl tail. Without Hedgehog, Patched represses Smoothened resulting in proteolysis of Cubitus interruptus to its repressor form. With Hedgehog, Patched repression of Smoothened is relieved and Cubitus interruptus is activated. Sex-lethal, the master switch for sex determination in Drosophila, has been shown to associate with Cubitus interruptus and the cytoplasmic components of the Hedgehog signaling pathway. Additionally, Sex-lethal responds to the presence of Hedgehog in a Patched-dependent manner. The latter prompted us to examine the role of Patched in signaling. We find that Cubitus interruptus, Sex-lethal, Patched and Smoothened co-immunoprecipitate and co-fractionate, suggesting a large complex of both membrane and cytoplasmic components of the Hedgehog pathway. The entire complex is present at the plasma membrane and the association of Patched changes depending on the activation state of the pathway; it also is not female specific. Colocalization analyses suggest that Sex-lethal alters the endocytic cycling of the Hedgehog components and may augment the Hedgehog signal in females by decreasing the proteolytic cleavage of Cubitus interruptus, availing more of it for activation.
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