The male tail of C. elegans is a complex structure composed of nine pairs of sensory rays embedded in a cuticular fan. Among the regulatory genes that define the patterning of these structures, mab-21 is required for the specification of the ray 6 identity. This thesis study aimed to define the regulation of the mab-21 gene at the molecular level....[ Read more ]

The male tail of C. elegans is a complex structure composed of nine pairs of sensory rays embedded in a cuticular fan. Among the regulatory genes that define the patterning of these structures, mab-21 is required for the specification of the ray 6 identity. This thesis study aimed to define the regulation of the mab-21 gene at the molecular level.

Several independent cis-regulatory elements were defined in the mab-21 locus. These elements included the hypodermal enhancer residing in the 5' flanking sequence; a neuronal enhancer in intron 4, the introns responsible for intron-mediated enhancement and the 1kb 3' enhancer for structural cell expression. This 3' enhancer, which efficiently boosts the mab-21 activity to revert the mutant phenotype into wild type phenotype, is believed to be essential for the specification of ray 6 identity.

Characterization of the 3' enhancer revealed that only 112bp of the 1kb 3' enhancer region was required for the specification of ray 6 identity. Within this 112bp, are three potential transcription factor binding sites, two are predicted to bind to homeodomain-containing factors and one to a forkhead domain-containing transcription factor. Eliminating of these sites by site directed mutagenesis either individually or in combination, revealed that all three sites contribute to the rescue of mab-21 tail phenotype. Moreover, interactions between these three sites were also uncovered.

The findings prompted me to link the transcriptional control of mab-21 with several known genetic partners/regulators of mab-21: egl-5, mab-5, mab-18 and unc-130. EGL-5, MAB-5 and MAB-18 are homeodomain-containing transcription factors and UNC-130 is a forkhead domain-containing transcription factor. Genetic analysis provided the evidence that UNC-130 acts through the 3' enhancer. When a construct containing full length mab-21 linked to green fluorescent protein was transformed in the wild type and unc-130, it is observed that fluorescent signal in the same tissues was much reduced in the mutants. This suggested that UNC-130 was a potential transcriptional regulatory factor for the expression of mab-21.

Besides studying the upstream regulatory factors, I also sought to identity the physical interacting partners for mab-21. Since mab-21's subcellular localization changes during development, some cellular components must be interacting with MAB21 to facilitate its changes in subcellular localization. To test this hypothesis, the yeast two-hybrid system was used to isolate the interacting partner(s) of MAB-21. A SIN3-like protein was isolated and was shown to interact with MAB-21. Ectopic expression of mab-21 by sin3-like promoter could rescue mab-21 mutant effectively; the expression pattern of the sin3-like gene resembled that of mab-21 as revealed by green fluorescent protein reporter; antisense blocking of the sin3-like gene and the expression of a dominant negative truncated SIN3-like protein induced ray phenotypes similar to those of mab-21 mutant. Taken together these results supported my model in which mab-21 is temporally and spatially co-regulated with the sin3-like gene. SIN3 is a molecule capable of interacting with many different transcription factors followed by repression of transcription through histone deacetylation. The establishment of a link between the novel protein MAB-21, and the SIN3-like protein should provide new insight into the role of mab-21, the regulation of MAB-21's subcellular localization and finally the importance of this gene in ray identity specification.