To determine sex in C. elegans, the early embryo must "count" the number of X chromosomes relative to the sets of autosomes (X:A ratio). The developmental switch gene, xol-1, assesses X chromosome number by responding to the dose of a small number of regulatory genes on the X chromosome, termed signal elements. A 2X dose of these signal elements repress xol-1, promoting hermaphrodite development, while a 1X dose allows xol-1 to remain active, promoting male development. We have identified and studied two X chromosome signal elements in detail. Repression of xol-1 by the nuclear hormone receptor, SEX-1, is conferred at the level of transcription and repression by the RNA binding protein, FOX-1, is conferred at the level of RNA splicing. Both modes of repression are required to maintain proper sex-specific expression of xol-1, and thus to ensure the fidelity of the X chromosome counting process. We have undertaken a promoter deletion analysis using xol-1::lacZ reporter gene arrays to identify cis-elements required for transcriptional repression by SEX-1 and other signal elements. In addition to identifying regions required for the sex-specific repression of the reporter gene, we have also uncovered a small region that may contain enhancer activity. We are further testing these regions for repressor/enhancer activity and asking if these activities are dependent on X chromosome signal elements. In vivo binding of SEX-1 to candidate repressor regions is underway (I. Carmi, this meeting). To understand the post-transcriptional repression of xol-1 by signal elements, we have investigated fox-1 function. xol-1 RNA is alternatively spiced at its terminal (sixth) intron. Retention of this intron, or inappropriate splicing of this intron results in loss of XOL-1 expression and activity. We found that FOX-1 functions in the nucleus to prevent proper splicing of xol-1's sixth intron. We show that this intron is both necessary and sufficient to confer fox-1-dependent repression in transgene studies. Furthermore, we show that FOX-1 acts through a small region contained within the sixth intron. This region of RNA is being tested for direct association with FOX-1. These data show that regulation of splicing is an integral part of the X chromosome counting process.

Affiliation:
- HHMI and Department of Molecular and Cellular Biology, University of California, Berkeley, CA. 94720