Worm Breeder's Gazette 14(5): 62 (February 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Dept. of MCD Biology, University of Colorado, Boulder, CO 80309-0347
We are continuing to investigate the control of her-1 expression, which results in a rare larger transcript encoding the HER-1A protein, necessary and sufficient for masculinization, and a moderately abundant smaller transcript of unknown function but with the potential to encode a HER-1B protein representing the C-terminal portion of HER-1A. These transcripts are driven by separate promoters, named P1 and P2, respectively (1), both under negative control by the sdc genes (2), which in turn are negatively regulated by xol-1 X and also are responsible for turning on dosage compensation in XX animals (see Ref. 3 and others from the Meyer lab). We have recent results consistent with the possibility that a her-1 gene product may feed back to help regulate dosage compensation.
In experiments related to those reported by Perry et al. (WBG 14(4): 60, 1996), we had independently noted that some constructs including the her-1 P2 promoter region, when carried as transgenic arrays in a him-8 strain, cause extensive embryonic lethality and a preponderance of males among surviving progeny, suggesting XX-specific lethality. In further experiments to understand this effect, we have obtained the following preliminary results, each in multiple independent transmitting lines. a) A P2-HER-1B construct including 3.4kb of promoter region, the HER-1B coding sequence, an HA tag sequence, and 0.4kb of 3' flanking region, causes the above described apparent XX lethality in him-8 transgenic lines. Unlike the effect described by Perry et al., we observe the skewed sex ratio primarily in the F2 and subsequent generations following injection. In xol-1 mutant lines, this construct causes much less embryonic lethality and also substantial masculinization of the xol-1 XX animals, which is dependent on presence of a functional her-1 gene. When mated to N2 males, hermaphrodites from these lines produce viable, mating XO male progeny, i.e., xol-1 XO lethality is strongly suppressed. b) We have begun similar experiments with a P2-HER-1B construct lacking the HA tag sequence but otherwise identical to the above. It appears to cause more severe XX lethality in him-8 strains (transmitting lines have not yet been recovered); it causes no apparent masculinization in xol-1 lines; but these animals produce viable mating xol-1 XO cross progeny. c) P2 promoter-only constructs (P2::lacZ and P2::GFP) with the same promoter region as above but producing no her-1 transcripts cause little apparent XX lethality in xol-1(+) lines and extensive masculinization of xol-1 lines, including production of mating XX males, in F2 and subsequent generations.
Thus the effects of P2 promoter-only arrays resemble those of weak or Tra sdc mutations, which result in low XX lethality and production of masculinized xol-1 XX animals, while the effects of P2-HER-1B arrays resemble those of some strong sdc mutations such as sdc-3 nulls, which result in high XX lethality and no masculinization (see Ref. 4 and references therein). These differences suggest that presence of the small her-1 transcript influences the effects seen. One possible model to explain our results so far is as follows: P2 promoter-only constructs include DNA sequences that in high copy number can titrate a negative regulator of her-1, such as maternally and embryonically produced SDC-1. P2-HER-1B constructs embryonically express the small her-1 transcript, which acts somehow to repress activation of dosage compensation, perhaps via an effect on SDC-3 (3). The differences seen between the HA-tag and no-HA-tag constructs could result from less repressing activity when the tag sequence is present. The lack of masculinization by the no-HA-tag construct could result from the stronger interference with dosage compensation, leading to repression of masculinization (presumably via repression of her-1), as observed earlier in genetic experiments (4). Such a function for the small transcript could possibly serve as a backup mechanism for repressing dosage compensation and for maintaining an appropriate level of the large her-1 transcript in normal XO animals.
One prediction of this model is that production of her-1 transcripts from the gf allele her-1(n695) [which is not well repressed by the sdc genes (2,6)] might allow survival of xol-1 XO animals by repressing dosage compensation. To test this possibility, we constructed him-8(e1489); her-1(n695); xol-1(y9). Analysis of this strain is still in progress; however, it is viable, producing some dead embryos, arrested larvae, and adults which are almost all partially to fully masculinized, including a high percentage (not yet accurately determined) of mating males. Based on (a still small number of) single-male matings, some of these are XX as expected (5), producing only a few XO male progeny (from nullo-X sperm resulting from him-8-induced nondisjunction). However, some appear to be XO, producing a much higher proportion (not yet accurately determined) of XO mating males among their cross progeny. If the triple mutant is indeed producing XO males, then her-1(n695) suppresses xol-1 XO lethality. More definitive tests of this suppression are in progress.
These results together suggest that one or more her-1 gene products may feed back on dosage compensation, possibly by regulating functions of SDC proteins. Further experiments are in progress to address some of many remaining questions, including whether HER-1A or its transcript are involved, whether translation of the small her-1 transcript occurs and is necessary, and whether SDC proteins are being directly regulated. In addition, we are using the masculinization of xol-1 XX animals as a preliminary assay for segments of the promoter region that interact with negative regulators of her-1.
References: 1) Perry et al. (1993) Genes & Dev. 7:216; 2) Trent et al. (1991) Mech. Dev. 34:43; 3) Davis and Meyer (1997) Devel., in press; 4) DeLong et al. (1993) Genetics 133:875; 5) Miller et al. (1988) Cell 55:167; 6) Perry et al. (1994) Genetics 138:317.