Molecular Determination of Gender in Drosophila

Transcription

1 Molecular Determination of Gender in Drosophila 5th edition Alternate Splicing of RNA p Proteins Involved in Control of Transcription: Transcription Factors p Hyperactivation of X Chromosomes in Drosophila p Sex Determination in Drosophila p Maternal-Effect Genes p : Q and P #7, 16, 17, 21, 29 21; BE #3; Q AND P #10, 11, 12, 13, 14, 16 GENERAL CONSIDERATIONS Gender development is controlled by the X/A Ratio in the cells of the early embryo. Gender development is Cell Autonomous Dosage Compensation involves hyperexpression of X-linked genes in males The text describes the process in a temporal sequence beginning with oogenesis and ending with gender-specific gene expression. These notes reverse that order. Otherwise, the information is equivalent.

2 Doublesex (dsx) DSX protein is the final downstream switch in the gender regulation scheme. dsx is an autosomal gene that is transcribed in all cells of both XX and XY embryos. DSX protein exists in either a male-specific (DSX-M) or female-specific (DSX-F) form. Which form of the Dsx protein is produced in a given cell is governed by alternate splicing of its pre-mrna. The default (unguided) splice apttern yields an mrna that is translated into DSX-M. DSX-M represses transcription of felmale-specific genes. The alternate splice pattern must be "guided" by the TRA protein, which is normally present in XX embryos. TRA-dependent splicing yields an mrna that is translated into DSX-F. DSX-F represses transcription of male-specific genes. Note that splicesomes regognize the boundaries of "exon 4" only in the presence of TRA. In the absence of the TRA protein, "exon 4" of the dsx pre-mrna excised, so the protein is terminated by a stop codon in exon 6. Both the DSX-F and DSX-M proteins are DNA-binding repressors of gender-specific gene expression. The dsx null phenotype is a true intersexual (sterile) individual for both XX and XY individuals because all cells express both male and female-specific genes. See Table These "true intersexual" individuals are not the same as the "intersexual mosaics" resulting when X/A = >0.5 <1.0) This begs the question of why TRA is normally present in XX, but not in XY embryos.

3 Transformer (tra) Production of TRA protein is also controlled by alternate splicing of its pre-mrna. Splicing of the tra pre-mrna is guided by the SXL protein, which is normally present only in XX embryos. In this case the protein, resulting from the unguided splice, has no function. Note that in this case the unguided (default) splice recognizes intron/exon boundaries that the SXL-guided splice ignores. This is the opposite of what happens in dsx pre-mrna splicing. The tra null phenotype is transformation off XX individuls to male gender. There is no effect (neutral) in XY embryos. autosomal, tra/tra converts XX to male (but no sperm production because that requires 6 Y-linked loci) This begs the question of why SXL is normally present in XX, but not in XY embryos.

4 Sex-Lethal (sxl) Production of SXL protein involves yet a third case of alternate pre-mrna splicing, but the situation is complicated by the fact that 2 different forms of sxl pre-mrna are produced by transcription of 2 different promoters. Also, SXL expression is ultimately controlled by the X/Autosome ratio. X-linked null phenotypes is XX embryonic lethal because X hyperexpression is not repressed two periods of transcription, from alternate promoters PE and PM MODEL FOR X/A RATIO COMPUTATION Control of sxl transcription in the fertilized egg by the X/A ratio requires 3 genes. (See text Fig. 21.3) daughterless (da) autosomal maternal effect- expressed from maternal autosome and transported to oocyte. XX offspring of da - /da - females, which would develop as males, die early in development due to imbalance caused by gene dosage compensation, which should occur only in XY embryos. sisterless (sis) ("numerator" protein) X-linked: expressed from X in all embryos 9-14 divisions post-fertilization sis XX null mutants develop as males; but hyperactivation of X-linked gene expression is lethal. sis is within the "scute complex"of genes involved in neurogenesis. sis expression in gender determination is temporally restricted but spatially unrestricted. Expression of sis also participates in neurogenesis, in which case its expression is temporally unrestricted but spatially restricted. level of SIS protein depends on sis gene dosage autosomal denominator (ad) ("denominator" protein) expressed at constant level from embryo autosome; These 3 genes all code for HLH-type transcription factor proteins. HLH transcription factors ordinarily operate as either homo or hetero dimers. DA and AD proteins compete for variable amounts of SIS protein to form hetero-dimers. The DA/SIS heterodimer activates transcription of sxl from its early promoter PE. Formation of the DA/SIS heterodimer normally occrs only in XX embryos.

5 The AD/SIS hetreodimer is formed in both XX and XY embryos, but has no function except to titrate out all the SIS monomer in XY embryos. Thus, the X:A chromosomal ratio is manifested by the relative amounts of SIS vs AD monomer in embryonic cells. SIS and AD compete fwith each other or dimerization with DA. Only DA/SIS heterodimernormally activates transcription of sxl from PE, leading to female development. In absence of sxl expression, development follows the male pathway because DSX-M will be produced. Early sxl expression from PE is DA/SISdependent. In the presence of DA/SIS the PE pre-mrna is spliced to allow production of the active SXL protein. Translation of the other form of mrna yields a non-functional protein. However, transcription from PE must be transitory, because the maternal DA protein is rapidly diluted by growth of the embryo. Later in embryogenesis transcription of sxl from PM is activated How?). Transcription of sxl from PM is independent of of DA/SIS activation and therefore occurs in both XX and XY embryos. This longer transcript is correctly spliced in the presence of preexisting SXL protein. Therefore, sxl pre-mrna is not correctly processed in XY embryos and translation terminates prematurely within intron containing a termination codon. No active SXL protein is made. In XX embryos, the PM pre-mrna is correctly spliced because of preexisting SXL protein. This system is a molecular toggle switch created by the self-regulation of sxl PM pre-mrna splicing. TOGGLE MECHANISM [What is the molecular basis for the SXL dependance of splice junction selection in PM premrna, that is not observed in in PE pre-mrna?] In addition to autoregulatory function, SXL is required for processing the tra transcript and for preventing expression of genes that, by default, hyperinduce most X-linked genes in males. See Fig for detals of sxl pre-mrna splicing.

6 DOSAGE COMPENSATION In Drosophila, dosage compensation occurs in males through the hyper transcription of X-linked genes. The elevated transcription means that the quantitative levels of X-linked gene products in male cells is comparable to that in females. XY embryos die in the absence of elevated X-transcription. XX embryos die in the presence of elevated X-transcription. MSL Genes 5 protein and 2 RNA gene products are required for elevated transcription of X in males. These evidently form a complex that binds to the X-chromosome chromatin. mle msl-2 msl-3: msl-4: msl-5: roxi rox2 a helicase a histone H4 transacetylase (see chromatin remodeling) MSL-dependent transcription of X-linked genes, Transcripts are rocessed as if they were pre-mrna's, but not translated. (see mammalian Xist RNA) Expression of MSL proteins is repressed by SXL. i.e. X hypertranscription is the "default" scenario, and must be actively turned off in XX embryos to prevent functional imbalance. This accounts for the inviability of female offspring of of da - /da - mothers.