Showing papers on "Dosage compensation published in 1999"

Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation.

Abstract: Conditional deletion of Xist disrupts histone macroH2A localization but not maintenance of X inactivation

Spatial Relationship between Transcription Sites and Chromosome Territories

Abstract: We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300–450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

Structural maintenance of chromosomes (SMC) proteins: conserved molecular properties for multiple biological functions.

Abstract: The evolutionarily-conserved eukaryotic SMC (structural maintenance of chromosomes) proteins are ubiquitous chromosomal components in prokaryotes and eukaryotes. The eukaryotic SMC proteins form two kind of heterodimers: the SMC1/SMC3 and the SMC2/SMC4 types. These heterodimers constitute an essential part of higher order complexes, which are involved in chromatin and DNA dynamics. The two most prominent and best-characterized complexes are cohesin and condensin, necessary for sister chromatid cohesion and chromosome condensation. Here we discuss these functions together with additional roles in gene dosage compensation and DNA recombination.

Histone Macroh2a1.2 Relocates to the Inactive X Chromosome after Initiation and Propagation of X-Inactivation

Abstract: The histone macroH2A1.2 has been implicated in X chromosome inactivation on the basis of its accumulation on the inactive X chromosome (Xi) of adult female mammals. We have established the timing of macroH2A1.2 association with the Xi relative to the onset of X-inactivation in differentiating murine embryonic stem (ES) cells using immuno-RNA fluorescence in situ hybridization (FISH). Before X-inactivation we observe a single macroH2A1.2-dense region in both undifferentiated XX and XY ES cells that does not colocalize with X inactive specific transcript (Xist) RNA, and thus appears not to associate with the X chromosome(s). This pattern persists through early stages of differentiation, up to day 7. Then the frequency of XY cells containing a macroH2A1.2-rich domain declines. In contrast, in XX cells there is a striking relocalization of macroH2A1.2 to the Xi. Relocalization occurs in a highly synchronized wave over a 2-d period, indicating a precisely regulated association. The timing of macroH2A1.2 accumulation on the Xi suggests it is not necessary for the initiation or propagation of random X-inactivation.

Xist RNA exhibits a banded localization on the inactive X chromosome and is excluded from autosomal material in cis.

Abstract: The propagation of X chromosome inactivation is thought to be mediated by the cis- limited spreading of the non-protein coding Xist transcript. In this report we have investigated the localization of Xist RNA on rodent metaphase chromosomes. We show that Xist RNA exhibits a banded pattern on the inactive X and is excluded from regions of constitutive heterochromatin. The banding pattern suggests a preferential association with gene-rich, G-light regions. Analysis of X:autosome rearrangements revealed that restricted propagation of X inactivation into cis -linked autosomal material is reflected by a corresponding limited spread of Xist RNA. We discuss these results in the context of models for the function of Xist RNA in the propagation of X inactivation.

Xp deletions associated with autism in three females.

Abstract: We report eight females with small deletions of the short arm of the X chromosome, three of whom showed features of autism. Our results suggest that there may be a critical region for autism in females with Xp deletions between the pseudoautosomal boundary and DXS7103. We hypothesise that this effect might be due either to the loss of function of a specific gene within the deleted region or to functional nullisomy resulting from X inactivation of the normal X chromosome.

Discovery of tetraploidy in a mammal

Abstract: The red viscacha rat is unaffected by having double the usual number of chromosomes. Polyploidy, or having more than a pair of each type of chromosome, is considered to be unlikely in mammals because it would disrupt the mechanism of dosage compensation that normally inactivates one X chromosome in females1. Also, any imbalance in chromosome number should affect the normal developmental processes and therefore constitute an evolutionary end, as in triploid humans2.

An apparent excess of sex- and reproduction related genes on the human X chromosome

Abstract: We describe here the results of a search of Mendelian inheritance in man, GENDIAG and other sources which suggest that, in comparison with autosomes 1, 2, 3, 4 and 11, the X chromosome may contain a significantly higher number of sex- and reproduction-related (SRR) genes. A similar comparison between X-linked entries and a subset of randomly chosen entries from the remaining autosomes also indicates an excess of genes on the X chromosome with one or more mutations affecting sex determination (e.g. DAX1), sexual differentiation (e.g. androgen receptor) or reproduction (e.g. POF1). A possible reason for disproportionate occurrence of such genes on the X chromosome could be that, during evolution, the 'choice' of a particular pair of homomorphic chromosomes for specialization as sex chromosomes may be related to the number of such genes initially present in it or, since sex determination and sexual dimorphism are often gene dose-dependent processes, the number of such genes necessary to be regulated in a dose-dependent manner. Further analysis of these data shows that XAR, the region which has been added on to the short arm of the X chromosome subsequent to eutherian-marsupial divergence, has nearly as high a proportion of SRR genes as XCR, the conserved region of the X chromosome. These observations are consistent with current hypotheses on the evolution of sexually antagonistic traits on sex chromosomes and suggest that both XCR and XAR may have accumulated SRR traits relatively rapidly because of X linkage.

Xist yeast artificial chromosome transgenes function as x-inactivation centers only in multicopy arrays and not as single copies

Abstract: X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation in cis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The Xist gene maps to the Xic region and has been shown to be essential for inactivation in cis. Transgenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid Xist-containing transgenes have previously been reported to be capable of cis inactivation and counting, the transgenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transgenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of Xist, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.

Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain.

Abstract: Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5′ sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.

Abstract: Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl-2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.

Dosage compensation proteins targeted to X chromosomes by a determinant of hermaphrodite fate.

Abstract: In many organisms, master control genes coordinately regulate sex-specific aspects of development. SDC-2 was shown to induce hermaphrodite sexual differentiation and activate X chromosome dosage compensation in Caenorhabditis elegans. To control these distinct processes, SDC-2 acts as a strong gene-specific repressor and a weaker chromosome-wide repressor. To initiate hermaphrodite development, SDC-2 associates with the promoter of the male sex-determining gene her-1 to repress its transcription. To activate dosage compensation, SDC-2 triggers assembly of a specialized protein complex exclusively on hermaphrodite X chromosomes to reduce gene expression by half. SDC-2 can localize to X chromosomes without other components of the dosage compensation complex, suggesting that SDC-2 targets dosage compensation machinery to X chromosomes.

Escapees on the X chromosome

Abstract: In many organisms, differentiation of the sex chromosome complement resulted in the coordinated regulation of genes on whole chromosomes to equalize gene expression between the sexes. In mammals, X inactivation evolved to restore equal expression of X-linked genes in males and females (1). Although X inactivation consists in the general repression of most genes on the X, some genes escape inactivation (reviewed in ref. 2). Recent advances in the Human Genome Project now allow the inactivation status of many X-linked genes to be systematically studied. In this issue of PNAS, Carrel et al. (3) report such a systematic analysis. Their data are so extensive that they are summarized in the paper but are fully accessible only as a file on the World Wide Web (www.pnas.org/supplementary.shtml).

X Inactive-Specific Transcript (Xist) Expression and X Chromosome Inactivation in the Preattachment Bovine Embryo

Abstract: Expression of the X inactive-specific transcript (Xist) is thought to be essential for the initiation of X chromosome inactivation and dosage compensation during female embryo development. In the present study, we analyzed the patterns of Xist transcription and the onset of X chromosome inactivation in bovine preattachment embryos. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of Xist transcripts in all adult female somatic tissues evaluated. In contrast, among the male tissues examined, Xist expression was detected only in testis. No evidence for Xist transcription was observed after a single round of RT-PCR from pools of in vitro-derived embryos at the 2- to 4-cell stage. Xist transcripts were detected as a faint amplicon at the 8-cell stage initially, and consistently thereafter in all stages examined up to and including the expanded blastocyst stage. Xist transcripts, however, were subsequently detected from the 2-cell stage onward after nested RT-PCR. Preferential [3H]thymidine labeling indicative of late replication of one of the X chromosomes was noted in female embryos of different developmental ages as follows: 2 of 7 (28.5%) early blastocysts, 6 of 13 (46.1%) blastocysts, 8 of 11 (72.1%) expanded blastocysts, and 14 of 17 (77.7%) hatched blastocysts. These results suggest that Xist expression precedes the onset of late replication in the bovine embryo, in a pattern compatible with a possible role of bovine Xist in the initiation of X chromosome inactivation.

Accelerated Telomere Shortening in the Human Inactive X Chromosome

Abstract: Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes, with important roles in the maintenance of genomic stability and in chromosome segregation. Normal somatic cells lose telomeric repeats with each cell division both in vivo and in vitro. To address a potential role of nuclear architecture and epigenetic factors in telomere-length dynamics, the length of the telomeres of the X chromosomes and the autosomes was measured in metaphases from blood lymphocytes of human females of various ages, by quantitative FISH with a peptide nucleic-acid telomeric probe in combination with an X-chromosome centromere-specific probe. The activation status of the X chromosomes was simultaneously visualized with antibodies against acetylated histone H4. We observed an accelerated shortening of telomeric repeats in the inactive X chromosome, which suggests that epigenetic factors modulate not only the length but also the rate of age-associated telomere shortening in human cells in vivo. This is the first evidence to show a differential rate of telomere shortening between and within homologous chromosomes in any species. Our results are also consistent with a causative role of telomere shortening in the well-documented X-chromosome aneuploidy in aging humans.

Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells

Abstract: Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.

A developmental switch in H4 acetylation upstream of Xist plays a role in X chromosome inactivation

Abstract: We have investigated the role of histone acetylation in X chromosome inactivation, focusing on its possible involvement in the regulation of Xist, an essential gene expressed only from the inactive X (Xi). We have identified a region of H4 hyperacetylation extending up to 120 kb upstream from the Xist somatic promoter P1. This domain includes the promoter P0, which gives rise to the unstable Xist transcript in undifferentiated cells. The hyperacetylated domain was not seen in male cells or in female XT67E1 cells, a mutant cell line heterozygous for a partially deleted Xist allele and in which an increased number of cells fail to undergo X inactivation. The hyperacetylation upstream of Xist was lost by day 7 of differentiation, when X inactivation was essentially complete. Wild-type cells differentiated in the presence of the histone deacetylase inhibitor Trichostatin A were prevented from forming a normally inactivated X, as judged by the frequency of underacetylated X chromosomes detected by immunofluorescence microscopy. Mutant XT67E1 cells, lacking hyperacetylation upstream of Xist, were less affected. We propose that (i) hyperacetylation of chromatin upstream of Xist facilitates the promoter switch that leads to stabilization of the Xist transcript and (ii) that the subsequent deacetylation of this region is essential for the further progression of X inactivation.

Heterogeneous X inactivation in trophoblastic cells of human full-term female placentas

Abstract: In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin.

Characterisation, phenotypic manifestations and X-inactivation pattern in 14 patients with X-autosome translocations.

Abstract: Here we describe a group of 14 patients carrying different X-autosome translocations and exhibiting phenotypes that demonstrate the range of alterations induced by such aberrations. All male carriers of an X-autosome translocation in our investigation group were infertile, whereas fertility in the female carriers was dependent on the position of the break-point in the X chromosome. Fertile women with translocation break-points outside of the critical region (Xq13-q26) in some cases passed on the translocation to their offspring. In balanced female carriers in our group, the normal X chromosome was usually inactivated, allowing full expression of genes on the translocated segments. In one case, disruption of the dystrophine gene in Xp21 led to the manifestation of Duchenne muscular dystrophy in a female carrier. Inactivation of the derivative X (Xt) in a balanced female carrier led to a partial monosomy of the autosome/disomy of the X chromosome and resulted in an aberrant phenotype. In unbalanced carriers, Xt is generally late-replicating/inactive, although failed spreading of inactivation to the autosomal segment often results in a partial trisomy, as evidenced by the case of an unbalanced translocation carrier in our group.

DNA methylation in transcriptional repression of two differentially expressed X-linked genes, GPC3 and SYBL1

Abstract: Methylation of CpG islands is an established transcriptional repressive mechanism and is a feature of silencing in X chromosome inactivation. Housekeeping genes that are subject to X inactivation exhibit differential methylation of their CpG islands such that the inactive alleles are hypermethylated. In this report, we examine two contrasting X-linked genes with CpG islands for regulation by DNA methylation: SYBL1, a housekeeping gene in the Xq pseudoautosomal region, and GPC3, a tissue-specific gene in Xq26 that is implicated in the etiology of the Simpson–Golabi–Behmel overgrowth syndrome. We observed that in vitro methylation of either the SYBL1 or the GPC3 promoter resulted in repression of reporter constructs. In normal contexts, we found that both the Y and inactive X alleles of SYBL1 are repressed and hypermethylated, whereas the active X allele is expressed and unmethylated. Furthermore, the Y and inactive X alleles of SYBL1 were derepressed by treatment with the demethylating agent azadeoxycytidine. GPC3 is also subject to X inactivation, and the active X allele is unmethylated in nonexpressing leukocytes as well as in an expressing cell line, suggesting that methylation is not involved in the tissue-specific repression of this allele. The inactive X allele, however, is hypermethylated in leukocytes, presumably reflecting early X inactivation events that become important for gene dosage in expressing lineages. These and other data suggest that all CpG islands on Xq, including the pseudoautosomal region, are subject to X inactivation-induced methylation. Additionally, methylation of SYBL1 on Yq may derive from a process related to X inactivation that targets large chromatin domains for transcriptional repression.

Imprinting and X-Chromosome Inactivation

Abstract: In normal female mammals one of the two X-chromosomes in every somatic cell is inactive i.e. it fails to transcribe RNA (reviews Gartler et al. 1992; Migeon 1994; Lyon 1996). The result of this is that chromosomally XX females and XY males both effectively have a single dosage of the products of X-linked genes. Thus X-chromosome inactivation fulfils the function of dosage compensation of X-linked genes. In eutherian mammals, typically either one of the two X-chromosomes in any cell, the maternally inherited Xm or the paternally inherited Xp, can be inactivated at random. Once the choice is made in each cell, it remains stable in all further cell generations in that individual and hence in the adult there are large clumps of cells with the same X-chromosome active. If the two X-chromosomes bear different alleles of a gene affecting some visible character, such as coat color, the clumps can be seen as a variegated effect. The best-known example of this is the tortoiseshell cat, in which the pattern results from the animal having a gene for ginger coat on one X-chromosome and black or tabby on the other. However, by contrast, in marsupials the same X-chromosome, the paternally derived Xp, becomes inactive in all cells (Cooper et al. 1993; Graves 1996). This preferential inactivation of Xp is seen also in some cells of the extraembryonic lineages of the embryo, which give rise to the placenta and other supporting tissues, in mice and rats (Takagi and Sasaki 1975), and probably, but less clearly, in humans also (Harrison 1989; Goto et al. 1997). Thus, in marsupials and in extraembryonic tissues of eutherians, the X-chromosome shows imprinting (Fig. 1). This imprinting has some similarities and some differences from autosomal imprinting. In order to understand the significance of imprinting in X-chromosome inactivation one must consider the mechanism by which the inactivation is brought about.

An N-terminal truncation uncouples the sex-transforming and dosage compensation functions of sex-lethal.

Abstract: Sex-lethal (Sxl) encodes an RNA recognition motif (RRM) class RNA binding protein that serves as the developmental switch for sex determination in Drosophila melanogaster (6). Sxl is expressed only in females, where it controls sexual differentiation and dosage compensation by posttranscriptional regulatory mechanisms that affect pre-mRNA splicing and mRNA translation. Misregulation of Sxl results in sex-specific lethality and sex transformations (see reference 15 and references therein). Female sexual identity is maintained by an autoregulatory feedback loop in which Sxl proteins promote their own synthesis by directing the female-specific splicing of Sxl pre-mRNAs (Fig. ​(Fig.1A;1A; references 5, 6, and 14). Functional female Sxl mRNA is generated by joining exon 2 to exon 4, skipping the third (male-specific) exon, which contains in-frame translation stop codons. Male identity is maintained by the default splicing machinery, which incorporates the third exon into the mature mRNAs, ensuring that no Sxl protein is produced. Sxl-dependent posttranscriptional regulation also controls the gene cascades that direct the different aspects of female or male development (Fig. ​(Fig.1A).1A). Sxl protein promotes female differentiation by directing the female-specific splicing of transformer (tra) pre-mRNAs (25, 45, 46). In the absence of Sxl protein, the default splicing of tra results in mRNAs that do not encode functional protein. Sxl also regulates dosage compensation, which is responsible for equalizing the expression of X-linked genes in the two sexes. One component of the dosage compensation system is the hyperactivation of X-chromosome gene expression in males by the male specific lethal (msl) genes (1, 7, 32, 33). Sxl proteins prevent hyperactivation in females by blocking both the splicing and translation of transcripts from one of these genes, msl-2 (3, 18, 28, 51). A second component of the dosage compensation system is msl independent (8, 19, 20) and is thought to function in females to reduce X-chromosome gene expression. Kelley et al. (28) recently suggested that Sxl itself mediates this dosage compensation by repressing the translation of mRNAs expressed from X-linked genes. FIG. 1 Regulatory activities of Sxl. (A) Models for Sxl splicing, tra splicing, and msl-2 translational control in Drosophila males (top) and females (below). The default splicing of both Sxl and tra occurs in males and results in transcripts with in-frame stop ... The posttranscriptional regulatory activities of the Sxl gene depend on direct interactions between Sxl proteins and target RNAs. RNA binding activity is provided by Sxl’s two RRM domains, R1 and R2 (26, 37, 43, 49). The two RRM domains recognize poly(U) runs of seven or more nucleotides, and all of the known Sxl regulatory targets have one or more of these arrays. In the case of tra, in vivo and in vitro studies indicate that Sxl protein directs female-specific splicing by binding to a poly(U) run in the polypyrimidine tract of the default 3′ splice site (Fig. ​(Fig.1A)1A) (25, 45, 46). It has been proposed that this prevents the generic splicing factor U2AF from binding to the default polypyrimidine tract and forces the assembly of a U2AF-U2 snRNP splicing complex on the weaker, female-specific 3′ splice site downstream (22, 48). While a direct competition for overlapping binding sites accounts for what is known about tra splicing, the RNA binding activity of Sxl is not sufficient to explain either Sxl autoregulation or the repression of msl-2 translation. The key targets for Sxl autoregulation are located in the introns upstream and downstream of the male exon at distances of 200 or more nucleotides from the regulated 3′ and 5′ splice sites (24). Hence, instead of a direct blockage mechanism, Sxl must indirectly prevent the assembly of productive splicing complexes at the male exon. One possibility is that homotypic interactions between Sxl proteins sequester the male exon from the splicing machinery (24, 35). Consistent with this possibility, Sxl proteins interact in vitro, and these interactions stabilize Sxl complexes on RNA (26, 36, 43, 49). These Sxl-Sxl interactions are mediated by the two RRM domains (37, 42, 50). A second model postulates that Sxl interacts with and inactivates components of the splicing machinery assembled at the male exon splice sites (e.g., U1 and U2 snRNPs [16, 40]). Consistent with this model, Sxl proteins in vivo are found in large complexes which contain both U1 and U2 snRNPs and Sxl pre-mRNAs (16, 43). In addition, mutations in the sans-fille (snf) gene, encoding the fly homologue of two mammalian snRNP proteins, U1A and U2B", disrupt autoregulation and exacerbate the female-lethal effects of Sxl mutations (38, 40). This synergism may be attributed to interactions between these two proteins; Snf-Sxl complexes can be detected in vivo and in vitro, and this interaction is mediated by the first Sxl RRM domain (16, 43). Finally, efficient translational repression of msl-2 mRNA requires Sxl protein binding sites in both the 5′ and 3′ untranslated regions (UTRs) (4, 18, 29). In a manner analogous to autoregulation, interactions between Sxl proteins upstream and downstream of the open reading frame could sequester the msl-2 mRNA from the translational machinery. Alternatively, Sxl might interact with and poison this machinery. While the two Sxl RRM domains have been implicated in both RNA binding and protein-protein interactions, much less is known about the functions of the N- and C-terminal domains. Though there are no known mutations in the N-terminal domain, there are some indications that it may be important for the regulatory functions of the Sxl protein. A truncated Sxl protein was detected in the heads of adult D. melanogaster males (11). This smaller isoform appears to result from translation initiation at an AUG codon in exon 4, downstream of the male exon (exon 3), and gives a protein lacking the first 40 amino acids (aa). A slightly larger male-specific protein is also detected in the related drosophilid, D. virilis (12). Although the D. melanogaster and D. virilis male proteins contain both RRM domains and appear to bind appropriate target RNAs, they do not have detectable feminizing activities. It was initially thought that the concentration of the truncated proteins might be too low to induce feminization. This explanation was called into question by Wang and Bell (49), who found that a truncated Sxl protein (SxlN1), similar to one observed in D. melanogaster male heads, was impaired in Sxl autoregulation when transiently expressed in tissue culture cells. To learn more about the regulatory functions of the different Sxl protein domains, we have compared the biological activities of the full-length Sxl protein (Sx.FL) and Sx-N in vivo. We were able to uncouple the splicing and translational regulatory activities of Sxl protein. Sx-N is impaired in autoregulatory function, and contrary to the expectations of the U2AF blockage model, the N terminus plays an essential role in the regulation of tra splicing. However, these amino acids are not required to regulate msl-dependent dosage compensation. Finally, we provide evidence that Sxl is controlled by both positive and negative autoregulation.