113 research outputs found
The Chromosomal High-Affinity Binding Sites for the Drosophila Dosage Compensation Complex
Dosage compensation in male Drosophila relies on the X chromosomeâspecific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called âhigh-affinity sitesâ (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features
Requirement of Male-Specific Dosage Compensation in Drosophila FemalesâImplications of Early X Chromosome Gene Expression
Dosage compensation equates between the sexes the gene dose of sex chromosomes that carry substantially different gene content. In Drosophila, the single male X chromosome is hypertranscribed by approximately two-fold to effect this correction. The key genes are male lethal and appear not to be required in females, or affect their viability. Here, we show these male lethals do in fact have a role in females, and they participate in the very process which will eventually shut down their functionâfemale determination. We find the male dosage compensation complex is required for upregulating transcription of the sex determination master switch, Sex-lethal, an X-linked gene which is specifically activated in females in response to their two X chromosomes. The levels of some X-linked genes are also affected, and some of these genes are used in the process of counting the number of X chromosomes early in development. Our data suggest that before the female state is set, the ground state is male and female X chromosome expression is elevated. Females thus utilize the male dosage compensation process to amplify the signal which determines their fate
Abnormal Dosage Compensation of Reporter Genes Driven by the Drosophila Glass Multiple Reporter (GMR) Enhancer-Promoter
In Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3Ⲡend. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex
Somatic sex-specific transcriptome differences in Drosophila revealed by whole transcriptome sequencing
<p>Abstract</p> <p>Background</p> <p>Understanding animal development and physiology at a molecular-biological level has been advanced by the ability to determine at high resolution the repertoire of mRNA molecules by whole transcriptome resequencing. This includes the ability to detect and quantify rare abundance transcripts and isoform-specific mRNA variants produced from a gene.</p> <p>The sex hierarchy consists of a pre-mRNA splicing cascade that directs the production of sex-specific transcription factors that specify nearly all sexual dimorphism. We have used deep RNA sequencing to gain insight into how the Drosophila sex hierarchy generates somatic sex differences, by examining gene and transcript isoform expression differences between the sexes in adult head tissues.</p> <p>Results</p> <p>Here we find 1,381 genes that differ in overall expression levels and 1,370 isoform-specific transcripts that differ between males and females. Additionally, we find 512 genes not regulated downstream of <it>transformer </it>that are significantly more highly expressed in males than females. These 512 genes are enriched on the Ă chromosome and reside adjacent to dosage compensation complex entry sites, which taken together suggests that their residence on the Ă chromosome might be sufficient to confer male-biased expression. There are no transcription unit structural features, from a set of features, that are robustly significantly different in the genes with significant sex differences in the ratio of isoform-specific transcripts, as compared to random isoform-specific transcripts, suggesting that there is no single molecular mechanism that generates isoform-specific transcript differences between the sexes, even though the sex hierarchy is known to include three pre-mRNA splicing factors.</p> <p>Conclusions</p> <p>We identify thousands of genes that show sex-specific differences in overall gene expression levels, and identify hundreds of additional genes that have differences in the abundance of isoform-specific transcripts. No transcription unit structural feature was robustly enriched in the sex-differentially expressed transcript isoforms. Additionally, we found that many genes with male-biased expression were enriched on the Ă chromosome and reside adjacent to dosage compensation entry sites, suggesting that differences in sex chromosome composition contributes to dimorphism in gene expression. Taken together, this study provides new insight into the molecular underpinnings of sexual differentiation.</p
Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila
<p>Abstract</p> <p>Background</p> <p>In male <it>Drosophila melanogaster</it>, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator.</p> <p>Results</p> <p>MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in <it>Drosophila</it>. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity <it>in vitro </it>and UAS-DsRed activation in <it>Drosophila</it>. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-<it>lacZ </it>and UAS-<it>arm-lacZ </it>reporter genes. The latter utilizes the constitutive promoter from the <it>arm </it>gene to drive <it>lacZ </it>expression. In contrast to the strong induction of UAS-DsRed expression, UAS-<it>arm-lacZ </it>expression increased by about 2-fold in both sexes.</p> <p>Conclusions</p> <p>Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-<it>DsRed </it>but not UAS-<it>arm-lacZ </it>genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.</p
Large introns in relation to alternative splicing and gene evolution: a case study of Drosophila bruno-3
Background:
Alternative splicing (AS) of maturing mRNA can generate structurally and functionally distinct transcripts from the same gene. Recent bioinformatic analyses of available genome databases inferred a positive correlation between intron length and AS. To study the interplay between intron length and AS empirically and in more detail, we analyzed the diversity of alternatively spliced transcripts (ASTs) in the Drosophila RNA-binding Bruno-3 (Bru-3) gene. This gene was known to encode thirteen exons separated by introns of diverse sizes, ranging from 71 to 41,973 nucleotides in D. melanogaster. Although Bru-3's structure is expected to be conducive to AS, only two ASTs of this gene were previously described.
Results:
Cloning of RT-PCR products of the entire ORF from four species representing three diverged Drosophila lineages provided an evolutionary perspective, high sensitivity, and long-range contiguity of splice choices currently unattainable by high-throughput methods. Consequently, we identified three new exons, a new exon fragment and thirty-three previously unknown ASTs of Bru-3. All exon-skipping events in the gene were mapped to the exons surrounded by introns of at least 800 nucleotides, whereas exons split by introns of less than 250 nucleotides were always spliced contiguously in mRNA. Cases of exon loss and creation during Bru-3 evolution in Drosophila were also localized within large introns. Notably, we identified a true de novo exon gain: exon 8 was created along the lineage of the obscura group from intronic sequence between cryptic splice sites conserved among all Drosophila species surveyed. Exon 8 was included in mature mRNA by the species representing all the major branches of the obscura group. To our knowledge, the origin of exon 8 is the first documented case of exonization of intronic sequence outside vertebrates.
Conclusion:
We found that large introns can promote AS via exon-skipping and exon turnover during evolution likely due to frequent errors in their removal from maturing mRNA. Large introns could be a reservoir of genetic diversity, because they have a greater number of mutable sites than short introns. Taken together, gene structure can constrain and/or promote gene evolution
Buffering and the evolution of chromosome-wide gene regulation
Copy number variation (CNV) in terms of aneuploidies of both entire chromosomes and chromosomal segments is an important evolutionary driving force, but it is inevitably accompanied by potentially problematic variations in gene doses and genomic instability. Thus, a delicate balance must be maintained between mechanisms that compensate for variations in gene doses (and thus allow such genomic variability) and selection against destabilizing CNVs. In Drosophila, three known compensatory mechanisms have evolved: a general segmental aneuploidy-buffering system and two chromosome-specific systems. The two chromosome-specific systems are the male-specific lethal complex, which is important for dosage compensation of the male X chromosome, and Painting of fourth, which stimulates expression of the fourth chromosome. In this review, we discuss the origin and function of buffering and compensation using Drosophila as a model
Buffering of Segmental and Chromosomal Aneuploidies in Drosophila melanogaster
Chromosomal instability, which involves the deletion and duplication of chromosomes or chromosome parts, is a common feature of cancers, and deficiency screens are commonly used to detect genes involved in various biological pathways. However, despite their importance, the effects of deficiencies, duplications, and chromosome losses on the regulation of whole chromosomes and large chromosome domains are largely unknown. Therefore, to explore these effects, we examined expression patterns of genes in several Drosophila deficiency hemizygotes and a duplication hemizygote using microarrays. The results indicate that genes expressed in deficiency hemizygotes are significantly buffered, and that the buffering effect is general rather than being mainly mediated by feedback regulation of individual genes. In addition, differentially expressed genes in haploid condition appear to be generally more strongly buffered than ubiquitously expressed genes in haploid condition, but, among genes present in triploid condition, ubiquitously expressed genes are generally more strongly buffered than differentially expressed genes. Furthermore, we show that the 4th chromosome is compensated in response to dose differences. Our results suggest general mechanisms have evolved that stimulate or repress gene expression of aneuploid regions as appropriate, and on the 4th chromosome of Drosophila this compensation is mediated by Painting of Fourth (POF)
A study of alternative splicing in the pig
<p>Abstract</p> <p>Background</p> <p>Since at least half of the genes in mammalian genomes are subjected to alternative splicing, alternative pre-mRNA splicing plays an important contribution to the complexity of the mammalian proteome. Expressed sequence tags (ESTs) provide evidence of a great number of possible alternative isoforms. With the EST resource for the domestic pig now containing more than one million porcine ESTs, it is possible to identify alternative splice forms of the individual transcripts in this species from the EST data with some confidence.</p> <p>Results</p> <p>The pig EST data generated by the Sino-Danish Pig Genome project has been assembled with publicly available ESTs and made available in the PigEST database. Using the Distiller package 2,515 EST clusters with candidate alternative isoforms were identified in the EST data with high confidence. In agreement with general observations in human and mouse, we find putative splice variants in about 30% of the contigs with more than 50 ESTs. Based on the criteria that a minimum of two EST sequences confirmed each splice event, a list of 100 genes with the most distinct tissue-specific alternative splice events was generated from the list of candidates. To confirm the tissue specificity of the splice events, 10 genes with functional annotation were randomly selected from which 16 individual splice events were chosen for experimental verification by quantitative PCR (qPCR). Six genes were shown to have tissue specific alternatively spliced transcripts with expression patterns matching those of the EST data. The remaining four genes had tissue-restricted expression of alternative spliced transcripts. Five out of the 16 splice events that were experimentally verified were found to be putative pig specific.</p> <p>Conclusions</p> <p>In accordance with human and rodent studies we estimate that approximately 30% of the porcine genes undergo alternative splicing. We found a good correlation between EST predicted tissue-specificity and experimentally validated splice events in different porcine tissue. This study indicates that a cluster size of around 50 ESTs is optimal for <it>in silico </it>detection of alternative splicing. Although based on a limited number of splice events, the study supports the notion that alternative splicing could have an important impact on species differentiation since 31% of the splice events studied appears to be species specific.</p
Lack of global meiotic sex chromosome inactivation, and paucity of tissue-specific gene expression on the Drosophila X chromosome
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