Murine HPV16 E7-expressing transgenic skin effectively emulates the cellular and molecular features of human high-grade squamous intraepithelial lesions.

Currently available vaccines prevent HPV infection and development of HPV-associated malignancies, but do not cure existing HPV infections and dysplastic lesions. Persistence of infection(s) in immunocompetent patients may reflect induction of local immunosuppressive mechanisms by HPV, providing a target for therapeutic intervention. We have proposed that a mouse, expressing HPV16 E7 oncoprotein under a Keratin 14 promoter (K14E7 mice), and which develops epithelial hyperplasia, may assist with understanding local immune suppression mechanisms that support persistence of HPV oncogene-induced epithelial hyperplasia. K14E7 skin grafts recruit immune cells from immunocompetent hosts, but consistently fail to be rejected. Here, we review the literature on HPV-associated local immunoregulation, and compare the findings with published observations on the K14E7 transgenic murine model, including comparison of the transcriptome of human HPV-infected pre-malignancies with that of murine K14E7 transgenic skin. We argue from the similarity of i) the literature findings and ii) the transcriptome profiles that murine K14E7 transgenic skin recapitulates the cellular and secreted protein profiles of high-grade HPV-associated lesions in human subjects. We propose that the K14E7 mouse may be an appropriate model to further study the immunoregulatory effects of HPV E7 expression, and can facilitate development and testing of therapeutic vaccines

Requirement of Male-Specific Dosage Compensation in Drosophila Females—Implications 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 function—female 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 3′ end. 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 × chromosome and reside adjacent to dosage compensation complex entry sites, which taken together suggests that their residence on the × 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 × 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

A new strategy for isolating genes controlling dosage compensation in Drosophila using a simple epigenetic mosaic eye phenotype

<p>Abstract</p> <p>Background</p> <p>The <it>Drosophila </it>Male Specific Lethal (MSL) complex contains chromatin modifying enzymes and non-coding <it>roX </it>RNA. It paints the male X at hundreds of bands where it acetylates histone H4 at lysine 16. This epigenetic mark increases expression from the single male X chromosome approximately twofold above what gene-specific factors produce from each female X chromosome. This equalises X-linked gene expression between the sexes. Previous screens for components of dosage compensation relied on a distinctive male-specific lethal phenotype.</p> <p>Results</p> <p>Here, we report a new strategy relying upon an unusual male-specific mosaic eye pigmentation phenotype produced when the MSL complex acts upon autosomal <it>roX1 </it>transgenes. Screening the second chromosome identified at least five loci, two of which are previously described components of the MSL complex. We focused our analysis on the modifier alleles of MSL1 and MLE (for 'maleless'). The MSL1 lesions are not simple nulls, but rather alter the PEHE domain that recruits the MSL3 chromodomain and MOF ('males absent on first') histone acetyltransferase subunits to the complex. These mutants are compromised in their ability to recruit MSL3 and MOF, dosage compensate the X, and support long distance spreading from <it>roX1 </it>transgenes. Yet, paradoxically, they were isolated because they somehow increase MSL complex activity immediately around <it>roX1 </it>transgenes in combination with wild-type MSL1 subunits.</p> <p>Conclusions</p> <p>We propose that these diverse phenotypes arise from perturbations in assembly of MSL subunits onto nascent <it>roX </it>transcripts. This strategy is a promising alternative route for identifying previously unknown components of the dosage compensation pathway and novel alleles of known MSL proteins.</p

Hypoxia Disruption of Vertebrate CNS Pathfinding through EphrinB2 Is Rescued by Magnesium

The mechanisms of hypoxic injury to the developing human brain are poorly understood, despite being a major cause of chronic neurodevelopmental impairments. Recent work in the invertebrate Caenorhabditis elegans has shown that hypoxia causes discrete axon pathfinding errors in certain interneurons and motorneurons. However, it is unknown whether developmental hypoxia would have similar effects in a vertebrate nervous system. We have found that developmental hypoxic injury disrupts pathfinding of forebrain neurons in zebrafish (Danio rerio), leading to errors in which commissural axons fail to cross the midline. The pathfinding defects result from activation of the hypoxia-inducible transcription factor (hif1) pathway and are mimicked by chemical inducers of the hif1 pathway or by expression of constitutively active hif1α. Further, we found that blocking transcriptional activation by hif1α helped prevent the guidance defects. We identified ephrinB2a as a target of hif1 pathway activation, showed that knock-down of ephrinB2a rescued the guidance errors, and showed that the receptor ephA4a is expressed in a pattern complementary to the misrouting axons. By targeting a constitutively active form of ephrinB2a to specific neurons, we found that ephrinB2a mediates the pathfinding errors via a reverse-signaling mechanism. Finally, magnesium sulfate, used to improve neurodevelopmental outcomes in preterm births, protects against pathfinding errors by preventing upregulation of ephrinB2a. These results demonstrate that evolutionarily conserved genetic pathways regulate connectivity changes in the CNS in response to hypoxia, and they support a potential neuroprotective role for magnesium

SU(VAR)3-7 Links Heterochromatin and Dosage Compensation in Drosophila

In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. We previously found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. In this study, we examine the impact of change in levels of SU(VAR)3-7 on dosage compensation. We first demonstrate that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, we show that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation

Progress and prospects toward our understanding of the evolution of dosage compensation

In many eukaryotic organisms, gender is determined by a pair of heteromorphic sex chromosomes. Degeneration of the non-recombining Y chromosome is a general facet of sex chromosome evolution. Selective pressure to restore expression levels of X-linked genes relative to autosomes accompanies Y-chromosome degeneration, thus driving the evolution of dosage compensation mechanisms. This review focuses on evolutionary aspects of dosage compensation, in light of recent advances in comparative and functional genomics that have substantially increased our understanding of the molecular mechanisms of dosage compensation and how it evolved. We review processes involved in sex chromosome evolution, and discuss the dynamic interaction between Y degeneration and the acquisition of dosage compensation. We compare mechanisms of dosage compensation and the origin of dosage compensation genes between different taxa and comment on sex chromosomes that apparently lack compensation mechanisms. Finally, we discuss how dosage compensation systems can also influence the evolution of well-established sex chromosomes

X chromosomal regulation in flies: when less is more

In Drosophila, dosage compensation of the single male X chromosome involves upregulation of expression of X linked genes. Dosage compensation complex or the male specific lethal (MSL) complex is intimately involved in this regulation. The MSL complex members decorate the male X chromosome by binding on hundreds of sites along the X chromosome. Recent genome wide analysis has brought new light into X chromosomal regulation. It is becoming increasingly clear that although the X chromosome achieves male specific regulation via the MSL complex members, a number of general factors also impinge on this regulation. Future studies integrating these aspects promise to shed more light into this epigenetic phenomenon