Studies on Cytochrome P450 genes in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e: Relationship between over expression and DDT resistance, and xenobiotic induction

Cytochrome P450 monooxygenases (CYPs), are involved in the metabolism of a diverse group of endogenous and xenobiotic compounds. In insects, CYPs are involved in conferring resistance against insecticides. In Drosophila, the expression of Cyp6a2, Cyp6a8, Cyp6g1, Cyp6w1 and Cyp12d1 is higher in the resistant compared to susceptible strains. Recent study by Daborn et al (2002, Science, 297, 2253-2256) showed that Cyp6g1 alone can confer resistance phenotype. The aim of my first objective was to re- examine this claim and second to examine the effect of common xenobiotic compounds on the transcriptome of Drosophila. In first objective, six strains of Drosophila were examined for DDT resistance and Cyp6g1 expression. Results showed that some of the highly susceptible strains showed high level of Cyp6g1 expression and Accord element in the Cyp6g1 upstream DNA. When Cyp6g1 allele of the resistant 91-R strain was substituted with that of the susceptible 91-C strain via recombination, the resulting three recombinant lines retained high level of resistance like the 91-R strain, but showed very low Cyp6g1 expression. This suggests that there is a correlation between overexpression of Cyp6g1 and the presence of Accord transposable element but not DDT resistance. In the second part of the first objective, I directly examined the role of the Cyp6a2 and Cyp6g1 in DDT resistance. Germ line transformation in susceptible strain showed that there was a two-fold increase in DDT resistance (LD50) in transformed flies showing two-fold higher expression of GAL4/UAS driven CYP6A2 or CYP6G1 cDNA. A cumulative increase (4-fold) in DDT resistance was observed when both cDNAs were overexpressed in the same fly. Results suggest that the expression of multiple Cyp genes may be needed to confer a high level of DDT resistance. In the second objective, microarray was used to examine the transcripts induced by caffeine and phenobarbital. Results showed that genes involved in detoxification, carbohydrate metabolism, signal transduction and Cyp genes are induced by caffeine and phenobarbital. These are the same group of genes overexpressed in the resistant 91-R and recombinant strains. These studies shed light on the molecular basis of induction of Cyp genes and insecticide resistance

The Drosophila gypsy Insulator Supports Transvection in the Presence of the vestigial Enhancer

Though operationally defined as cis-regulatory elements, enhancers can also communicate with promoters on a separate homolog in trans, a mechanism that has been suggested to account for the ability of certain alleles of the same gene to complement one another in a process otherwise known as transvection. This homolog-pairing dependent process is facilitated in Drosophila by chromatin-associated pairing proteins, many of which remain unknown and their mechanism of action uncharacterized. Here we have tested the role of the gypsy chromatin insulator in facilitating pairing and communication between enhancers and promoters in trans using a transgenic eGFP reporter system engineered to allow for targeted deletions in the vestigial Boundary Enhancer (vgBE) and the hsp70 minimal promoter, along with one or two flanking gypsy elements. We found a modest 2.5-3x increase in eGFP reporter levels from homozygotes carrying an intact copy of the reporter on each homolog compared to unpaired hemizygotes, although this behavior was independent of gypsy. However, detectable levels of GFP protein along the DV wing boundary in trans-heterozygotes lacking a single enhancer and promoter was only observed in the presence of two flanking gypsy elements. Our results demonstrate that gypsy can stimulate enhancer-promoter communication in trans throughout the genome in a context-dependent manner, likely through modulation of local chromatin dynamics once pairing has been established by other elements and highlights chromatin structure as the master regulator of this phenomenon. DOI: 10.1371/journal.pone.008133

The <i>vg</i>BE can drive <i>eGFP</i> reporter expression in the absence of a functional promoter but this does not correlate with GFP protein levels.

<p>QPCR analysis (top graph) and immunostaining (bottom panels) of wing discs from intact hemizygotes (P⁺E⁺/+) and “promoterless” homozygotes (P-/P^-) for 2-insulator (A), 1-insulator (B) and 0-insulator (C) lines. For microscopy, images were normalized to P⁺E⁺/+ for each respective line and minimum/maximum level corrections were applied equally to both genotypes using ImageJ and false-colored green. Error bars represent standard error of the mean (S.E.M) and scalebars are 50 μm. </p

The Drosophila <i>gypsy</i> Insulator Supports Transvection in the Presence of the <i>vestigial</i> Enhancer

<div><p>Though operationally defined as <i>cis-</i>regulatory elements, enhancers can also communicate with promoters on a separate homolog in <i>trans</i>, a mechanism that has been suggested to account for the ability of certain alleles of the same gene to complement one another in a process otherwise known as transvection. This homolog-pairing dependent process is facilitated in Drosophila by chromatin-associated pairing proteins, many of which remain unknown and their mechanism of action uncharacterized. Here we have tested the role of the <i>gypsy</i> chromatin insulator in facilitating pairing and communication between enhancers and promoters in <i>trans</i> using a transgenic <i>eGFP</i> reporter system engineered to allow for targeted deletions in the <i>vestigial</i> Boundary Enhancer (<i>vg</i>BE) and the <i>hsp70</i> minimal promoter, along with one or two flanking <i>gypsy</i> elements. We found a modest 2.5-3x increase in <i>eGFP</i> reporter levels from homozygotes carrying an intact copy of the reporter on each homolog compared to unpaired hemizygotes, although this behavior was independent of <i>gypsy</i>. However, detectable levels of GFP protein along the DV wing boundary in trans-heterozygotes lacking a single enhancer and promoter was only observed in the presence of two flanking <i>gypsy</i> elements. Our results demonstrate that <i>gypsy</i> can stimulate enhancer-promoter communication in <i>trans</i> throughout the genome in a context-dependent manner, likely through modulation of local chromatin dynamics once pairing has been established by other elements and highlights chromatin structure as the master regulator of this phenomenon. </p> </div

Pairing can stimulate <i>eGFP</i> transcription more than two-fold independently of <i>gypsy</i> presence.

<p>QPCR analysis (top graph) and immunostaining (bottom panels) of wing discs from intact homozygotes (P⁺E⁺/P⁺E⁺) and intact hemizygotes (P⁺E⁺/+) for 2-insulator (A), 1-insulator (B) and 0-insulator (C) lines. For microscopy, images were normalized to P⁺E⁺/P⁺E⁺ for each respective line and minimum/maximum level corrections were applied equally to both genotypes using ImageJ and false-colored green. Error bars represent standard error of the mean (S.E.M) and scalebars are 50 μm. </p

Flanking <i>gypsy</i> insulators promote enhancer-promoter communication in <i>trans</i>.

<p>QPCR analysis (top graph) and immunostaining (bottom panels) of wing discs from all seven 2-insulator genotypes. For microscopy, all images were normalized to P⁺E⁺/P⁺E⁺ and minimum/maximum level corrections were applied equally to all genotypes using ImageJ and false-colored green. Error bars represent standard error of the mean (S.E.M) and scalebars are 50 μm.</p

Absence of <i>gypsy</i> fails to promote enhancer-promoter communication in <i>trans</i>.

<p>QPCR analysis (top graph) and immunostaining (bottom panels) of wing discs from all seven 0-insulator genotypes. For microscopy, all images were normalized to P⁺E⁺/P⁺E⁺ and minimum/maximum level corrections were applied equally to all genotypes using ImageJ and false-colored green. Error bars represent standard error of the mean (S.E.M) and scalebars are 50 μm.</p

A model for <i>gypsy</i> insulator function in transvection.

<p>Looping in <i>cis</i> between flanking insulators in a wildtype background prevents silencing of regulatory elements located on opposite homologs. Mutations in <i>su</i>(Hw) disrupt <i>cis</i> looping contacts and alter the local chromatin landscape to a more repressive state, preventing stable communication between regulatory elements even if pairing between homologs is maintained. </p