Engrailed cooperates directly with Extradenticle and Homothorax on a distinct class of homeodomain binding sites to repress sloppy paired.

Even skipped (Eve) and Engrailed (En) are homeodomain-containing transcriptional repressors with similar DNA binding specificities that are sequentially expressed in Drosophila embryos. The sloppy-paired (slp) locus is a target of repression by both Eve and En. At blastoderm, Eve is expressed in 7 stripes that restrict the posterior border of slp stripes, allowing engrailed (en) gene expression to be initiated in odd-numbered parasegments. En, in turn, prevents expansion of slp stripes after Eve is turned off. Prior studies showed that the two tandem slp transcription units are regulated by cis-regulatory modules (CRMs) with activities that overlap in space and time. An array of CRMs that generate 7 stripes at blastoderm, and later 14 stripes, surround slp1 (Fujioka and Jaynes, 2012). Surprisingly given their similarity in DNA binding specificity and function, responsiveness to ectopic Eve and En indicates that most of their direct target sites are either in distinct CRMs, or in different parts of coregulated CRMs. We localized cooperative binding sites for En, with the homeodomain-containing Hox cofactors Extradenticle (Exd) and Homothorax (Hth), within two CRMs that drive similar expression patterns. Functional analysis revealed two distinct, redundant sites within one CRM. The other CRM contains a single cooperative site that is both necessary and sufficient for repression in the en domain. Correlating in vivo and in vitro analysis suggests that cooperativity with Exd and Hth is a key ingredient in the mechanism of En-dependent repression, and that apparent affinity in vitro is an unreliable predictor of in vivo function

Activation of the HH pathway upregulates PDGF.

<p>Larvae were incubated in 30μM purmorphamine, a HH receptor agonist, at 2dpf. (<b>A</b>) PED-6 gallbladder uptake shows decreased gallbladder staining in incubated larvae (p≤.00001) (<b>B</b>) PDGF-AA protein expression of purmorphamine-incubated larvae relative to the control (ctrl) at 5dpf. (<b>B’</b>) PDGF-AA protein quantification of two independent experiments with standard deviations. (<b>C,D</b>) Confocal projections of cytokeratin-18 immunostaining of livers from 5dpf control (ctrl) and purmorphamine (pur) incubated larvae. Numbers refer to individuals exhibiting comparable morphology as representative image.</p

Methylation of Hedgehog pathway genes.

<p>Methylation of Hedgehog pathway genes.</p

Quantification of duct characteristics in zebrafish larvae injected with control vs. PDGF-AA peptide.

<p>Quantification of duct characteristics in zebrafish larvae injected with control vs. PDGF-AA peptide.</p

DNA hypomethylation in BA samples.

<p>(A) Summary of the data, showing the difference in methylation for all points, for those with statistical significance. The Chi-square value for each comparison and the corresponding p-value, although all p-values were considerably less than 0.0001. (B) Shown is a scatter plot where probe sets are plotted for the relative amount of methylation in control (C, x-axis) and BA (y-axis). The diagonal line represents equal methylation, so points below are relatively hypomethylated in BA, while those above the line are hypermethylated. Only those points with greater than 15% difference and a statistically significant (p<0.05) difference are shown.</p

Increased PDGFA in BA samples.

<p>Immunostaining of liver biopsy specimens from disease and non-disease control and BA subjects shows increased staining of PDGF in BA cholangiocytes. Duct outlined in white.</p

Most significantly hypomethylated targets identified in the microarray, with most closely associated gene(s).

<p>Most significantly hypomethylated targets identified in the microarray, with most closely associated gene(s).</p

Methylation Microarray Studies Highlight <i>PDGFA</i> Expression as a Factor in Biliary Atresia

<div><p>Biliary atresia (BA) is a progressive fibro-inflammatory disorder that is the leading indication for liver transplantation in children. Although there is evidence implicating genetic, infectious, environmental, and inflammatory causes, the etiology of BA remains unknown. We have recently reported that cholangiocytes from BA patients showed decreased DNA methylation relative to disease- and non-disease controls, supporting a potential role for DNA hypomethylation in BA etiopathogenesis. In the current study, we examined the methylation status of specific genes in human BA livers using methylation microarray technology. We found global DNA hypomethylation in BA samples as compared to disease- and non-disease controls at specific genetic loci. Hedgehog pathway members, <i>SHH</i> and <i>GLI2</i>, known to be upregulated in BA, were both hypomethylated, validating this approach as an investigative tool. Another region near the <i>PDGFA</i> locus was the most significantly hypomethylated in BA, suggesting potential aberrant expression. Validation assays confirmed increased transcriptional and protein expression of PDGFA in BA livers. We also show that PDGF-A protein is specifically localized to cholangiocytes in human liver samples. Injection of PDGF-AA protein dimer into zebrafish larvae caused biliary developmental and functional defects. In addition, activation of the Hedgehog pathway caused increased expression of PDGF-A in zebrafish larvae, providing a previously unrecognized link between PDGF and the Hedgehog pathway. Our findings implicate DNA hypomethylation as a specific factor in mediating overexpression of genes associated with BA and identify PDGF as a new candidate in BA pathogenesis.</p></div