RNA-Interference Components Are Dispensable for Transcriptional Silencing of the <i>Drosophila</i> Bithorax-Complex

<div><p>Background</p><p>Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.</p><p>Principal Findings</p><p>Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (<i>BX-C</i>). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the <i>BX-C</i>. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.</p><p>Conclusions</p><p>We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in <i>Drosophila</i>.</p></div

PcG-occupied promoters are not enriched in AGO2-bound proximal promoter-derived small RNA sequences.

<p>No significant enrichment in small RNAs observed in PcG-bound promoters relative to promoters without PcG-binding (Kolmogorov–Smirnov test, p-value = 1.0); tag counts are normalized to tags per ten million tags (tptm). Promoter regions were defined as the genome region 50 nucleotides upstream and downstream of the Refseq-defined transcriptional start site, which were linked to PcG occupancy as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065740#pone.0065740-Enderle1" target="_blank">[50]</a>.</p

Contribution of the RNAi pathway to H3K27 methylation.

<p>Histones were acid extracted from mock treated S2 cells (control transfection without the dsRNA) or S2 cells treated with Dcr2 dsRNA, separated by SDS-PAGE, isolated from the gel and analyzed by MALDI-TOF mass spectrometry. The graph shows the amount of methylation within the peptide containing aminoacids 27–40 of H3 from two independent experiments.</p

<i>BX-C</i> higher order interactions are not impaired after Dcr2 depletion.

<p>(<b>A</b>) The scheme shows the Bithorax Complex (<i>BX-C</i>), including transcription units and genetically characterized regulatory regions (black). (<b>B–C</b>) Chromosome Conformation Capture (3C) on S2 cells. Cells were treated with EGFP-dsRNA (control) and Dcr2-dsRNA. Crosslinking frequencies between PcG targets are shown. All data points were generated from an average of at least three independent experiments. The standard error of the mean is indicated. Two-tailed <i>t</i>-test was applied for statistical analysis. Asterisks indicate statistically relevant differences; α = 0.05. (<b>B</b>) Crosslinking frequencies, normalized on the control, between the fixed fragments spanning homeotic promoters (<i>Abd-Bγ</i>; <i>abd-A</i>) and <i>BX-C</i> PREs. (<b>C</b>) PRE/PRE crosslinking frequencies, normalized on the control. Standard error of the mean is indicated.</p

Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function

Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function

Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function

Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function

Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function

Structural and Functional Characterization of MppR, an Enduracididine Biosynthetic Enzyme from <i>Streptomyces hygroscopicus</i>: Functional Diversity in the Acetoacetate Decarboxylase-like Superfamily

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant <i>Staphylococcus aureus</i>. Enduracididine is produced in <i>Streptomyces hygroscopicus</i> by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5′-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of “4-hydroxy-2-ketoarginine” shows the correct 4<i>R</i> enantiomer of “2-ketoenduracididine” bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function