A PWWP Domain-Containing Protein Targets the NuA3 Acetyltransferase Complex via Histone H3 Lysine 36 trimethylation to Coordinate Transcriptional Elongation at Coding Regions

Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5′-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with identifier PXD001156

HPLC-Based Mass Spectrometry Characterizes the Phospholipid Alterations in Ether-Linked Lipid Deficiency Models Following Oxidative Stress

<div><p>Despite the fact that the discovery of ether-linked phospholipids occurred nearly a century ago, many unanswered questions remain concerning these unique lipids. Here, we characterize the ether-linked lipids of the nematode with HPLC-MS/MS and find that more than half of the phosphoethanolamine-containing lipids are ether-linked, a distribution similar to that found in mammalian membranes. To explore the biological role of ether lipids <i>in vivo</i>, we target fatty acyl-CoA reductase (<i>fard-1)</i>, an essential enzyme in ether lipid synthesis, with two distinct RNAi strategies. First, when <i>fard-1</i> RNAi is initiated at the start of development, the treated animals have severely reduced ether lipid abundance, resulting in a shift in the phosphatidylethanolamine lipid population to include more saturated fatty acid chains. Thus, the absence of ether lipids during development drives a significant remodeling of the membrane landscape. A later initiation of <i>fard-1</i> RNAi in adulthood results in a dramatic reduction of new ether lipid synthesis as quantified with ¹⁵N-tracers; however, there is only a slight decrease in total ether lipid abundance with this adult-only <i>fard-1</i> RNAi. The two RNAi strategies permit the examination of synthesis and ether lipid abundance to reveal a relationship between the amount of ether lipids and stress survival. We tested whether these species function as sacrificial antioxidants by directly examining the phospholipid population with HPLC-MS/MS after oxidative stress treatment. While there are significant changes in other phospholipids, including polyunsaturated fatty acid-containing species, we did not find any change in ether-linked lipids, suggesting that the role of ether lipids in stress resistance is not through their general consumption as free radical sinks. Our work shows that the nematode will be a useful model for future interrogation of ether lipid biosynthesis and the characterization of phospholipid changes in various stress conditions.</p></div

Quantification of Membrane Dynamics With ¹³C-Labeling and GC-MS in Young Adults.

<p>(A) Day 3 adult nematodes were fed a diet of 50% ¹³C-labeled <i>E</i>. <i>coli</i> for 6 hours to introduce ¹³C into their lipids and mark them as newly added or modified. The ¹³C can be traced into the major membrane FAs (>2% abundance) by GC-MS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s011" target="_blank">S1 Table</a>). The amount of new FA measured by ¹³C-incorporation, was determined for purified PLs (black) and NLs (white). C20s with 4 or more double bonds were excluded in the analysis due to insufficient isotopomers. C20:0 and C20:2 FAs were profiled despite low abundance in the membrane to increase the representation of long-chain FAs. (B) After combining the amount of new FA found in all major C15 to C19 FAs, the overall contribution of new FA/hour was significantly greater in the PLs versus the NLs. (C) The relative amount of new FA derived from <i>de novo</i> FA synthesis was not significantly different for PLs (black) and NLs (white) except for C16:0. Numbers shown represent the mean of at least four experiments ± SEM. Statistical significance was calculated by two-tailed unpaired t-tests where significance (*p<0.05, **p<0.01, ***p<0.001) is noted by the asterisks.</p

LC-MS/MS Analysis of PL Composition in <i>fat-7</i> RNAi Treated Animals.

<p>The major PL species (>2%) in control animals are represented by X:Y (where X is the number of carbons in both FAs tails and Y indicates the total double bonds). For a complete list of species, see source data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s002" target="_blank">S2 Dataset</a>). For P-PlsEtn, the LC-MS/MS detection allowed for both chains to be accurately reported. Control RNAi is represented with white bars, and <i>fat-7</i> RNAi is shown in red. The fatty acid tails for each species can be qualitatively assessed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s009" target="_blank">S7 Fig</a>). Significant changes as are indicated by * (p<0.05) and ** (p<0.01) determined by two-tailed unpaired t-tests. Numbers shown represent the mean of at least three experiments ± SEM.</p

¹³C- and ¹⁵N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in <i>C</i>. <i>elegans</i>

<div><p>Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in <i>C</i>. <i>elegans</i> combining ¹³C and ¹⁵N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with <i>SCD</i> RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism.</p></div

Dietary ¹³C Allows for Modeling of Individual FA Dynamics.

<p>(A) Day 3 adult nematodes are grown on ¹²C (gray)-<i>E</i>.<i>coli</i> (OP50) before the diet is switched to 50% ¹³C (green)-<i>E</i>. <i>coli</i>. After 6 hours, the C18:1n7 in the nematode was a combination of old fat from the original (pre-¹³C) diet, FA absorbed directly from the diet, and FA derived from lipogenesis with a random but statistically definable incorporation of single carbon molecules. (B) The C18:1n7 isotopomers were assessed by GC-MS. The natural abundance of ¹³C in the environment as well as background were subtracted from the C18:1n7 isotopomers derived from ¹³C-fed animals. The contributions of <i>de novo</i> FA synthesis (light green) and dietary absorption (dark green) can be mathematically determined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#sec014" target="_blank">Material and Methods</a>, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.ref009" target="_blank">9</a>]). (C) For any given fatty acid species including the C18:1n7 shown here, the total abundance of the fatty acid is determined by integrating the area under its peak in the gas chromatograph (total abundance outlined in dashed line). Further, we can divide the area as follows with gray representing ¹²C species and green indicating the presence of at least one ¹³C molecule. The unlabeled fatty acids absorbed during the labeling period were accounted for as new. The percentage of the population from absorption (dark green) and synthesis (light green) can be quantified to ultimately define that 29.3 ± 1.5% of the C18:1n7 peak is generated from new fatty acid.</p

Modeling the Impact of <i>SCD</i> Depletion on Membrane Dynamics.

<p>(A) ¹³C-tracers allow for the quantification of <i>de novo</i> synthesis as well as incorporation of FAs into PLs (measurements from ¹³C-tracers shown in green). Our implementation of ¹⁵N to measure PL headgroup turnover defines the rates that the two most abundant species within each major PL class are replaced (shown in blue); for example, 40:10 and 38:6 are the largest contributors to the PtdCho pool and are replaced at a rate of 1.3% new PL/hour and 1.5% new PL/hour respectively. A weighted average of the PL replacement calculated for the PL species reported here predicts that 1.5% of the total PLs are replaced each hour. We combined the PL replacement rates with those calculated for FA tails where we found that 4.5% of measured fatty acid tails are new each hour. The quantification after 24 hours revealed a stable population of membrane lipid (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s005" target="_blank">S3 Fig</a>); thus, we reduced our average fatty acid predictions by 30% to account for that population. Taken together, we use the ¹³C and ¹⁵N-tracers to define how the individual lipid components (FA tails and polar headgroups) are replaced over time, and predict that at least 60% of the membrane is new each day when considering each fatty acid tail and headgroup as individual entities. (B) Upon <i>fat-7</i> RNAi treatment, there is a buildup of C18:0 and a depletion of C18:1n9, which in turn reduces the activation of <i>de novo</i> FA synthesis. Subsequently, FAs provided to PL synthesis and ultimately provided to the membrane, are severely compromised.</p

¹⁵N-Tracers and LC-MS/MS Measure PL Dynamics in Wild-Type and <i>fat-7</i> RNAi Animals.

<p>(A) Representation of the major PLs in the nematode: phosphatidylethanolamine (PtdEtn), phosphatidylcholine (PtdCho), and plasmenylethanolamine (P-PlsEtn) where R₁ and R₂ represent aliphatic carbon chains. The single nitrogen is highlighted in blue. (B) LC-MS/MS showed successful incorporation of ¹⁵N into the headgroups of PtdEtn (36:2) in labeled (blue) compared to unlabeled (white) control animals. Incorporation of ¹⁵N was compromised in this representative example for animals treated with <i>fat-7</i> RNAi (red). (C) The enrichment of ¹⁵N seen in each PL was modeled by % ¹⁵N-labeled PL per hour. The incorporation of ¹⁵N was significantly lower in <i>fat-7</i> RNAi treated animals (red) compared to control (blue). (D) The amount of new FAs for the most abundant species from each PL class is shown for control (blue) and <i>fat-7</i> RNAi treatment (red) as well. Data shown represent SEM and a minimum of n of 3. Significance was defined by two-tailed unpaired t-tests (*p<0.05, **p<0.01).</p

Adult-Only <i>fard-1</i> RNAi Treatment Causes Reduced Ether Lipid Production.

<p>(A) ¹⁵N-incorporation assays allowed for the measurement of new phospholipid headgroup incorporation. Phospholipid species are identified here by the summated molecular species that comprise both fatty acid tails. For example O-PE (36:1) represents a plasmanylethanolamine with 36 carbons and one double bond in the associated fatty acids. In <i>fard-1</i> RNAi-treated adults (yellow), there is depletion in newly synthesized O-PE (36:1) and P-PE (36:1), but not PC (36:2) or PE (36:2) as compared to control animals (black). Data was generated from at least 4 independent biological replicates with SEM shown. *p<0.05 was determined by unpaired t-tests using Holm-Sidak corrections for multiple comparisons. (B) Here we present a summary of the lipid biochemical data from developmental and adult-only <i>fard-1</i> RNAi treatments. Developmental <i>fard-1</i> RNAi results in stronger phenotypes across all categories including synthesis where there were not sufficient O-PE and P-PE lipids for analysis.</p