Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.

BackgroundΔ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA.Methodology/Principal FindingsThe substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C20 and C22 polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3.ConclusionsThe competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.

The health benefits of the (n-3) PUFA, EPA, and DHA have created a demand for fish and fish oil, the main sources of these PUFA. Production animals, such as poultry, are potential alternate and sustainable sources of EPA and DHA, provided these fatty acids can be synthesized from plant-derived α-linolenic acid [ALA, 18:3(n-3)]. Because elongases are potential control points in the conversion of ALA to DHA in rats, we examined the chicken elongases, ELOVL2 and ELOVL5, which had not been characterized. ELOVL2 activity was limited to C20-22 PUFA substrates and the major product of ELOVL2 metabolism of EPA was 24:5(n-3). This indicates that ELOVL2 can sequentially elongate EPA to docosapentaenoic acid [DPA, 22:5(n-3)] and then onto 24:5(n-3). ELOVL5 selectivity was broader with elongation of C18-22 PUFA substrates. The ability of chicken ELOVL5 to efficiently synthesize 24:5(n-3) is unique compared with ELOVL5 enzymes from other species. The expression of ELOVL5 was higher than ELOVL2 in livers of broiler chickens and their expression did not change when dietary ALA was increased from 0.6 to 1.3% of dietary energy for 42 d. The expression of both genes was higher than previously seen in rats. The chicken elongase enzymes are unlike those of any species studied to date, because both ELOVL2 and ELOVL5 have the ability to efficiently elongate DPA. In addition, the relative abundance of ELOVL2 and ELOVL5 in the liver suggests that chickens may be able to metabolize more DPA through to 24:5(n-3), the precursor of DHA, compared with other species such as rats.

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and Elovl2, which have been overlooked as regulators of DHA synthesis. Using a yeast expression system, it was apparent that the substrate specifi cities of the two rat elongases had some overlap, but that only Elovl2 could convert endogenously formed C 22 PUFA docosapentaenoic acid (DPA) (22:5n-3) to 24:5n-3, which is the penultimate precursor of DHA ( 5 ). Elovl2 performs the sequential elongation of EPA to DPA followed by further elongation to 24:5n-3.Thus, Elovl2 is crucial for DHA synthesis at least in the rat where Elovl5 cannot elongate DPA to 24:5n-3 ( 5 ). This probably explains the poor or absent ability to produce DHA in species that do not have detectable Elovl2 such as barramundi ( 6, 7 ) or in species such as the rat in which Elovl2 is expressed at low levels ( 5 ). However, there is not an absolute Elovl2 dependence for DHA synthesis in all species because the sea bream, cobia, Atlantic bluefi n tuna, and chicken Elovl5 have a small but measurable ability to elongate DPA ( 8-11 ). In order to better understand the potential for these elongases to be involved in DHA synthesis, we have sought the molecular reasons for the differences between Elovl5 and Elovl2 in their ability to elongate DPA to 24:5n-3.Purifi cation of membrane-bound elongases to determine the substrate binding pocket has proven to be unsuccessful ( 12 ). However, chimeric elongase proteins from yeast ( 13 ), the moss Physcomitrella patens ( 14 ), and the fungi Pythium irregulare and Phytophthore infestans ( 15 ) have been used to investigate the regions involved in C 18 and C 20 PUFA substrate specifi city and product chain length determination. Therefore, we have constructed a series of rat Elovl2/Elovl5 chimeras and point mutations to examine the Elovl2 residues responsible for DPA substrate specifi city using a yeast expression system.Abstract Functional characterization of the rat elongases, Elovl5 and Elovl2, has identifi ed that Elovl2 is crucial for omega-3 docosahexaenoic acid (DHA) (22:6n-3) synthesis. While the substrate specifi cities of the rat elongases had some overlap, only Elovl2 can convert the C 22 omega-3 PUFA docosapentaenoic acid (DPA) (22:5n-3) to 24:5n-3, which is the penultimate precursor of DHA. In order to better understand the potential for these elongases to be involved in DHA synthesis, we have examined the molecular reasons for the differences between Elovl5 and Elovl2 in their ability to elongate DPA to 24:5n-3. We identifi ed a region of heterogeneity between Elovl5 and Elovl2 spanning transmembrane domains 6 and 7. Using a yeast expression system, we examined a series of Elovl2/Elovl5 chimeras and point mutations to identify Elovl2 residues within this region which are responsible for DPA substrate specifi city. The results indicate that the cysteine at position 217 in Elovl2 and a tryptophan at the equivalent position in Elovl5 explain their differing abilities to elongate DPA to 24:5n-3. Further studies confi rmed that Elovl2 C217 is a critical residue for elongation of DPA at the level observed i...