Role of histone variants H2BC1 and H2AZ.2 in H2AK119ub nucleosome organization and Polycomb gene silencing.

Ubiquitination of histone H2A at lysine 119 residue (H2AK119ub) plays critical roles in a wide range of physiological processes, including Polycomb gene silencing 1,2 , replication 3-5 , DNA damage repair 6-10 , X inactivation 11,12 , and heterochromatin organization 13,14 . However, the underlying mechanism and structural basis of H2AK119ub remains largely elusive. In this study, we report that H2AK119ub nucleosomes have a unique composition, containing histone variants H2BC1 and H2AZ.2, and importantly, this composition is required for H2AK119ub and Polycomb gene silencing. Using the UAB domain of RSF1, we purified H2AK119ub nucleosomes to a sufficient amount and purity. Mass spectrometry analyses revealed that H2AK119ub nucleosomes contain the histone variants H2BC1 and H2AZ.2. A cryo-EM study resolved the structure of native H2AK119ub nucleosomes to a 2.6A resolution, confirming H2BC1 in one subgroup of H2AK119ub nucleosomes. Tandem GST-UAB pulldown, Flag-H2AZ.2, and HA-H2BC1 immunoprecipitation revealed that H2AK119ub nucleosomes could be separated into distinct subgroups, suggesting their composition heterogeneity and potential dynamic organization. Knockout or knockdown of H2BC1 or H2AZ.2 reduced cellular H2AK119ub levels, establishing H2BC1 and H2AZ.2 as critical determinants of H2AK119ub. Furthermore, genomic binding profiles of H2BC1 and H2AZ.2 overlapped significantly with H2AK119ub binding, with the most significant overlapping in the gene body and intergenic regions. Finally, assays in developing embryos reveal an interaction of H2AZ.2, H2BC1, and RING1A in vivo . Thus, this study revealed, for the first time, that the H2AK119ub nucleosome has a unique composition, and this composition is required for H2AK119ub and Polycomb gene silencing.

RNA Sensing and Innate Immunity Constitutes a Barrier for Interspecies Chimerism.

Interspecies chimera formation with human pluripotent stem cells (PSCs) holds great promise to generate humanized animal models and provide donor organs for transplant. However, the approach is currently limited by low levels of human cells ultimately represented in chimeric embryos. Different strategies have been developed to improve chimerism by genetically editing donor human PSCs. To date, however, it remains unexplored if human chimerism can be enhanced in animals through modifying the host embryos. Leveraging the interspecies PSC competition model, here we discovered retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling, an RNA sensor, in "winner" cells plays an important role in the competitive interactions between co-cultured mouse and human PSCs. We found that genetic inactivation of Ddx58/Ifih1-Mavs-Irf7 axis compromised the "winner" status of mouse PSCs and their ability to outcompete PSCs from evolutionarily distant species during co-culture. Furthermore, by using Mavs-deficient mouse embryos we substantially improved unmodified donor human cell survival. Comparative transcriptome analyses based on species-specific sequences suggest contact-dependent human-to-mouse transfer of RNAs likely plays a part in mediating the cross-species interactions. Taken together, these findings establish a previously unrecognized role of RNA sensing and innate immunity in "winner" cells during cell competition and provides a proof-of-concept for modifying host embryos, rather than donor PSCs, to enhance interspecies chimerism.

Generalized nuclear localization of retroelement transcripts.

BACKGROUND: LINE-1s, Alus and SVAs are the only retrotransposition competent elements in humans. Their mobilization followed by insertional mutagenesis is often linked to disease. Apart from these rare integration events, accumulation of retrotransposition intermediates in the cytoplasm is potentially pathogenic due to induction of inflammatory response pathways. Although the retrotransposition of LINE-1 and Alu retroelements has been studied in considerable detail, there are mixed observations about the localization of their RNAs. RESULTS: We undertook a comprehensive and unbiased approach to analyze retroelement RNA localization using common cell lines and publicly available datasets containing RNA-sequencing data from subcellular fractions. Using our customized analytic pipeline, we compared localization patterns of RNAs transcribed from retroelements and single-copy protein coding genes. Our results demonstrate a generalized characteristic pattern of retroelement RNA nuclear localization that is conserved across retroelement classes as well as evolutionarily young and ancient elements. Preferential nuclear enrichment of retroelement transcripts was consistently observed in cell lines, in vivo and across species. Moreover, retroelement RNA localization patterns were dynamic and subject to change during development, as seen in zebrafish embryos. CONCLUSION: The pronounced nuclear localization of transcripts arising from ancient as well as de novo transcribed retroelements suggests that these transcripts are retained in the nucleus as opposed to being re-imported to the nucleus or degraded in the cytoplasm. This raises the possibility that there is adaptive value associated with this localization pattern to the host, the retroelements or possibly both.

Distinct p53 isoforms code for opposing transcriptional outcomes.

p53 genes are conserved transcriptional activators that respond to stress. These proteins can also downregulate genes, but the mechanisms are not understood and are generally assumed to be indirect. Here, we investigate synthetic and native cis-regulatory elements in Drosophila to examine opposing features of p53-mediated transcriptional control in vivo. We show that transcriptional repression by p53 operates continuously through canonical DNA binding sites that confer p53-dependent transactivation at earlier developmental stages. p53 transrepression is correlated with local H3K9me3 chromatin marks and occurs without the need for stress or Chk2. In sufficiency tests, two p53 isoforms qualify as transrepressors and a third qualifies as a transcriptional activator. Targeted isoform-specific knockouts dissociate these opposing transcriptional activities, highlighting features that are dispensable for transactivation but critical for repression and for proper germ cell formation. Together, these results demonstrate that certain p53 isoforms function as constitutive tissue-specific repressors, raising important implications for tumor suppression by the human counterpart.

Silencing of LINE-1 retrotransposons is a selective dependency of myeloid leukemia.

Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.

p53 directly represses human LINE1 transposons.

p53 is a potent tumor suppressor and commonly mutated in human cancers. Recently, we demonstrated that p53 genes act to restrict retrotransposons in germline tissues of flies and fish but whether this activity is conserved in somatic human cells is not known. Here we show that p53 constitutively restrains human LINE1s by cooperatively engaging sites in the 5'UTR and stimulating local deposition of repressive histone marks at these transposons. Consistent with this, the elimination of p53 or the removal of corresponding binding sites in LINE1s, prompted these retroelements to become hyperactive. Concurrently, p53 loss instigated chromosomal rearrangements linked to LINE sequences and also provoked inflammatory programs that were dependent on reverse transcriptase produced from LINE1s. Taken together, our observations establish that p53 continuously operates at the LINE1 promoter to restrict autonomous copies of these mobile elements in human cells. Our results further suggest that constitutive restriction of these retroelements may help to explain tumor suppression encoded by p53, since erupting LINE1s produced acute oncogenic threats when p53 was absent.

Follow-Up Care and 6-Month Continuation Rates for Long-Acting Reversible Contraceptives in Adolescents and Young Adults: A Retrospective Chart Review.

STUDY OBJECTIVE: Describe follow-up care patterns and continuation rates during the first 6 months after initiating a long-acting reversible contraceptive (LARC) device among adolescent and young adult women. DESIGN: Retrospective chart review among patients who had an intrauterine device (IUD) or subdermal implant placed between January 2015 and December 2016. SETTING: Urban adolescent specialty care clinic. PARTICIPANTS: Women ages 13-23 years. MAIN OUTCOME MEASURES: Follow-up encounters were defined as scheduled and unscheduled phone calls, outpatient clinic visits, or emergency department visits during the 6 months after device placement. Continuation was defined as not having the device removed or expelled during the 6 months after initiation. Frequencies were calculated, and logistic regression was used to determine predictors of follow-up encounters and continuation. RESULTS: Among the 177 patients, 180 LARC devices were placed. Most were 13-17 years of age (56%), non-Hispanic black (64%), publicly insured (57%), and had an IUD placed (57%). Most (86%) had 1 or more clinical encounters during the 6 months: 70% attended a scheduled encounter and 53% had an unscheduled encounter. Approximately half (45%) attended the scheduled 2-week office visit; only 6% attended the 6-month office visit. The 6-month LARC continuation rate was 92% (n = 166), with most discontinuations among IUD users (n = 12; 7%). CONCLUSION: LARC continuation rates were high in our study population. Most adolescent and young adult women have at least 1 follow-up encounter in the 6 months after LARC placement. Clinical practices should be prepared to address issues that arise during follow-up encounters, whether in person or by phone.

Drosophila p53 directs nonapoptotic programs in postmitotic tissue.

TP53 is the most frequently mutated gene in human cancers, and despite intensive research efforts, genome-scale studies of p53 function in whole animal models are rare. The need for such in vivo studies is underscored by recent challenges to established paradigms, indicating that unappreciated p53 functions contribute to cancer prevention. Here we leveraged the Drosophila system to interrogate p53 function in a postmitotic context. In the developing embryo, p53 robustly activates important apoptotic genes in response to radiation-induced DNA damage. We recently showed that a p53 enhancer (p53RErpr) near the cell death gene reaper forms chromatin contacts and enables p53 target activation across long genomic distances. Interestingly, we found that this canonical p53 apoptotic program fails to activate in adult heads. Moreover, this failure to exhibit apoptotic responses was not associated with altered chromatin contacts. Instead, we determined that p53 does not occupy the p53RErpr enhancer in this postmitotic tissue as it does in embryos. Through comparative RNA-seq and chromatin immunoprecipitation-seq studies of developing and postmitotic tissues, we further determined that p53 regulates distinct transcriptional programs in adult heads, including DNA repair, metabolism, and proteolysis genes. Strikingly, in the postmitotic context, p53-binding landscapes were poorly correlated with nearby transcriptional effects, raising the possibility that p53 enhancers could be generally acting through long distances.

Transposons, p53 and Genome Security.

p53, the most commonly mutated tumor suppressor, is a transcription factor known to regulate proliferation, senescence, and apoptosis. Compelling studies have found that p53 may prevent oncogenesis through effectors that are unrelated to these canonical processes and recent findings have uncovered ancient roles for p53 in the containment of mobile elements. Together, these developments raise the possibility that some p53-driven cancers could result from unrestrained transposons. Here, we explore evidence linking conserved features of p53 biology to the control of transposons. We also show how p53-deficient cells can be exploited to probe the behavior of transposons and illustrate how unrestrained transposons incited by p53 loss might contribute to human malignancies.

Retrotransposons Mimic Germ Plasm Determinants to Promote Transgenerational Inheritance.

Retrotransposons are a pervasive class of mobile elements present in the genomes of virtually all forms of life [1, 2]. In metazoans, these are preferentially active in the germline, which, in turn, mounts defenses that restrain their activity [3, 4]. Here we report that certain classes of retrotransposons ensure transgenerational inheritance by invading presumptive germ cells before they are formed. Using sensitized Drosophila and zebrafish models, we found that diverse classes of retrotransposons migrate to the germ plasm, a specialized region of the oocyte that prefigures germ cells and specifies the germline of descendants in the fertilized egg. In Drosophila, we found evidence for a "stowaway" model, whereby Tahre retroelements traffic to the germ plasm by mimicking oskar RNAs and engaging the Staufen-dependent active transport machinery. Consistent with this, germ plasm determinants attracted retroelement RNAs even when these components were ectopically positioned in bipolar oocytes. Likewise, vertebrate retrotransposons similarly migrated to the germ plasm in zebrafish oocytes. Together, these results suggest that germ plasm targeting represents a fitness strategy adopted by some retrotransposons to ensure transgenerational propagation.

Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing.

Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions.

p53 in the game of transposons.

Throughout the animal kingdom, p53 genes function to restrain mobile elements and recent observations indicate that transposons become derepressed in human cancers. Together, these emerging lines of evidence suggest that cancers driven by p53 mutations could represent "transpospoathies," i.e. disease states linked to eruptions of mobile elements. The transposopathy hypothesis predicts that p53 acts through conserved mechanisms to contain transposon movement, and in this way, prevents tumor formation. How transposon eruptions provoke neoplasias is not well understood but, from a broader perspective, this hypothesis also provides an attractive framework to explore unrestrained mobile elements as inciters of late-onset idiopathic disease. Also see the video abstract here.

p53 genes function to restrain mobile elements.

Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, we show that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53(-) germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, we also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5' sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

In vitro and in vivo assays for studying histone ubiquitination and deubiquitination.

Posttranslational histone modifications play important roles in regulating chromatin structure and function (Rando, Curr Opin Genet Dev 22:148-155, 2012; Zentner and Henikoff, Nat Struct Mol Biol 20:259-266, 2013). One example of such modifications is histone ubiquitination, which occurs predominately on H2A and H2B. Recent studies have highlighted important regulatory roles of H2A ubiquitination in Polycomb group protein-mediated gene silencing and DNA damage repair (de Napoles et al., Dev Cell 7:663-676, 2004; Wang et al., Nature 431:873-878, 2004; Doil et al., Cell 136:435-446, 2009; Gatti et al., Cell Cycle 11:2538-2544, 2012; Mattiroli et al., Cell 150:1182-1195, 2012; Stewart et al., Cell 136:420-434, 2009; Bergink et al., Genes Dev 20:1343-1352, 2006; Facchino et al., J Neurosci 30:10096-10111, 2010; Ginjala et al., Mol Cell Biol 31:1972-1982, 2011; Ismail et al., J Cell Biol 191:45-60, 2010), H2B ubiquitination in transcription initiation and elongation (Xiao et al., Mol Cell Biol 25:637-651, 2005; Kao et al., Genes Dev 18:184-195, 2004; Pavri et al., Cell 125:703-717, 2006; Kim et al., Cell 137:459-471, 2009), pre-mRNA splicing (Jung et al. Genome Res 22:1026-1035, 2012; Shieh et al., BMC Genomics 12:627, 2011; Zhang et al., Genes Dev 27:1581-1595, 2013), nucleosome stabilities (Fleming et al., Mol Cell 31:57-66, 2008; Chandrasekharan et al., Proc Natl Acad Sci U S A 106:16686-16691, 2009), H3 methylation (Sun and Allis, Nature 418:104-108, 2002; Briggs et al., Nature 418:498, 2002; Dover et al., J Biol Chem 277:28368-28371, 2002; Ng et al., J Biol Chem 277:34655-34657, 2002), and DNA methylation (Sridhar et al., Nature 447:735-738, 2007). Here we describe methods for in vitro histone ubiquitination and deubiquitination assays. We also describe approaches to investigate the in vivo function of putative histone ubiquitin ligase(s) and deubiquitinase(s). These experimental procedures are largely based on our studies in mammalian cells. These methods should provide useful tools for studying this bulky histone modification.

The histone H2A deubiquitinase Usp16 regulates embryonic stem cell gene expression and lineage commitment.

Polycomb Repressive Complex 1 and histone H2A ubiquitination (ubH2A) contribute to embryonic stem cell (ESC) pluripotency by repressing lineage-specific gene expression. However, whether active deubiquitination co-regulates ubH2A levels in ESCs and during differentiation is not known. Here we report that Usp16, a histone H2A deubiquitinase, regulates H2A deubiquitination and gene expression in ESCs, and importantly, is required for ESC differentiation. Usp16 knockout is embryonic lethal in mice, but does not affect ESC viability or identity. Usp16 binds to the promoter regions of a large number of genes in ESCs, and Usp16 binding is inversely correlated with ubH2A levels, and positively correlates with gene expression levels. Intriguingly, Usp16(-/-) ESCs fail to differentiate due to ubH2A-mediated repression of lineage-specific genes. Finally, Usp16, but not a catalytically inactive mutant, rescues the differentiation defects of Usp16(-/-) ESCs. Therefore, this study identifies Usp16 and H2A deubiquitination as critical regulators of ESC gene expression and differentiation.

Binding of different histone marks differentially regulates the activity and specificity of polycomb repressive complex 2 (PRC2).

The polycomb repressive complex 2 (PRC2) is the major methyltransferase for H3K27 methylation, a modification critical for maintaining repressed gene expression programs throughout development. It has been previously shown that PRC2 maintains histone methylation patterns during DNA replication in part through its ability to bind to H3K27me3. However, the mechanism by which PRC2 recognizes H3K27me3 is unclear. Here we show that the WD40 domain of EED, a PRC2 component, is a methyllysine histone-binding domain. The crystal structures of apo-EED and EED in complex respectively with five different trimethyllysine histone peptides reveal that EED binds these peptides via the top face of its ß-propeller architecture. The ammonium group of the trimethyllysine is accommodated by an aromatic cage formed by three aromatic residues, while its aliphatic chain is flanked by a fourth aromatic residue. Our structural data provide an explanation for the preferential recognition of the Ala-Arg-Lys-Ser motif-containing trimethylated H3K27, H3K9, and H1K26 marks by EED over lower methylation states and other histone methyllysine marks. More importantly, we found that binding of different histone marks by EED differentially regulates the activity and specificity of PRC2. Whereas the H3K27me3 mark stimulates the histone methyltransferase activity of PRC2, the H1K26me3 mark inhibits PRC2 methyltransferase activity on the nucleosome. Moreover, H1K26me3 binding switches the specificity of PRC2 from methylating H3K27 to EED. In addition to determining the molecular basis of EED-methyllysine recognition, our work provides the biochemical characterization of how the activity of a histone methyltransferase is oppositely regulated by two histone marks.

Variability in drug metabolizing enzyme activity in HIV-infected patients.

AIMS: To evaluate variability in cytochrome P450 (CYP) 1A2, CYP2D6, CYP3A, N-acetyltransferase 2 (NAT2), and xanthine oxidase (XO) activity in HIV-infected patients and compare this with data from uninfected, healthy volunteers. METHODS: Ten HIV-infected men and seven women on medication affecting CYP enzyme activity were phenotyped four times over 2 months using caffeine, dextromethorphan, and midazolam. Urinary caffeine and dextromethorphan metabolite ratios were used to phenotype CYP1A2, NAT2, XO, and CYP2D6 activity and midazolam plasma clearance was used to phenotype CYP3A activity. Plasma and urine samples were analyzed by validated LC/UV or LC/MS methods for midazolam, caffeine, and dextromethorphan. Noncompartmental pharmacokinetics and nonparametric statistical analyses were performed, and the data compared with those of healthy volunteer historic controls. RESULTS: Compared with age and sex-matched healthy volunteers, HIV-infected subjects had 18% lower hepatic CYP3A4 activity, 90% lower CYP2D6 activity, 53% lower NAT2 activity, and 22% higher XO activity. No significant difference was found in CYP1A2 activity. Additionally, 25% genotype-phenotype discordance in CYP2D6 activity was noted in HIV-infected subjects. Intraindividual variability in enzyme activity increased by 42-62% in HIV-infected patients for CYP1A2, NAT2, and XO, and decreased by 33% for CYP2D6. Interindividual variability in enzyme activity increased by 27-63% in HIV-infected subjects for CYP2D6, CYP1A2, and XO, and decreased by 38% for NAT2. Higher plasma TNFalpha concentrations correlated with lower CYP2D6 and CYP3A4 activity. CONCLUSIONS: Infection with HIV or stage of HIV infection may alter Phase I and II drug metabolizing enzyme activity. HIV infection was related to an increase in variability of these drug-metabolizing enzymes. Altered metabolism may be a consequence of immune activation and cytokine exposure.

Chylomicron particle size and number, factor VII activation and dietary monounsaturated fatty acids.

This study evaluated the effects of substituting dietary saturated fatty acids (SFAs) with monounsaturated fatty acids (MUFAs) on postprandial chylomicron (triacylglycerol (TAG), apolipoprotein B-48 (apo B-48) and retinyl ester (RE)), chylomicron particle size and factor VII (FVII) response when subjects were given a standard meal. In a controlled sequential design, 51 healthy young subjects followed an SFA-rich diet (Reference diet) for 8 weeks after which half of the subjects followed a moderate MUFA diet (n=25) and half followed a high MUFA diet (n=26) for 16 weeks. Fasting lipoprotein and lipid measurements were evaluated at baseline and at 8-week intervals during the Reference and MUFA diets. In 25 of the subjects (n=12 moderate MUFA, n=13 high MUFA), postprandial responses to a standard test meal containing RE and 13C-tripalmitin were investigated at the end of the Reference and the MUFA diet periods. Although there were no differences in the postprandial lipid markers (TAG, RE, 13C-TAG) on the two diets, the postprandial apo B-48 response (incremental area under the curve (IAUC)) was reduced by 21% on the moderate MUFA diet (NS) and by 54% on the high MUFA diet (P<0.01). The postprandial peak concentrations of apo B-48 were reduced by 33% on the moderate MUFA diet (P<0.01) and 48% on the high MUFA diet (P<0.001). Fasting values for factor VII activity (FVIIc), activated factor VII (FVIIa) or factor VII antigen (FVIIag) did not differ significantly when subjects were transferred from Reference to MUFA diets. However, the postprandial increases in coagulation FVII activity (FVIIc) were 18% lower and of activated FVII (FVIIa) were 17% lower on the moderate MUFA diet (NS). Postprandial increases in FVIIc and FVIIa were 50% (P<0.05) and 29% (P<0.07) lower on the high MUFA diet and the area under the postprandial FVIIc response curve (AUC) was also lower on the high MUFA diet (P<0.05). Significantly higher ratios of RE:apo B-48 (P<0.001) and 13C-palmitic acid:apo B-48 (P<0.01) during both MUFA diets suggest that the CMs formed carry larger amounts of dietary lipids per particle, reflecting an adaptation to form larger lipid droplets in the enterocyte when increased amounts of dietary MUFAs are fed. Smaller numbers of larger chylomicrons may explain attenuated activation of factor VII during the postprandial state when the background diet is rich in MUFA.

Eicosapentaenoic and docosapentaenoic acids are the principal products of alpha-linolenic acid metabolism in young men*.

The capacity for conversion of alpha-linolenic acid (ALNA) to n-3 long-chain polyunsaturated fatty acids was investigated in young men. Emulsified [U-13C]ALNA was administered orally with a mixed meal to six subjects consuming their habitual diet. Approximately 33 % of administered [13C]ALNA was recovered as 13CO2 on breath over the first 24 h. [13C]ALNA was mobilised from enterocytes primarily as chylomicron triacylglycerol (TAG), while [13C]ALNA incorporation into plasma phosphatidylcholine (PC) occurred later, probably by the liver. The time scale of conversion of [13C]ALNA to eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) suggested that the liver was the principal site of ALNA desaturation and elongation, although there was some indication of EPA and DPA synthesis by enterocytes. [13C]EPA and [13C]DPA concentrations were greater in plasma PC than TAG, and were present in the circulation for up to 7 and 14 d, respectively. There was no apparent 13C enrichment of docosahexaenoic acid (DHA) in plasma PC, TAG or non-esterified fatty acids at any time point measured up to 21 d. This pattern of 13C n-3 fatty acid labelling suggests inhibition or restriction of DHA synthesis downstream of DPA. [13C]ALNA, [13C]EPA and [13C]DPA were incorporated into erythrocyte PC, but not phosphatidylethanolamine, suggesting uptake of intact plasma PC molecules from lipoproteins into erythrocyte membranes. Since the capacity of adult males to convert ALNA to DHA was either very low or absent, uptake of pre-formed DHA from the diet may be critical for maintaining adequate membrane DHA concentrations in these individuals.