Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Regulation of ARTD1 by SET7/9-dependent methylation

Proteins are essential structural and functional components of cells and catalyze a multitude of biochemical reactions. Many proteins are subject to post-translational modifications in order to regulate their function and enzymatic activities. ADP- ribosylation of proteins is catalyzed by ADP-ribosyltransferases, which transfer one or more ADP-ribose molecules from their co-substrate NAD+ to their target proteins. The best-studied mammalian ADP-ribosyltransferase is ARTD1 (also called PARP1), a nuclear enzyme, which binds to and is activated by damaged DNA and poly(ADP- ribosyl)ates itself and other proteins. SET7/9 is a lysine methyltransferase that was initially identified as a mono- methyltransferase for H3K4, but also methylates many non-histone proteins such as p53 and DNMT1. SET7/9-dependent methylation has variable functional outcomes e.g. protein stabilization or crosstalk with other post-translational modifications. In this thesis, ARTD1 and the linker histone H1 have been identified as new targets for SET7/9 and the influence of SET7/9 on their cellular functions has been characterized. We found in vitro and in vivo that SET7/9 methylates ARTD1 at K508. This modification was strongly inhibited by prior poly(ADP-ribosyl)ation of ARTD1, while the methylation did not inhibit the automodification of ARTD1 nor its acetylation by p300. In cells, H2O2-induced poly(ADP-ribose) formation was decreased after SET7/9 knockdown and increased after SET7/9 overexpression. Mutation of K508 to arginine reduced the activity of ARTD1 under certain conditions, indicating that direct methylation of ARTD1 by SET7/9 is involved in the regulation of its enzymatic activity. Furthermore, we showed that SET7/9 methylates several lysine residues (K121, K129, K159, K171, K177 and K192) in the C-terminal domain of linker histone variant H1.4. Mutation of these residues, which are located at the terminal position of six KAKme motifs, did not alter the nuclear distribution of H1.4. The functional consequence of H1 methylation by SET7/9 has to be further elucidated. Finally, we observed that ARTD1-dependent histone modifications shift the substrate specificity of SET7/9 from H3 to H1. This crosstalk supports a role of poly(ADP-ribosyl)ation as an additional component of the histone code. Taken together, the identification of these two novel SET7/9 target proteins further underlines the important role of SET7/9 during the cellular stress response. Proteine verleihen Zellen ihre Struktur und Funktion und steuern alle biochemischen Prozesse. Viele Proteine werden posttranslational modifiziert, wodurch ihre Funktion und enzymatische Aktivität reguliert werden. Die ADP-Ribosylierung von Proteinen wird von ADP-Ribosyltransferasen katalysiert, welche ADP-Ribose von ihrem Coenzym NAD+ auf ihre Zielproteine übertragen. Die bekannteste ist ARTD1 (auch PARP1), ein nukleäres Enzym, welches durch DNA-Schäden aktiviert wird und Polymere aus ADP-Ribose auf sich selbst und andere Proteine überträgt. SET7/9 wurde ursprünglich als Monomethyltransferase für H3K4 entdeckt, aber methyliert auch Lysine von vielen Nicht-Histon-Proteinen wie p53 und DNMT1. Dies hat unterschiedliche Folgen für die Substrate, z. B. ihre Stabilisierung oder die Beeinflussung von anderen posttranslationalen Modifikationen. In der vorliegenden Arbeit wurden ARTD1 und das Linkerhiston H1 als neue Substrate für SET7/9 identifiziert und der Einfluss der Methylierung auf deren Funk- tionen in der Zelle untersucht. Wir fanden in vitro und in vivo, dass SET7/9 ARTD1 an K508 methyliert. Dies wurde durch vorherige Poly(ADP-Ribosyl)ierung von ARTD1 stark gehemmt, während die Methylierung selbst weder die Automodifikation von ARTD1 noch seine Acetylierung durch p300 verringerte. In Zellen führte SET7/9-Knockdown zu verringerter Synthese von Poly(ADP-Ribose) nach H2O2- Behandlung, während SET7/9-Überexpression den gegenteiligen Effekt hatte. Auch Mutation von K508 zu Arginin verringerte die Aktivität von ARTD1 unter bestimmten Bedingungen, was darauf hindeutet, dass die Methylierung durch SET7/9 die enzymatische Aktivität von ARTD1 direkt beeinflusst. Desweiteren haben wir gezeigt, dass SET7/9 mehrere Lysine (K121, K129, K159, K171, K177 and K192) im C-Terminus der Linkerhiston-Variante H1.4 methyliert. Alle sechs Lysine liegen am Ende der Konsensus-Sequenz KAKme und ihre Mutation verändert die Verteilung von H1.4 im Zellkern nicht. Die genaue Funktion dieser H1-Methylierung muss noch erforscht werden. Interessanterweise methyliert SET7/9 nach ARTD1-abhängigen Histonmodifikationen bevorzugt H1 statt H3, was die Rolle der Poly(ADP-Ribosyl)ierung als Teil des Histoncodes untermauert. Die Identifikation von zwei neuen SET7/9-Substraten in dieser Dissertation hebt die wichtige Rolle von SET7/9 in zellulären Stressreaktionen hervor

Crosstalk between SET7/9-dependent methylation and ARTD1-mediated ADP-ribosylation of histone H1.4

BACKGROUND: Different histone post-translational modifications (PTMs) fine-tune and integrate different cellular signaling pathways at the chromatin level. ADP-ribose modification of histones by cellular ADP-ribosyltransferases such as ARTD1 (PARP1) is one of the many elements of the histone code. All 5 histone proteins were described to be ADP-ribosylated in vitro and in vivo. However, the crosstalk between ADP-ribosylation and other modifications is little understood. RESULTS: In experiments with isolated histones, it was found that ADP-ribosylation of H3 by ARTD1 prevents H3 methylation by SET7/9. However, poly(ADP-ribosyl)ation (PARylation) of histone H3 surprisingly allowed subsequent methylation of H1 by SET7/9. Histone H1 was thus identified as a new target for SET7/9. The SET7/9 methylation sites in H1.4 were pinpointed to the last lysine residues of the six KAK motifs in the C-terminal domain (K121, K129, K159, K171, K177 and K192). Interestingly, H1 and the known SET7/9 target protein H3 competed with each other for SET7/9-dependent methylation. CONCLUSIONS: The results presented here identify H1.4 as a novel SET7/9 target protein, and document an intricate crosstalk between H3 and H1 methylation and PARylation, thus implying substrate competition as a regulatory mechanism. Thereby, these results underline the role of ADP-ribosylation as an element of the histone code

SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein-protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo. ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo. Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organspecific regulation of ubiquitin activation

Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function

Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor B (NF-B) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like modifiers (SUMOs). Here, we characterize PARP1 as a substrate for modification by SUMO1 and SUMO3, both in vitro and in vivo. PARP1 is sumoylated at the single lysine residue K486 within its automodification domain. Interestingly, modification of PARP1 with SUMO does not affect its ADP-ribosylation activity but completely abrogates p300-mediated acetylation of PARP1, revealing an intriguing crosstalk of sumoylation and acetylation on PARP1. Genetic complementation of PARP1-depleted cells with wildtype and sumoylation-deficient PARP1 revealed that SUMO modification of PARP1 restrains its transcriptional coactivator function and subsequently reduces gene expression of distinct PARP1-regulated target genes. Messner, S., Schuermann, D., Altmeyer, M., Kassner, I., Schmidt, D., Scha¨r, P., Mu¨ller, S., and Hottiger, M. O. Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function

Effect of Omega-3 Fatty Acid Supplementation on Oxylipins in a Routine Clinical Setting

Omega-6 polyunsaturated fatty acid (n-6 PUFA) is the predominant polyunsaturated fatty acid (PUFA), especially in Western diet. A high omega-6/omega-3 ratio in Western diets is implicated in the development of cardiovascular diseases and inflammatory processes. Studies in animal models and in humans have demonstrated beneficial effects of omega-3 PUFA (n-3 PUFA) in a variety of diseases, including cardiac arrhythmias and inflammatory diseases, as well as breast and colon cancer. The molecular mechanisms underlying the effects of n-3 PUFA are still not well understood. Possible mechanisms include competition between n-3 and n-6 PUFAs at the cyclooxygenase (COX) and lipoxygenase (LOX) and cytochrome P450 levels, and subsequent formation of oxylipins with specific anti-inflammatory or anti-arrhythmic effects. In this study, we report the impact of routine long-term treatment with prescription-grade n-3 PUFA (either 840 mg or 1680 mg per day) on blood cell membrane fatty acid composition, as well as plasma oxylipin patterns, in a patient population with severe hyperlipidemia and cardiovascular disease who are on standard lipid-lowering and cardioprotective medications. Lipidomics analyses were performed by LC/ESI-MS/MS. Supplementation led to a dose-dependent increase in n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the blood cell fraction. We also observed a dose-dependent increase in EPA- and DHA-derived epoxy metabolites, whereas the effect of n-3 PUFA supplementation on LOX-dependent EPA- and DHA-derived hydroxy metabolites was less pronounced, with a tendency towards lower metabolites in subjects with higher n-3 PUFA levels. These data thus generally confirm effects of n-3 PUFA supplementation observed previously in healthy individuals. Additionally, they indicate a suppressive effect of high n-3 PUFA supplementation on the formation of LOX metabolites in the context of concomitant aspirin medication

Inflammasome-activated caspase 7 cleaves PARP1 to enhance the expression of a subset of NF-κB target genes

Caspase 1 is part of the inflammasome, which is assembled upon pathogen recognition, while caspases 3 and/or 7 are mediators of apoptotic and nonapoptotic functions. PARP1 cleavage is a hallmark of apoptosis yet not essential, suggesting it has another physiological role. Here we show that after LPS stimulation, caspase 7 is activated by caspase 1, translocates to the nucleus, and cleaves PARP1 at the promoters of a subset of NF-κB target genes negatively regulated by PARP1. Mutating the PARP1 cleavage site D214 renders PARP1 uncleavable and inhibits PARP1 release from chromatin and chromatin decondensation, thereby restraining the expression of cleavage-dependent NF-κB target genes. These findings propose an apoptosis-independent regulatory role for caspase 7-mediated PARP1 cleavage in proinflammatory gene expression and provide insight into inflammasome signaling

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: Non-standard errors (NSEs). We study NSEs by letting 164 teams test the same hypotheses on the same data. NSEs turn out to be sizable, but smaller for better reproducible or higher rated research. Adding peer-review stages reduces NSEs. We further find that this type of uncertainty is underestimated by participants

Non-Standard Errors

In statistics, samples are drawn from a population in a data-generating process (DGP). Standard errors measure the uncertainty in sample estimates of population parameters. In science, evidence is generated to test hypotheses in an evidence-generating process (EGP). We claim that EGP variation across researchers adds uncertainty: non-standard errors. To study them, we let 164 teams test six hypotheses on the same sample. We find that non-standard errors are sizeable, on par with standard errors. Their size (i) co-varies only weakly with team merits, reproducibility, or peer rating, (ii) declines significantly after peer-feedback, and (iii) is underestimated by participants