Universiẗat, Diss., 2004--Freiburg (Breisgau).

Techn. Universiẗat, Diss., 2006--München.

Histone methyltransferases are key players in eukaryotic gene regulation. The goal of this thesis was to study the role of the histone methyltransferase Mll2 in developing embryos and adult mice. Targeting of mouse ES cells with a multipurpose allele and blastocyst injection had previously generated a mouse line allowing analysis of Mll2 function by knock-out and conditional mutagenesis using Cre/loxP. The first part of the thesis comprised the analysis of the Mll2-/- phenotype, and included the cloning of a targeting construct to generate an ubiquitous, ligand-regulated Cre line. In the second part, we did conditional mutagenesis using the Rosa26-CreER(T2) line obtained from collaborators, and achieved complete knock-out of Mll2 in most tissues of embryos, neonates and adult mice. Mll2 is essential during embryonic development, as mutant embryos were severely growth retarded, had significant increases in apoptosis, and failed in gestation between E 9.5 and E11. Conditional removal of Mll2 protein at gastrulation (E 6.5) produced a similar phenotype at E 11. In contrast, the absence of Mll2 function after E 11 did not result in obvious defects at E16 and indicates an essential role for Mll2 between E6 and E11. Indeed, we identified a loss of expression of 3 important developmental regulators in mutants of this developmental stage: Hoxb1, Mox1 and Six3 are candidate targets for Mll2 regulation that encode homeobox type transcription factors involved in specifying cellular identity. We observed correct establishment of their developmental expression patterns, which than decay in Mll2-/- mutants at E9.5. These data concord with and extend current thoughts about the fly orthologue of Mll2, Trithorax, which suggest that it acts as an epigenetic lock in chromatin to maintain expression of certain transcription factors key to respective cellular identities, after their expression patterns have been established. After birth, Mll2 is dispensable in most tissues, as conditional knock out in neonates and adult mice did not produce any pathological findings except infertility of mutant males and females. Histological analysis of testis revealed progressive loss of spermatogonia, associated with increases in apoptosis but without overt proliferation, meiotic or differentiation defects or loss of the supporting Sertoli cells. Consequently, in addition to its regulation of homeotic genes during development, Mll2 is required for the maintenance of various mitotic cell populations including ES cells, embryonal cells and germ cells.

Histone methyltransferases are key players in eukaryotic gene regulation. The goal of this thesis was to study the role of the histone methyltransferase Mll2 in developing embryos and adult mice. Targeting of mouse ES cells with a multipurpose allele and blastocyst injection had previously generated a mouse line allowing analysis of Mll2 function by knock-out and conditional mutagenesis using Cre/loxP. The first part of the thesis comprised the analysis of the Mll2-/- phenotype, and included the cloning of a targeting construct to generate an ubiquitous, ligand-regulated Cre line. In the second part, we did conditional mutagenesis using the Rosa26-CreER(T2) line obtained from collaborators, and achieved complete knock-out of Mll2 in most tissues of embryos, neonates and adult mice. Mll2 is essential during embryonic development, as mutant embryos were severely growth retarded, had significant increases in apoptosis, and failed in gestation between E 9.5 and E11. Conditional removal of Mll2 protein at gastrulation (E 6.5) produced a similar phenotype at E 11. In contrast, the absence of Mll2 function after E 11 did not result in obvious defects at E16 and indicates an essential role for Mll2 between E6 and E11. Indeed, we identified a loss of expression of 3 important developmental regulators in mutants of this developmental stage: Hoxb1, Mox1 and Six3 are candidate targets for Mll2 regulation that encode homeobox type transcription factors involved in specifying cellular identity. We observed correct establishment of their developmental expression patterns, which than decay in Mll2-/- mutants at E9.5. These data concord with and extend current thoughts about the fly orthologue of Mll2, Trithorax, which suggest that it acts as an epigenetic lock in chromatin to maintain expression of certain transcription factors key to respective cellular identities, after their expression patterns have been established. After birth, Mll2 is dispensable in most tissues, as conditional knock out in neonates and adult mice did not produce any pathological findings except infertility of mutant males and females. Histological analysis of testis revealed progressive loss of spermatogonia, associated with increases in apoptosis but without overt proliferation, meiotic or differentiation defects or loss of the supporting Sertoli cells. Consequently, in addition to its regulation of homeotic genes during development, Mll2 is required for the maintenance of various mitotic cell populations including ES cells, embryonal cells and germ cells.

Die Verknüpfung einer exergonischen mit einer endergonischen Reaktion zur Ermöglichung der letzteren ist eine in biologischen Systemen weit verbreitete Strategie. Energetisch benachteiligte Elektronenübertragungsreaktionen im Rahmen der reduktiven Aktivierung von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen, der zu diesen verwandten Benzoyl-CoA-Reduktasen und diversen Cobalamin-abhängigen Methyltransferasen sind gekoppelt an die Hydrolyse von ATP. Der Methylgruppentransfer des reduktiven Acetyl-CoA-Weges von Carboxydothermus hydrogenoformans erfordert den Co(I)-Zustand des Corrinoid/Eisen-Schwefel Proteins (CoFeSP). Um diese superreduzierte Form nach einer oxidativen Inaktivierung zu regenerieren ist ein „Reparaturmechanismus“ erforderlich. Ein offenes Leseraster (orf7), welches möglicherweise für eine reduktive Aktivase von Corrinoid Enzymen (RACE) kodiert, wurde in dem Gencluster der am reduktiven Acetyl-CoA-Weg beteiligten Proteine entdeckt. Im Rahmen dieser Arbeit wurde dieses potenzielle RACE Protein biochemisch und strukturell charakterisiert und die ATP-abhängige reduktive Aktivierung von CoFeSP untersucht. Auf Grundlage der in dieser Arbeit gewonnenen Ergebnisse wurde ein Mechanismus für die ATP-abhängige Aktivierung entworfen. Dieser gibt Einblicke wie die durch ATP-Hydrolyse bereitgestellte Energie einen energetisch ungünstigen Elektronentransfer ermöglichen kann. Hierzu kombiniert RACo das Ausgleichen von Bindungsenergien mit Modulationen am Elektronenakzeptor. Eine vergleichbare Strategie wurde bisher in keinem anderen ATP-abhängigen Elektronenübertragungssystem wie dem von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen oder Benzoyl-CoA-Reduktasen beobachtet und könnte ein für RACE Proteine allgemein gültige Eigenschaft darstellen. / The principle of coupling an exergonic to an endergonic reaction to enable the latter is a widespread strategy in biological systems. Unfavoured electron transfer reactions in the reductive activation of nitrogenases, radical-dependent β,α-dehydratases and the related benzoyl- CoA reductases, as well as different cobalamin-dependent methyltransferases are coupled to the hydrolysis of ATP. The reductive acetyl-CoA pathway of Carboxydothermus hydrogenoformans relies on the superreduced Co(I)-state of the corrinoid/iron-sulfur protein (CoFeSP) that requires a “repair mechanism” in case of incidental oxidation. An open reading frame (orf7) coding for a putative reductive activase of corrinoid enzymes (RACE) was discovered in the gene cluster of proteins involved in the reductive acetyl-CoA pathway. In this work, this putative RACE protein was biochemically and structurally characterised and the ATP-dependent reductive activation of CoFeSP was investigated. Based on the results of this study, a mechanism for the ATP-dependent reactivation of CoFeSP was deduced providing insights into how the energy provided by ATP could trigger this unfavourable electron transfer. The reductive activator of CoFeSP combines balance of binding energies and modulations of the electron acceptor to promote the uphill electron transfer to CoFeSP. A comparable strategy has not been observed in other ATP-dependent electron transfer systems like nitrogenases, radical-dependent β,α-dehydratases and benzoyl- CoA reductases and could be a universal feature of RACE proteins.