No description available.

Transcription elongation regulator 1 (TCERG1) has previously been demonstrated to be an inhibitor of the transactivation and growth arrest activities of CCAAT/Enhancer binding protein alpha (C/EBPα). Furthermore, TCERG1 had been demonstrated to become relocalized from nuclear speckles to the pericentromeric regions where C/EBPα resides when both proteins are co-expressed in the cell. This thesis demonstrates that the deletion of a unique, imperfect series of 38 glutamine-alanine (QA) repeats near the amino terminus of TCERG1 is able to abrogate the ability of TCERG1 to inhibit C/EBPα-mediated growth arrest, the physical interaction between TCERG1 and C/EBPα, and the relocalization of TCERG1 from nuclear speckles when C/EBPα is co-expressed in the cell. The deletion of the QA domain demonstrated that there was a threshold amount of QA repeats required in TCERG1 for the relocalization and growth arrest inhibitory activities between TCERG1 and C/EBPα. It was demonstrated that between 11 and 20 QA repeats were required in TCERG1 to produce the relocalization from nuclear speckles or to be able to inhibit C/EBPα-mediated growth arrest. The physical interaction of TCERG1 and C/EBPα as examined by co-immunoprecipitation was also found to be QA dependent, with a diminishing interaction observed as the number of QA repeats in TCERG1 were reduced. However, experiments examining the isolated QA domain indicated that it was insufficient to relocalize an mCherry fluorescent protein fusion to either the nucleus or to pericentromeric regions where C/EBPα is concentrated. This inability to produce relocalization suggests that the QA domain requires another domain or domains from TCERG1 to mediate the relocalization activity. When expressed with the WT TCERG1, ΔQA TCERG1 was able to act in a dominant negative manner, preventing the relocalization of the WT TCERG1 protein to pericentromeric domains. Interestingly, the transactivation inhibitory activities of TCERG1 on C/EBPα do not appear to require the QA domain, but rather are localized to the carboxy half of TCERG1, somewhere within amino acids 612-1098. The data obtained provides the first report of a role for this unique QA repeat domain.

We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...

CCAAT/Enhancer Binding Protein Alpha (C/EBPa) is transcription factor protein involved in the differentiation of many cell types, including granulocytes and pulmonary cells. Studies have found that downregulation of C/EBPa leads to tumor formation in the hematopoietic system, bones, lungs, liver, and other organs. Mutations and post translational modifications can also reduce the function of C/EBPa in humans, leading to cancers. Recent studies have made progress in treating acute promyelocytic leukemia (APL), an M3 subtype of acute myeloid leukemia (AML), by incorporating all trans retinoic acid (ATRA) in targeted treatments. ATRA increases C/EBPa expression levels, thus promoting cell differentiation and subsequent apoptosis of leukemia cells. Still, survival rates of AML patients are low. In patients diagnosed with AML subtypes M4 or higher, ATRA does not work. In addition, patients can become resistant to ATRA, making it essential to find an alternative therapy. Therefore, novel drug treatments are necessary. Through a high throughput screening method, we have determined a potent chemical compound, ICCB280, that can enhance C/EBPa expression levels. However, ICCB280’s effective concentration for cell differentiation is relatively high, so we performed a structural-activity relationship (SAR) analysis and discovered a more potent chemical, styryl quinazolinone CCAAT/Enhancer Binding Protein Compound 73 (CEBP- 73). We tested CEBP-73 with two cell lines, HL-60 and A549, which represent leukemia and lung cancer models, respectively. We found that CEBP-73 increased C/EBPa expression levels in a time-dependent, dose-responsive manner in both leukemia cells and lung cancer cells. In western blot analyses, while both ICCB280 and CEBP-73 upregulated C/EBPa protein expression, more protein was expressed in leukemia and lung cancer cells treated with CEBP-73 in a dose-dependent manner than in cells treated with ICCB280. Next, we investigated CEBP-73’s effectiveness in upregulating C/EBPa’s downstream genes. We observed enhanced expression of CEBPe (HL-60 specific downstream gene) in HL-60 cells, and enhanced SPC, NKX2-1 (codes for TTF-1), and HIF-1a (A549 specific downstream genes) expression levels in A549 cells. To investigate the mechanisms of increased C/EBPa expression, we asked whether expression of an extra coding CEBPA (ecCEBPA), a noncoding RNA for C/EBPa that prevents methylation at the CEBPA gene promoter site, will increase. We found that CEBP-73 increased not only C/EBPa expression, but also ecCEBPA in HL-60 and A549 cells. This is the first study to our knowledge that confirms styryl quinazolinone CEBP- 73 can increase ecCEBPA expression. To examine the effectiveness of CEBP-73 in vivo, EGFR-L858R-T790M (EGFRTL/CCSP-rtTA) mice were administered a vehicle solution (control), 1 mg/kg of CEBP-73, or 10 mg/kg CEBP-73. The results showed a trend in CEBP-73 concentrations; higher doses of CEBP-73 induce higher levels of C/EBPa expression in lung tissue. Fewer and smaller tumors were present in lungs treated with CEBP-73 than lungs treated with a control. These findings support the role of CEBP-73 in enhancing C/EBPa expression, including upregulation at the promoter region of the CEBPA gene and at downstream gene loci. In addition, the study’s results affirm the role of C/EBPa as an inducer of cell differentiation in leukemia and lung cancer by showing neutrophils with segmented lobes and granules, indications of cell maturity, in cells treated with compounds that enhanced C/EBPa expression. These data suggest that CEBP-73 could provide novel therapeutic approaches in treating leukemia and lung cancer and could potentially be modified to treat other cancers in targeted drug therapies.

We did not find mutation in coding areas of genes for components of TGFbeta1 signaling pathway but we detected decreased or undetectable expression of these analysed genes.The decreased expression is probably caused by epigenetic changes, so by hypermethylation and deacetylation of promoter regionsof these genes.Antiproliferative and apoptotic effect of TGF1 was analysed in AML cell lines (ML1, ML2, CTV1 and Kasumi1). ML2 cells rezistence to inhibition of DNA synthesis by TGFβ1 is not caused by mutations of genes for components of TGFβ1 signaling pathway. We found that increased SnoN (Ski-like novel gene) expression on the level of coresponding mRNA and protein is probably accountable for this rezistence. Kasumi1 and M2 cells were sensitive to induction of apoptózis caused by TGFβ1 treatment but in less extent than by proteazome inhibitor bortezomib. The difference of AML cells of different lines answers shows a great heterogeneity AML in AML patients. Prognostic factors analysis in AML with normal karyotype confirmed that CEBPA (CCAAT/enhancer binding protein alpha) mutations predict favourable prognosis but the elevated EVI1 ("Ecotropic Virus Integration Site 1") and ERG ("ETS-related gene") expression are connected with unfavourable prognosis. EVI1 is a negative marker for MDS as well. We did not confirm...

La leucémie aiguë myéloïde est une hémopathie maligne génétiquement hétérogène caractérisée par de fréquents réarrangements impliquant la bande chromosomique 21q22 et le gène RUNX1. Dans ce groupe d’anomalies, les translocations t(8;21)(q22;q22) et t(3;21)(q26;q22), associées respectivement à un pronostic favorable et défavorable, sont les mieux étudiées. Or, plus de la moitié des réarrangements ciblant RUNX1 ne sont toujours pas caractérisés au niveau clinique et moléculaire. Les principaux objectifs de cette thèse sont de caractériser quatre nouvelles translocations ciblant RUNX1 et d’étudier la dérégulation transcriptionnelle associée à ces anomalies au niveau de cibles plus spécifiques ayant un rôle dans l’auto-renouvellement ou dans la différenciation hématopoïétique. À l’aide des techniques de cytogénétique et de biologie moléculaire, deux nouveaux partenaires de RUNX1, soit CLCA2 et SV2B, ont été identifiés au sein des t(1;21)(p22.3;q22) et t(15;21)(q26.1;q22) et la récurrence des partenaires USP42 et TRPS1 a été démontrée suite à l’étude des t(7;21)(p22.1;q22) et t(8;21)(q23.3;q22). Ce travail a permis de confirmer l’existence de divers modes de dérégulation de RUNX1 dans les leucémies aiguës. L’expression présumée de protéines chimériques et/ou d’isoformes tronquées de RUNX1, un dosage aberrant des transcrits de RUNX1 et la surexpression des gènes partenaires sont des conséquences révélées par l’étude de ces fusions. Le séquençage et l’analyse des jonctions génomiques des fusions récurrentes RUNX1-USP42/USP42-RUNX1 et RUNX1-TRPS1/TRPS1-RUNX1 ont démontré la présence de signatures moléculaires caractéristiques du mode de recombinaison non-homologue de type NHEJ. En raison de la structure et de la composition différente des jonctions, l’implication de composantes distinctes du mécanisme NHEJ a été proposée. Enfin, des analyses par PCR quantitative en temps réel nous ont permis de démontrer l’existence de cibles de dérégulation partagées par les fusions récurrentes et plus rares de RUNX1. Nous avons démontré que CEBPA est moins exprimé dans la majorité des spécimens étudiés présentant une fusion de RUNX1 par rapport aux spécimens avec un caryotype normal alors que JUP, une composante effectrice de la voie Wnt, est plutôt surexprimé. Malgré l’activation transcriptionnelle de JUP dans l’ensemble de ces spécimens, certaines cibles de la voie Wnt telles que CCND1 et MYC sont différemment exprimées dans ces cellules, appuyant l’hétérogénéité décrite dans ce groupe de leucémies. Malgré l’implication de partenaires variés, nos données d’expression démontrent que les chimères et les protéines tronquées de RUNX1 partagent des cibles communes d’activation et de répression transcriptionnelle et établissent, pour la première fois, des évidences moléculaires suggérant l’existence de similitudes entre la fusion récurrente RUNX1-RUNX1T1 et quatre fusions plus rares de RUNX1. Puisque des rechutes surviennent fréquemment dans ce groupe génétique, l’inhibition de JUP pourrait être une option thérapeutique intéressante et ceci est appuyé par les bénéfices observés lors de l’inhibition de la voie Wnt dans d’autres groupes génétiques de leucémies aiguës. / Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by frequent rearrangements of the RUNX1 gene located at chromosomal band 21q22. In this subtype of leukemias, t(8;21)(q22;q22) and t(3;21)(q26;q22) translocations are among the most studied rearrangements, being respectively associated with a favourable and poor prognosis. However, approximately half of RUNX1 translocations remain uncharacterized at the clinical and molecular levels at the present time. The main objectives of this thesis are to characterize four novel RUNX1 translocations in adult patients with acute leukemias and to study the expression profiles of specific transcriptional targets of RUNX1 fusions involved in self-renewal or differentiation of hematopoietic cells. Using molecular techniques, we identified CLCA2 and SV2B genes as novel fusion partners of RUNX1 in t(1;21)(p22;q22) and t(15;21)(q26;q22) translocations. We also described the recurrence of the USP42 and TRPS1 genes involved in t(7;21)(p22;q22) and t(8;21)(q23.3;q22) translocations. Chimeric fusion proteins, truncated isoforms of RUNX1, alteration of RUNX1 transcripts expression and overexpression of the fusion partner were possible outcomes of these various fusions, thus demonstrating the diversity of RUNX1 alterations in acute leukemias. Genomic breakpoints of the recurrent RUNX1-UPS42/USP42-RUNX1 and RUNX1-TRPS1/TRPS1-RUNX1 fusions were cloned and analyzed revealing typical signatures of the non-homologous end joining recombination mechanism at fusion junctions. Since variation in the structure and composition of these junctions was observed, we proposed that distinct cellular machineries would be involved in the genesis of these abnormalities. Quantitative real-time PCR was performed on primary leukemic cells expressing these rare RUNX1 fusions. We demonstrated, for the first time, that similar downregulation of CEBPA and upregulation of JUP, an effector of the Wnt pathway, are detected in most samples studied presenting either recurrent or rare RUNX1 fusions. Despite an overexpression of JUP detected in each RUNX1 positive sample studied, other targets of the Wnt pathway like CCND1 and MYC genes were differently expressed in these cells, thus confirming the heterogeneity of this group of leukemias. Our expression data show that similar transcriptional targets, activated or repressed, are detected in cells expressing either chimeric or truncated RUNX1 proteins and establish the first molecular evidences suggesting that the recurrent RUNX1-RUNX1T1 and four rare RUNX1 fusions share common molecular deregulations. As relapse frequently occurs in RUNX1 positive leukemias, JUP overexpression could be of particular interest with regard to targeted-therapy, as demonstrated by previous work showing potential benefits of inhibiting the Wnt pathway in other genetic groups of acute leukemias.

La leucémie aiguë myéloïde est une hémopathie maligne génétiquement hétérogène caractérisée par de fréquents réarrangements impliquant la bande chromosomique 21q22 et le gène RUNX1. Dans ce groupe d’anomalies, les translocations t(8;21)(q22;q22) et t(3;21)(q26;q22), associées respectivement à un pronostic favorable et défavorable, sont les mieux étudiées. Or, plus de la moitié des réarrangements ciblant RUNX1 ne sont toujours pas caractérisés au niveau clinique et moléculaire. Les principaux objectifs de cette thèse sont de caractériser quatre nouvelles translocations ciblant RUNX1 et d’étudier la dérégulation transcriptionnelle associée à ces anomalies au niveau de cibles plus spécifiques ayant un rôle dans l’auto-renouvellement ou dans la différenciation hématopoïétique. À l’aide des techniques de cytogénétique et de biologie moléculaire, deux nouveaux partenaires de RUNX1, soit CLCA2 et SV2B, ont été identifiés au sein des t(1;21)(p22.3;q22) et t(15;21)(q26.1;q22) et la récurrence des partenaires USP42 et TRPS1 a été démontrée suite à l’étude des t(7;21)(p22.1;q22) et t(8;21)(q23.3;q22). Ce travail a permis de confirmer l’existence de divers modes de dérégulation de RUNX1 dans les leucémies aiguës. L’expression présumée de protéines chimériques et/ou d’isoformes tronquées de RUNX1, un dosage aberrant des transcrits de RUNX1 et la surexpression des gènes partenaires sont des conséquences révélées par l’étude de ces fusions. Le séquençage et l’analyse des jonctions génomiques des fusions récurrentes RUNX1-USP42/USP42-RUNX1 et RUNX1-TRPS1/TRPS1-RUNX1 ont démontré la présence de signatures moléculaires caractéristiques du mode de recombinaison non-homologue de type NHEJ. En raison de la structure et de la composition différente des jonctions, l’implication de composantes distinctes du mécanisme NHEJ a été proposée. Enfin, des analyses par PCR quantitative en temps réel nous ont permis de démontrer l’existence de cibles de dérégulation partagées par les fusions récurrentes et plus rares de RUNX1. Nous avons démontré que CEBPA est moins exprimé dans la majorité des spécimens étudiés présentant une fusion de RUNX1 par rapport aux spécimens avec un caryotype normal alors que JUP, une composante effectrice de la voie Wnt, est plutôt surexprimé. Malgré l’activation transcriptionnelle de JUP dans l’ensemble de ces spécimens, certaines cibles de la voie Wnt telles que CCND1 et MYC sont différemment exprimées dans ces cellules, appuyant l’hétérogénéité décrite dans ce groupe de leucémies. Malgré l’implication de partenaires variés, nos données d’expression démontrent que les chimères et les protéines tronquées de RUNX1 partagent des cibles communes d’activation et de répression transcriptionnelle et établissent, pour la première fois, des évidences moléculaires suggérant l’existence de similitudes entre la fusion récurrente RUNX1-RUNX1T1 et quatre fusions plus rares de RUNX1. Puisque des rechutes surviennent fréquemment dans ce groupe génétique, l’inhibition de JUP pourrait être une option thérapeutique intéressante et ceci est appuyé par les bénéfices observés lors de l’inhibition de la voie Wnt dans d’autres groupes génétiques de leucémies aiguës. / Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by frequent rearrangements of the RUNX1 gene located at chromosomal band 21q22. In this subtype of leukemias, t(8;21)(q22;q22) and t(3;21)(q26;q22) translocations are among the most studied rearrangements, being respectively associated with a favourable and poor prognosis. However, approximately half of RUNX1 translocations remain uncharacterized at the clinical and molecular levels at the present time. The main objectives of this thesis are to characterize four novel RUNX1 translocations in adult patients with acute leukemias and to study the expression profiles of specific transcriptional targets of RUNX1 fusions involved in self-renewal or differentiation of hematopoietic cells. Using molecular techniques, we identified CLCA2 and SV2B genes as novel fusion partners of RUNX1 in t(1;21)(p22;q22) and t(15;21)(q26;q22) translocations. We also described the recurrence of the USP42 and TRPS1 genes involved in t(7;21)(p22;q22) and t(8;21)(q23.3;q22) translocations. Chimeric fusion proteins, truncated isoforms of RUNX1, alteration of RUNX1 transcripts expression and overexpression of the fusion partner were possible outcomes of these various fusions, thus demonstrating the diversity of RUNX1 alterations in acute leukemias. Genomic breakpoints of the recurrent RUNX1-UPS42/USP42-RUNX1 and RUNX1-TRPS1/TRPS1-RUNX1 fusions were cloned and analyzed revealing typical signatures of the non-homologous end joining recombination mechanism at fusion junctions. Since variation in the structure and composition of these junctions was observed, we proposed that distinct cellular machineries would be involved in the genesis of these abnormalities. Quantitative real-time PCR was performed on primary leukemic cells expressing these rare RUNX1 fusions. We demonstrated, for the first time, that similar downregulation of CEBPA and upregulation of JUP, an effector of the Wnt pathway, are detected in most samples studied presenting either recurrent or rare RUNX1 fusions. Despite an overexpression of JUP detected in each RUNX1 positive sample studied, other targets of the Wnt pathway like CCND1 and MYC genes were differently expressed in these cells, thus confirming the heterogeneity of this group of leukemias. Our expression data show that similar transcriptional targets, activated or repressed, are detected in cells expressing either chimeric or truncated RUNX1 proteins and establish the first molecular evidences suggesting that the recurrent RUNX1-RUNX1T1 and four rare RUNX1 fusions share common molecular deregulations. As relapse frequently occurs in RUNX1 positive leukemias, JUP overexpression could be of particular interest with regard to targeted-therapy, as demonstrated by previous work showing potential benefits of inhibiting the Wnt pathway in other genetic groups of acute leukemias.

Der Transkriptionsfaktor CEBPA reguliert Differenzierung und Proliferation in verschiedenen Zelltypen und spielt eine herausragende Rolle in der Hämatopoese. Die CEBPA RNA kann in die lange P42-Isoform oder die N-terminal verkürzte P30-Isoform translatiert werden. Während P42-CEBPA differenzierungsinduzierend wirkt, ist P30 als Inhibitor von P42 und als Onkogen in akuter myeloider Leukämie beschrieben. Die Modularität und Multifunktionalität von CEBPA, die ihn zahlreichen Studien beobachtet wurde, lässt sich möglicherweise durch differentielle Protein–Protein-Interaktionen erklären. Zahlreiche post-translationale Modifikationen (PTMs) und die intrinsisch ungeordnete, flexible Struktur von CEBPA stellen jedoch eine Herausforderung für traditionelle Ansätze in Proteininteraktionsstudien dar. In der vorliegenden Arbeit wird ein neuer, alternativer Ansatz präsentiert, der auf einem in vitro Proteininteraktions-screen auf einer Peptidmatrix (PRISMA) und Biotinligase proximity labelling (BioID) in lebenden Zellen basiert. In einem PRISMA-screen wurden 120 CEBPA Peptide auf Proteininteraktionen mit Proteinextrakt aus myeloiden Zellen untersucht. Im Screen wurden 40 verschiedene CEBPA PTMs inkludiert, unter anderem auch die hier erstmals neu beschriebenen Methylierungen der CEBPA Argininreste R12 und R142. Daten aus dem PRISMA-screen wurden mit BioID Experimenten in myeloiden Zellen validiert, um eine Proteininteraktionslandkarte von CEBPA zu generieren, die 52 bekannte und 68 neue CEBPA Proteininteraktoren umfasst. Hotspots für Proteininteraktionen fallen in evolutionär konservierte CEBPA Regionen und der Vergleich des Bindungsprofils mit publizierten Daten zeigt Ähnlichkeiten zu verwandten Transkriptionsfaktoren der CEBP Familie. Die Ergebnisse legen nahe, dass die Multifunktionalität von CEBPA von multivalenten Proteininteraktionen in Abhängigkeit von PTMs koordiniert wird, um CEBPA mit dem epigenetischen und transkriptionellen Apparat der Zelle verknüpfen. / The pioneering transcription factor CEBPA plays a lineage-instructing role during haematopoiesis and also regulates proliferation and differentiation in many other cell types. The CEBPA RNA can be translated into a full length (P42-CEBPA) or N-terminally truncated isoform (P30-CEBPA). While P42 induces differentiation in various cell types, the P30 isoform is mostly regarded as a dominant inhibitor of P42-CEBPA and acts as an oncogene in acute myeloid leukaemia. Protein interactions may be the key to explaining the functional plasticity and modularity of CEBPA that has been demonstrated in diverse experimental settings. However, the disordered structure and the numerous post-translational modification sites (PTMs) of CEBPA pose a challenge to traditional protein interaction studies. In the present work, a novel alternative approach is presented that combines an in vitro protein interaction screen on a peptide matrix (PRISMA) with biotin ligase proximity labelling (BioID) in living cells. To this end, 120 CEBPA peptides were probed for protein interactions with PRISMA. The screen comprised 40 different PTMs, including newly identified CEBPA arginine methylation sites. PRISMA data was validated with BioID experiments and generated a detailed CEBPA protein interaction map in myeloid cells. The interactome presented here contains 52 known and 68 novel CEBPA interactors that can now be mapped across the CEBPA sequence in a PTM dependent fashion. Hotspots of protein interaction correlated with conserved regions and comparison with previously published data revealed related binding profiles of homologous CEBP regions. Taken together, the data indicates that the functional plasticity of CEBPs is orchestrated by multivalent protein interactions and PTMs to configure a dynamic CEBP hub that interacts with many partners of the transcriptional and epigenetic machinery.

La calidad de la carne está determinada por cualidades como el marmoleo, el sabor, la terneza y la composición, entre otras. Estas cualidades están reguladas a distintos niveles, y uno de ellos es la genética. Hoy en día se conoce buena parte de las vías metabólicas que regulan estas características, y se han propuesto "genes candidatos" que codifican factores importantes dentro de estas vías. Los genes PPARG, CEBPA, FABP4, LIPE y RXRA son parte de las vías de diferenciación adipocítica y del metabolismo lipídico. El objetivo de este proyecto fue caracterizar la variabilidad genética en estos genes en razas bovinas con diferente calidad carnicera. Los datos se obtuvieron por medio de técnicas moleculares (reacción en cadena de la polimerasa, re-secuenciación) aplicadas a muestras de ADN extraídas de animales pertenecientes a diferentes razas criadas alrededor del mundo. Luego se realizaron una serie de análisis a través de programas bioinformáticos y herramientas web. Algunos de los polimorfismos detectados en los genes y otros disponibles en las bases de datos de internet fueron seleccionados para realizar estudios de validación a nivel poblacional y análisis estadísticos de asociación a caracteres de calidad carnicera en una población de ganado local. Los resultados fueron diversos: PPARG y CEBPA presentaron una variabilidad moderada, y FABP4 y LIPE presentaron una variabilidad alta. Algunos de los polimorfismos analizados sugieren una asociación a la composición lipídica de la carne y otros caracteres de engrasamiento, como espesor de grasa dorsal. Algunas de las posibles explicaciones biológicas para estas asociaciones fueron analizadas con diferentes herramientas bioinformáticas y se observaron algunos fenómenos interesantes. El conocimiento de la variabilidad existente en estos genes es de importancia para complementar los métodos de selección genética tradicionales y mejorar la calidad del ganado.

Die myeloische Entwicklung wird durch die Familie der Transkriptionsfaktoren CCAAT/Enhancer-Binding-Protein (C/EBP) reguliert. Eine aberrante Expression oder Funktion von C/EBPs stört die normale myeloische Differenzierung und wird bei vielen Arten hämatopoetischer Malignome beobachtet. Mutationen von CEBPA führen zu einem veränderten Expressionsanteil der verkürzten Isoform C/EBPa p30 und werden bei etwa 15% der AML-Patienten (akute myeloische Leukämie) nachgewiesen. Obwohl die verkürzte Isoform C/EBPα p30 als Onkogen identifiziert wurde da sie die Proliferation myeloischer Vorläufer fördert, behält sie dennoch eine Differenzierungsfunktion. Unser Interesse gilt der Frage, wie diese beiden Funktionen von C/EBPα p30 reguliert werden. Die C/EBP-Familie gehört der Gruppe intrinsisch ungeordneter Proteine an, die zudem viele posttranslationale Modifikationen (PTMs) aufweisen. PTMs auf C/EBPα verändern seine biologische Funktionsweise stark. Frühere Forschungsarbeiten haben drei Argininreste am N-Terminus von C/EBPα p30 identifiziert, die aufgrund des Methylierungsstatus differentiell mit anderen Proteinen interagieren. In dieser Arbeit untersuchen wir den Einfluss der C/EBPα p30 Arginin-Methylierung auf seine pro-leukämische Aktivität sowie dessen Fähigkeit zur Neuausrichtung der hämatopoietischen Differenzierungslinie. Mit Hilfe von Aminosäuresubstitutionen fanden wir heraus, dass C/EBPα p30 Mutanten der Methylierungsmimesis oder Ladungsabschaffung die myeloische Differenzierung verstärkt, während Ladungserhalt-Mutanten die Erneuerung und Proliferation hämatopoetischer Stamm-/Vorläuferzellen unterstützt. Transkriptionelles Profiling von Zellen, die mutierte C/EBPα -p30-Varianten exprimieren, deutet auf potenzielle Ziele der methyliertem bzw. unmethyliertem C/EBPα p30 hin. Die Ergebnisse legen nahe, dass der Arginin-Methylierungsstatus das Leukämie- und Differenzierungs-Potenzial von C/EBPα p30 verändert und somit ein neues Ziel der Leukämietherapie darstellen könnten. / Myeloid development is regulated by the family of transcription factors CCAAT/enhancer-binding-protein (C/EBP). Aberrant expression or functioning of C/EBPs disturbs normal myeloid differentiation and is found in many types of hematopoietic malignancies. Mutations of CEBPA lead to imbalanced expression of the truncated isoform C/EBPα p30 and are found in approximately 15% of AML (acute myeloid leukemia) patients. Yet, how C/EBPα participates in leukemic progression remains to be discovered. More specifically, the truncated isoform C/EBPα p30, although being identified as an oncogenic isoform that promotes proliferation of myeloid progenitors, still retains differentiation function. The question of how both functions of C/EBPα p30 are regulated, is of our interest. C/EBP family also represents a group of intrinsically disordered proteins, which contain many post-translational modifications (PTMs). PTMs on C/EBPα greatly alter its functioning. Previous works have identified three arginine residues at the N-terminus of C/EBPα p30 that interact differently with others protein dependent on their methylation status. We hypothesize, that methylation of these arginine residues plays important roles in the biology of C/EBPα p30. In this study, we used a lymphoid-to-myeloid transdifferentiation (LMT) system to investigate the influence of arginine-methylation on C/EBPα-induced lineage switch and its pro-leukemic activity. Using amino acid substitution, we found that C/EBPα p30 mutants that resemble arginine-methylated p30 enhanced myeloid differentiation, while the charge-retention mutant, resembling arginine-unmethylated p30, supported renewability and proliferation of hematopoietic progenitors. Transcriptional profiling of cells expressing C/EBPα p30 variants suggested potential targets of either methylated or unmethylated p30. The results implied that arginine methylations alter C/EBPα p30’s leukemic potential and might comprise novel targets of leukemia therapy.