Sepsis-induced myeloid-derived suppressor cells (MDSCs) contribute to immunosuppression associated with sepsis. We reported that the CCAAT enhancer-binding protein C/EBPβ activates microRNA (miR)-21 and miR-181b expressions, which induce transcription factor NFI-A to support the generation and expansion of MDSCs in the bone marrow and spleens of septic mice. Here, using a conditional knockout mouse model lacking C/EBPβ in the myeloid lineage, we find that without C/EBPβ, myeloid progenitor cells could not express miR-21 or miR-181b, and ectopic expression of C/EBPβ in the C/EBPβ-deficient myeloid progenitors activated the expression of the two miRNAs. Moreover, C/EBPβ-reconstituted myeloid cells expressed IL-10 and reduced T cell proliferation and function, similar to control MDSCs that express C/EBPβ. Exogenous expression of miR-21 and miR-181b in the C/EBPβ-deficient myeloid progenitors from septic mice produced similar results. Notably, NFI-A-dependent transactivation of NF-kB MDSC generating pathway was reversed in the C/EBPβ-deficient myeloid progenitors from septic mice. Together, these results support that decreasing C/EBPβ expression prevents MDSC generation and decreases immunosuppression in septic mice, providing a target for sepsis treatment.

Der CCAAT enhancer binding protein beta (C/EBPβ) Transkriptionsfaktor reguliert die Differenzierung, Proliferation und Funktion vieler Zelltypen, einschließlich verschiedener Zellen des Immunsystems. Eine detaillierte molekulare Analyse des Mechanismus, wie C/EBPβ alternative Zellschicksale steuert, wurde jedoch bisher noch nicht unternommen. Es wurde gezeigt, dass die ektopische Expression von C/EBPβ in determinierten B- Vorläuferzellen diese zu inflammatorischen Makrophagen reprogrammieren kann. Wir haben dieses Reprogrammierungsystem verwendet, um die Strukturelemente in C/EBPβ, die für die Regulation der (Trans)Differenzierung durch C/EBPβ wichtig sind, zu untersuchen. Um die maßgeblichen C/EBPβ Proteinmodule für die Reprogrammierung zu bestimmen, wurden entweder C/EBPβ Wildtyp Isoformen oder Mutanten in primären murinen B-Vorläuferzellen ektopisch exprimiert. Die Analysen ergaben, dass die translational regulierten langen Isoformen LAP* and LAP, jedoch nicht die kurze Isoform LIP lymphoide Zellen zu myeloischen Zellen reprogrammieren können. Des weiteren haben wir gezeigt, dass die konservierten Regionen 2, 3 und 4 der C/EBPβ Transaktivierungsdomäne essentiell und ausreichend für die Konvertierung von B Zellen zu myeloischen Zellen sind. Die reprogrammierten myeloischen Zellen setzten sich aus einer heterogenen Population verschiedener myeloischer Zelltypen zusammen. Detaillierte Analysen von CD11b+ reprogrammierten Zellen zeigten, dass diskrete konservierte Regionen von C/EBPβ verschiedene pro- und anti-inflammatorische Gene und divergente Entwicklungsprogramme aktivierten. Des Weiteren führten nicht nur strukturelle C/EBPβ Mutanten sondern auch Puktmutationen an Stellen, die posttranslationalen Modifikationen (PTM) unterliegen, zu verschiedenen Reprogrammierungsergebnissen. Diese Daten zeigen, dass die C/EBPβ abhängige myeloische Diversifikation durch die Integration von strukturellen C/EBPβ Proteinmodulen und deren signalabhängigen PTMs erreicht wird. / The CCAAT enhancer binding protein beta (C/EBPβ) transcription factor regulates differentiation, proliferation, and functionality of many cell types, including various cells of the immune system. A detailed molecular understanding of how C/EBPβ directs alternative cell fates remains largely elusive. Ectopic expression of C/EBPβ has been previously shown to reprogram committed B cell progenitors into inflammatory macrophages. We took advantage of this reprogramming system in order to examine how C/EBPβ regulates (trans)differentiation. To determine which C/EBPβ protein modules are important for reprogramming, C/EBPβ wild type isoforms and mutants were ectopically expressed in primary mouse B cell progenitors. The data showed that the translationally regulated long isoforms LAP* and LAP, but not the N-terminally truncated isoform LIP can reprogram lymphoid cells into myeloid cells. Furthermore, we found that conserved regions 2,3 and 4 in the C/EBPβ protein transactivation domain are necessary and sufficient for B-to-myeloid cell conversion. Interestingly, the reprogrammed myeloid cells were found to represent a heterogeneous mixture of different myeloid cell types. Detailed analyses of the reprogrammed CD11b+ cells revealed that discrete conserved regions in C/EBPβ activated distinct pro- and anti-inflammatory genes and triggered divergent differentiation programs. Moreover, not only structural C/EBPβ mutants, but also post-translational modification (PTM) site mutations led to different reprogramming outcomes. These data suggest that C/EBPβ orchestrates myeloid diversification by integrating PTMs with structural plasticity as signal dependent adaptable modular properties to determine cell fate.

Regeneration of adult skeletal muscle relies upon a population of quiescent myogenic progenitor cells, called satellite cells (SCs). Upon injury, SCs activate, proliferate, differentiate and fuse to make new myofibers or to repair damaged ones. SCs can also self-renew to repopulate the SC niche. The balance between differentiation and self-renewal is critical to maintain muscle homeostasis and changes in this equilibrium can lead to chronic muscle degeneration. For example, Duchenne’s muscular dystrophy (DMD) is characterized by rounds of muscle degeneration and regeneration leading to increased muscle wasting. One approach to treat DMD is transplantation of SCs. For this treatment to be viable, transplanted cells must contribute to repairing injured muscle and repopulating the SC niche. Here, we show that the transcription factor CCAAT/Enhancer Binding Protein beta (C/EBPβ) regulates SC function. C/EBPβ is down-regulated during differentiation and persistent expression of C/EBPβ inhibits differentiation and expression of the myogenic regulatory factors MyoD and Myogenin. C/EBPβ also promotes Pax7 expression by directly binding to and regulating Pax7 transcription. Using genetic tools to conditionally excise C/EBPβ expression in SCs, we found that C/EBPβ-null SCs lose quiescence and precociously differentiate at the expense of self-renewal. After a single injury, C/EBPβ-deficient SCs failed to self-renew, resulting in impaired muscle repair after a second injury. C/EBPβ-induced quiescence also requires upregulation of caveolin-1. Furthermore, pharmacological manipulation of C/EBPβ expression with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), increased the number of cells available for transplantation into dystrophic muscle and enhanced the expression of stem cell markers in a C/EBPβ-dependent fashion. IBMX treatment improved cell survival and migration, engraftment into the SC niche and repair of dystrophic muscle. Together, these results demonstrate that C/EBPβ is an important regulator of SC function and that pharmacological manipulation of C/EBPβ improves culture conditions for the expansion and selection of SCs available for cell therapy for the treatment of muscular dystrophies.

Adult skeletal muscles have remarkable regenerative capacity. Muscle regeneration occurs when muscle tissue experiences injury, causing a population of normally quiescent muscle-resident stem cells, called satellite cells, to become activated. The CCAAT/enhancer binding proteins known as C/EBPs are transcription factors belonging to the bZIP family. Previous work from our lab has identified C/EBPβ as an important negative regulator of myogenesis. C/EBPβ expression is localized to muscle satellite cells and is downregulated upon induction to differentiate, mirroring the loss of Pax7 expression in early myogenesis. C/EBPβ expression also negatively regulates MyoD protein expression. Leaky ribosomal scanning of the Cebpb mRNA produces three C/EBPβ isoforms: LAP*, LAP and LIP, though the individual role of each of these isoforms has not been investigated in myoblasts. This thesis focuses on determining the role of each of the C/EBPβ isoforms during skeletal muscle differentiation. Forced expression of the C/EBPβ-LIP isoform in myoblasts led to a decrease in Myf5, MyoD, and myogenin expression under differentiation conditions when compared to empty vector controls. Further, the fusion of cells was greatly reduced following differentiation. C/EBPβ-LIP expressing cells also demonstrated a growth defect, with pronounced G1 arrest and features of senescence. In contrast, myoblasts expressing the C/EBPβ-LAP isoform has impaired differentiation, though this was not as pronounced as in C/EBPβ-LIP expressing cells and proliferated normally. While LIP is not normally expressed in primary myoblasts from healthy muscle, the ratio of LIP:LAP was increased in primary myoblasts isolated from mdx mice, an animal model for Duchenne muscular dystrophy. These findings suggest that the regulation of C/EBPβ isoform expression could regulate stem cell stamina and may contribute to defects in muscle regeneration in disease.

C-reactive protein (CRP), a plasma protein of the innate immune system, is produced by hepatocytes. A critical regulatory region (-42 to -57) on the CRP promoter contains binding site for the IL-6-activated transcription factor C/EBPβ. The IL-1β-activated transcription factor NF-κB binds to a κB site located nearby (-63 to -74). The κB site overlaps an octamer motif (-59 to -66) which is the binding site for the constitutively active transcription factor Oct-1. Oct-1 is known to function both as a transcriptional repressor and as an activator depending upon the promoter context. Also, Oct-1 can regulate gene expression either by binding directly to the promoter or by interacting with other transcription factors bound to the promoter. The aim of this study was to investigate the functions of Oct-1 in regulating CRP expression. In luciferase transactivation assays, overexpressed Oct-1 inhibited (IL-6 + IL-1β)-induced CRP expression in Hep3B cells. Deletion of the Oct-1 site from the promoter drastically reduced the cytokine response because the κB site was altered as a consequence of deleting the Oct-1 site. Surprisingly, overexpressed Oct-1 inhibited the residual (IL-6 + IL-1β)-induced CRP expression through the promoter lacking the Oct-1 site. Similarly, deletion of the Oct-1 site reduced the induction of CRP expression in response to overexpressed C/EBPβ, and overexpressed Oct-1 inhibited C/EBPβ-induced CRP expression through the promoter lacking the Oct-1 site. We conclude that Oct-1 acts as a transcriptional repressor of CRP expression and it does so by occupying its cognate site on the promoter and also via other transcription factors by an as yet undefined mechanism.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17978号 / 医博第3842号 / 新制||医||1001(附属図書館) / 80822 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長澤 丘司, 教授 河本 宏, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Der Transkriptionsfaktor CCAAT/enhancer-binding Protein β (C/EBPβ) reguliert die Expression zahlreicher Gene, welche die Proliferation, Differenzierung und Seneszenz in hämatopoietischen Zellen, Adipozyten und Leukämiezellen kontrollieren. Um diese mannigfaltigen Aufgaben zu erfüllen interagiert C/EBPβ mit zahlreichen Kofaktoren und Proteinen der Transkriptionsregulations-Maschinerie. Da das funktionale Netzwerk von C/EBPβ und seinen zahlreichen Kooperationspartnern bis heute nicht vollständig entziffert ist, ist es das Ziel dieser Arbeit das Netzwerk aus Interaktionspartnern und C/EBPβ regulierten Proteinen in Leukämiezelllinien und darüber hinaus zu erforschen und aufzudecken. Das Interaktom von C/EBPβ wurde mittels einer Kombination aus einem membranbasierten Peptid-Interaktions Testverfahrens (APS) und endogener Immunprezipitationen mit gekoppelter MS-Analyse untersucht. Außerdem wurde die Proteinmenge von C/EBPβ und von potentiell von C/EBPβ regulierten Proteinen mittels proteomischer MS-Analyse in C/EBPβ Knock-out- und Leukämiezelllinien untersucht. Die Protein-Interaktionsversuche ergaben epigenetische und allgemeine transkriptionsregulierende Proteine, sowie Chromatinstruktur modellierende Faktoren, die mit C/EBPβ interagieren. Zusätzlich konnten neue Interaktionen von C/EBPβ mit Kondensin- und Kinetochorproteinen beobachtet werden. Die Versuchsergebnisse eröffnen überdies neue Interaktionen von C/EBPβ mit DNA Reparatur und Apoptose assoziierten Proteinen. Interessanterweise konnten auch Komponenten des Spliceosomes und RNA-prozessierende Proteine als Interaktoren von C/EBPβ identifiziert werden. Zusammenfassend ermöglicht diese Studie nicht nur die Verifikation von bereits bekannten Proteininteraktionen von C/EBPβ, sondern eröffnet zahlreiche weitere zukünftige Forschungsfelder bezüglich des Interaktionsnetzwerkes von C/EBPβ in Leukämien, sowie anderen Zellarten und Geweben. / The basic leucine zipper transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) regulates the expression of various genes that control the proliferation, differentiation and senescence of haematopoietic cells, adipocytes and leukemia cells. To facilitate its multifaceted functions C/EBPβ interacts with a collection of cofactors and proteins of the transcription regulation machinery. As the functional network of C/EBPβ and its numerous cooperation partners is still incomplete this study attempted to analyze interaction partners and downstream proteins of C/EBPβ in leukemia cells and beyond. A combinatory approach of an array based peptide-interaction screening (APS) and endogenous shotgun IP-MS from leukemia cell lines was applied to elucidate the interactome of C/EBPβ. Moreover, C/EBPβ abundance and potential C/EBPβ regulated proteins were determined by MS proteomics in C/EBPβ knockout and leukemia cell lines. The interaction screenings revealed proteins associated with the general and epigenetic regulation of transcription, with chromatin remodeling and mitotic chromatin organization as well as cell cycle regulation. Additionally, new interactions of C/EBPβ with condensin and kinetochore proteins could be elucidated. The data reports of novel C/EBPβ interactors involved in DNA repair and apoptosis. In addition, components of the spliceosome and RNA-processing were detected. Altogether this study verifies known and reveals various novel interactions of the transcription factor C/EBPβ and augments the network of previous reported interactions and potential cooperation partners. The here collected data discloses new subjects for further research concerning the interaction network of C/EBPβ during cell differentiation and in leukemia.

C/EBPα und C/EBPβ sind Transkriptionsfaktoren, die die Zellproliferation und Zelldifferenzierung in vielen Geweben regulieren. C/EBPα und C/EBPβ spielen auch eine onkogene Rolle in akuter myeloischer Leukämie und anaplastisch-großzelligen Lymphomen (ALCL). In dieser Studie wird C/EBPα auf neue posttranslationale Modifikationen, wie z. B. Arginin-Methylierungen und Citrullinierungen, sowie Lysin-Methylierungen und Acetylierungen, mit massenspektrometrischen Mitteln untersucht. Es werden eine modifizierbare C/EBPα-Arginin-Citrullinierungsposition und die C/EBPα-Lysinsumoylierung eingehend auf ihren Einfluss auf C/EBPα-Proteininteraktionsnetzwerk überprüft. Außerdem wurde im Verlauf dieser Studie ein Hochdurchsatz-Screening-Verfahren entwickelt, das wir Protein Interaktions-Screening auf einer Peptid Matrix (PrISMa) nennen. Dieses Verfahren dient der Aufklärung des modifikationsabhängigen Proteininteraktionsnetzwerkes von C/EBPβ. PrISMa basiert auf einer Peptidmembran, auf der C/EBPβ-Peptide synthetisiert sind. Viele dieser Peptide enthalten methylierte Arginine und Lysine, acetylierte Lysine, citrullinierte Arginine und phosphorylierte Serine, Tyrosine und Threonine. Mittels PrISMa konnten Interaktionen von C/EBPβ mit Histonacetyltransferasen, dem Mediatorkomplex, Proteinen des nukleären Transports und RNA bindenden und spleißenden Proteinen verifiziert und kartiert werden. Des Weiteren konnte mit Hilfe von PrISMa eine große Anzahl von publizierten C/EBPβ Interaktionspartner spezifischen C/EBPβ-Sequenzen zugeordnet werden. C/EBPβ wird in ALCL in hohem Maße exprimiert und ist für die Zellproliferation dieser Krebsart wichtig. In dieser Studie wird das Proteininteraktionsnetzwerk C/EBPβ in einer ALCL Zelllinie aufgeklärt, um tiefere Einsichten über die Funktion von C/EBPβ als Onkogen zu erlangen. / C/EBPα and C/EBPβ regulate cell proliferation and differentiation in multiple cell types. Moreover, C/EBPα and C/EBPβ are known to play oncogenic roles in acute myeloid leukemia and anaplastic large cell lymphomas (ALCLs). In this study C/EBPα is screened for novel posttranslational modifications (PTMs), such as arginine methylation and citrullination, as well as lysine methylation and acetylation by using mass spectrometry. A in this survey identified C/EBPα site of arginine citrullination and the C/EBPα lysine sumoylation are scrutinized for their impact on C/EBPα protein interaction network. A new high-throughput method named Protein Interaction Screen on peptide Matrices (PrISMa) is introduced in this study. This method was developed to determine the C/EBPβ protein interaction network in a PTM dependent manner. The PrISMa survey is based on a peptide membrane spotted with C/EBPβ peptides. Many of these C/EBPβ peptide sequences contain amino acid sequences comprising arginine and lysine methylation, lysine acetylation, arginine citrullination and serine, tyrosine and threonine phosphorylation. By means of PrISMa the C/EBPβ interplay with histone acetyltransferases, the mediator complex, proteins of the nucleoplasmic transport and RNA processing proteins is verified and specified by mapping these interactions to C/EBPβ amino acid sequences. Furthermore, PrISMa provides a map of C/EBPβ protein interaction patterns for a great number of the up to date published C/EBPβ protein interaction partners. C/EBPβ is highly expressed in ALCL cell lines and is essential for the cell proliferation of this type of cancer. In this study the C/EBPβ protein-protein interaction pattern in an ALCL cell line is unraveled providing valuable insight into the protein interaction network of C/EBPβ as an oncogene.

<p>Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. Previous studies have indicated that many of these changes are dependent on the activity of transcription factors belonging to the AP-1, STAT and Ets families. This study took advantage of transformation-deficient and temperature sensitive mutants of the Rous sarcoma virus to characterize transformation-induced patterns of gene expression of two primary cell types, chicken embryo fibroblasts (CEF) and chicken neuroretina (CNR). In these cells, v-Src alters the expression of up to 6% of the protein coding genes. Comparison of this program with independent breast carcinoma data sets identified a group of 42 v-Src inducible genes associated with reduced disease-free survival. Pathway and ontological analyses of the genes differentially regulated by transformation in CEF and CNR indicated a generalized program of de-differentiation induced by Src.</p> <p>To investigate the role of AP-1 inSrc-mediated transformation, a gene profiling study was conducted to characterize the transcriptomes of v-Src-transformed CEF expressing the <em>Jun</em> dominant-negative allele or the <em>JunD</em> short-hairpin RNA (shRNA). Microarray data analysis indicated a cluster of 18 co-regulated probe-sets activated in v-Src-transformed CEF with repressed AP-1 activity but not activated in normal CEF or CEF transformed only by v-Src. One gene, <em>death-associated protein kinase 1</em> (<em>DAPK1</em>), is a C/EBPβ-regulated mediator of apoptosis in IFN-γ-induced cell death. Inhibition of DAPK1 abrogated cell-death in v-Src-transformed CEF expressing the JunD shRNA and expression of DAPK1 was dependent on C/EBPβ but antagonized by AP-1. Chromatin immunoprecipitation indicated that C/EBPβ, but not JunD, is recruited to the DAPK1 promoter. In conclusion, JunD promotes survival by indirectly antagonizing C/EBPβ-dependent expression of DAPK1.</p> / Doctor of Philosophy (PhD)