Funktionelle Charakterisierung der CK2-vermittelten Phosphorylierung des Transkriptionsfaktors TIF-IA bei der Regulation der Synthese ribosomaler RNA

In Säugern wird die ribosomale RNA von RNA-Polymerase I (Pol I) synthetisiert. Die Interaktion der Pol I-spezifischen Faktoren TIF-IA und TIF-IB miteinander sowie die Bindung von TIF-IB an den rDNA-Promotor sind für die Transkriptionsinitiation notwendig. Ein Guanin an Position -7 (G-7) im core promoter element des rDNAPromotors ist nicht für die Bindung von TIF-IB, jedoch für eine effiziente rRNA Synthese notwendig. Wie im Rahmen dieser Arbeit gezeigt wurde, führt eine G-7->A Transition in vitro und in vivo zu einer starken Inhibierung der Promotoraktivität. Transkriptionsanalysen mit immobilisierten rDNA-Matrizen sowie abortive Transkriptionsexperimente wiesen auf eine Hemmung der Assoziation von TIF-IA mit dem rDNA-Promotor durch die G-7 Mutation hin. Jedoch haben Transkriptionsanalysen unter single round-Bedingungen und nach Depletierung von TIF-IA gezeigt, daß TIF-IA für die Transkription an der mutierten Matrize erforderlich ist. Somit scheint ein bislang unbekanntes Protein an der Ausbildung des Prä-Initiationskomplexes beteiligt zu sein, das an G-7 bindet. TIF-IA ist ein Phospho-Protein, das an mehreren Serinresten, unter anderem auch an Serin 170 und 172, phosphoryliert wird. Ziel dieser Arbeit war es, die Phosphorylierung dieser Serinreste funktionell aufzuklären und die entsprechende Kinase zu identifizieren. Durch Herstellung einer nicht-phosphorylierbaren Mutante (TIF-IAS170A/S172A) konnte gezeigt werden, daß die Serine 170 und 172 durch Casein-Kinase 2 (CK2) in vitro und in vivo phosphoryliert werden. Die Analyse der transkriptionellen Aktivität sowie die Expression von TIF-IAS170A/S172A in TIF-IA knockout Zellen ergaben, daß TIF-IA ohne die CK2-vermittelte Phosphorylierung nicht funktionell ist. In Co- Immunpräzipitationsexperimenten wurde nachgewiesen, daß die Inhibierung der Phosphorylierung von Serin 170 und 172 eine verstärkte Bindung von TIF-IA an Pol I bewirkt, während die Interaktion mit TIF-IB nicht beeinflußt wird. Mit Hilfe eines Antiserums, das spezifisch Phospho-Serin 170 und 172 in TIF-IA erkennt, wurde gezeigt, daß die CK2-vermittelte Phosphorylierung die Bindung von TIF-IA an Pol I verhindert. Aufgrund dieser Daten ist anzunehmen, daß TIF-IA nach der Transkriptionsinitiation von CK2 phosphoryliert wird und daß diese Phosphorylierung die Dissoziation von TIF-IA vom Elongationskomplex induziert. Diese CK2-vermittelte, reversible Phosphorylierung von TIF-IA ist somit für die produktive Elongation bzw. eine effiziente Re-Initiation essentiell

Dichotomous Impact of Myc on rRNA Gene Activation and Silencing in B Cell Lymphomagenesis

A major transcriptional output of cells is ribosomal RNA (rRNA), synthesized by RNA polymerase I (Pol I) from multicopy rRNA genes (rDNA). Constitutive silencing of an rDNA fraction by promoter CpG methylation contributes to the stabilization of these otherwise highly active loci. In cancers driven by the oncoprotein Myc, excessive Myc directly stimulates rDNA transcription. However, it is not clear when during carcinogenesis this mechanism emerges, and how Myc-driven rDNA activation affects epigenetic silencing. Here, we have used the Eµ-Myc mouse model to investigate rDNA transcription and epigenetic regulation in Myc-driven B cell lymphomagenesis. We have developed a refined cytometric strategy to isolate B cells from the tumor initiation, promotion, and progression phases, and found a substantial increase of both Myc and rRNA gene expression only in established lymphoma. Surprisingly, promoter CpG methylation and the machinery for rDNA silencing were also strongly up-regulated in the tumor progression state. The data indicate a dichotomous role of oncogenic Myc in rDNA regulation, boosting transcription as well as reinforcing repression of silent repeats, which may provide a novel angle on perturbing Myc function in cancer cells

Genetic mutations linked to Parkinson's disease differentially control nucleolar activity in pre-symptomatic mouse models

Genetic mutations underlying neurodegenerative disorders impair ribosomal DNA (rDNA) transcription suggesting that nucleolar dysfunction could be a novel pathomechanism in polyglutamine diseases and in certain forms of amyotrophic lateral sclerosis/frontotemporal dementia. Here, we investigated nucleolar activity in pre-symptomatic digenic models of Parkinson's disease (PD) that model the multifactorial aetiology of this disease. To this end, we analysed a novel mouse model mildly overexpressing mutant human alpha-synuclein (hA53T-SNCA) in a PTEN-induced kinase 1 (PINK1/ PARK6) knockout background and mutant mice lacking both DJ-1 (also known as PARK7) and PINK1. We showed that overexpressed hA53T-SNCA localizes to the nucleolus. Moreover, these mutants show a progressive reduction of rDNA transcription linked to a reduced mouse lifespan. By contrast, rDNA transcription is preserved in DJ-1/PINK1 double knockout (DKO) mice. mRNA levels of the nucleolar transcription initiation factor 1A (TIF-IA, also known as RRN3) decrease in the substantia nigra of individuals with PD. Because loss of TIF-IA, as a tool to mimic nucleolar stress, increases oxidative stress and because DJ-1 and PINK1 mutations result in higher vulnerability to oxidative stress, we further explored the synergism between these PD-associated genes and impaired nucleolar function. By the conditional ablation of TIF-IA, we blocked ribosomal RNA (rRNA) synthesis in adult dopaminergic neurons in a DJ-1/PINK1 DKO background. However, the early phenotype of these triple knockout mice was similar to those mice exclusively lacking TIF-IA. These data sustain a model in which loss of DJ-1 and PINK1 does not impair nucleolar activity in a pre-symptomatic stage. This is the first study to analyse nucleolar function in digenic PD models. We can conclude that, at least in these models, the nucleolus is not as severely disrupted as previously shown in DA neurons from PD patients and neurotoxin-based PD mouse models. The results also show that the early increase in rDNA transcription and nucleolar integrity may represent specific homeostatic responses in these digenic pre-symptomatic PD models.Peer reviewe

Role of nucleolar dysfunction in neurodegenerative disorders: a game of genes?

Within the cell nucleus the nucleolus is the site of rRNA transcription and ribosome biogenesis and its activity is clearly essential for a correct cell function, however its specific role in neuronal homeostasis remains mainly unknown. Here we review recent evidence that impaired nucleolar activity is a common mechanism in different neurodegenerative disorders. We focus on the specific causes and consequences of impaired nucleolar activity to better understand the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). In particular, we discuss the genetic and epigenetic factors that might regulate nucleolar function in these diseases. In addition, we describe novel animal models enabling the dissection of the context-specific series of events triggered by nucleolar disruption, also known as nucleolar stress. Finally, we suggest how this novel mechanism could help to identify strategies to treat these still incurable disorders

The nucleolus as a stress sensor: JNK2 inactivates the transcription factor TIF-IA and down-regulates rRNA synthesis

Cells respond to a variety of extracellular and intracellular forms of stress by down-regulating rRNA synthesis. We have investigated the mechanism underlying stress-dependent inhibition of RNA polymerase I (Pol I) transcription and show that the Pol I-specific transcription factor TIF-IA is inactivated upon stress. Inactivation is due to phosphorylation of TIF-IA by c-Jun N-terminal kinase (JNK) at a single threonine residue (Thr 200). Phosphorylation at Thr 200 impairs the interaction of TIF-IA with Pol I and the TBP-containing factor TIF-IB/SL1, thereby abrogating initiation complex formation. Moreover, TIF-IA is translocated from the nucleolus into the nucleoplasm. Substitution of Thr 200 by valine as well as knock-out of Jnk2 prevent inactivation and translocation of TIF-IA, leading to stress-resistance of Pol I transcription. Our data identify TIF-IA as a downstream target of the JNK pathway and suggest a critical role of JNK2 to protect rRNA synthesis against the harmful consequences of cellular stress

Phosphorylation by Casein Kinase 2 Facilitates rRNA Gene Transcription by Promoting Dissociation of TIF-IA from Elongating RNA Polymerase I ▿

The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription

lncRNA-Induced Nucleosome Repositioning Reinforces Transcriptional Repression of rRNA Genes upon Hypotonic Stress

The activity of rRNA genes (rDNA) is regulated by pathways that target the transcription machinery or alter the epigenetic state of rDNA. Previous work has established that downregulation of rRNA synthesis in quiescent cells is accompanied by upregulation of PAPAS, a long noncoding RNA (lncRNA) that recruits the histone methyltransferase Suv4-20h2 to rDNA, thus triggering trimethylation of H4K20 (H4K20me3) and chromatin compaction. Here, we show that upregulation of PAPAS in response to hypoosmotic stress does not increase H4K20me3 because of Nedd4-dependent ubiquitinylation and proteasomal degradation of Suv4-20h2. Loss of Suv4-20h2 enables PAPAS to interact with CHD4, a subunit of the chromatin remodeling complex NuRD, which shifts the promoter-bound nucleosome into the transcriptional “off” position. Thus, PAPAS exerts a “stress-tailored” dual function in rDNA silencing, facilitating either Suv4-20h2-dependent chromatin compaction or NuRD-dependent changes in nucleosome positioning

Epigenetic Erosion in Adult Stem Cells: Drivers and Passengers of Aging

In complex organisms, stem cells are key for tissue maintenance and regeneration. Adult stem cells replenish continuously dividing tissues of the epithelial and connective types, whereas in non-growing muscle and nervous tissues, they are mainly activated upon injury or stress. In addition to replacing deteriorated cells, adult stem cells have to prevent their exhaustion by self-renewal. There is mounting evidence that both differentiation and self-renewal are impaired upon aging, leading to tissue degeneration and functional decline. Understanding the molecular pathways that become deregulate in old stem cells is crucial to counteract aging-associated tissue impairment. In this review, we focus on the epigenetic mechanisms governing the transition between quiescent and active states, as well as the decision between self-renewal and differentiation in three different stem cell types, i.e., spermatogonial stem cells, hematopoietic stem cells, and muscle stem cells. We discuss the epigenetic events that channel stem cell fate decisions, how this epigenetic regulation is altered with age, and how this can lead to tissue dysfunction and disease. Finally, we provide short prospects of strategies to preserve stem cell function and thus promote healthy aging