Biomedical subjects

Ingrid Grummt

Publications and source records attributed to Ingrid Grummt.

At least 19 recordsLinked to original sources

PubMed2006

Development of a species-specific RNA polymerase I-based shRNA expression vector.

RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions.

Animals↗

PubMed2006

NoRC-dependent nucleosome positioning silences rRNA genes.

Previous studies have established that the Snf2h-containing chromatin remodeling complex NoRC mediates epigenetic silencing of a subset of rRNA genes (rDNA) by recruiting enzymatic activities that modify histones and methylate DNA. Here we have analyzed nucleosome positions at the murine rDNA promoter and show that active and silent rDNA copies are characterized not only by specific epigenetic marks but also by differently positioned nucleosomes. At active genes the promoter-bound nucleosome covers nucleotides from -157 to -2, whereas at silent genes the nucleosome is positioned 25 nucleotides further downstream. We provide evidence that NoRC is the molecular machine that shifts the promoter-bound nucleosome downstream of the transcription start site into a translational position that is unfavorable for transcription complex formation.

3T3-L1 Cells↗

PubMed2006

Intergenic transcripts regulate the epigenetic state of rRNA genes.

Transcripts originating from the intergenic spacer (IGS) that separates rRNA genes (rDNA) have been known for two decades; their biological role, however, is largely unknown. Here we show that IGS transcripts are required for establishing and maintaining a specific heterochromatic configuration at the promoter of a subset of rDNA arrays. The mechanism of action appears to be mediated through the interaction of TIP5, the large subunit of the chromatin remodeling complex NoRC, with 150-300 nucleotide RNAs that are complementary in sequence to the rDNA promoter. Mutations that abrogate RNA binding of TIP5 impair the association of NoRC with rDNA and fail to promote H3K9&H4K20 methylation and HP1 recruitment. Knockdown of IGS transcripts abolishes the nucleolar localization of NoRC, decreases DNA methylation, and enhances rDNA transcription. The results reveal an important contribution of processed IGS transcripts in chromatin structure and epigenetic control of the rDNA locus.

Animals↗

PubMed2006

Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription.

We investigated the role of SIRT7, one of the seven members of the mammalian sirtuin family. We show that SIRT7 is a widely expressed nucleolar protein that is associated with active rRNA genes (rDNA), where it interacts with RNA polymerase I (Pol I) as well as with histones. Overexpression of SIRT7 increases Pol I-mediated transcription, whereas knockdown of SIRT7 or inhibition of the catalytic activity results in decreased association of Pol I with rDNA and a reduction of Pol I transcription. Depletion of SIRT7 stops cell proliferation and triggers apoptosis. Our findings suggest that SIRT7 is a positive regulator of Pol I transcription and is required for cell viability in mammals.

Animals↗

PubMed2006

Acetylation of UBF changes during the cell cycle and regulates the interaction of UBF with RNA polymerase I.

The upstream binding factor UBF, an activator of RNA polymerase I transcription, is posttranslationally modified by phosphorylation and acetylation. We found that in NIH3T3 cells, UBF is acetylated in S-phase but not in G1-phase. To assess the role of acetylation in regulation of UBF activity, we have established an NIH3T3 cell line that inducibly overexpresses HDAC1. Both in vivo and in vitro, HDAC1 efficiently hypoacetylates UBF. Immunoprecipitation with antibodies against the Pol I-associated factor PAF53 co-precipitated UBF in mock cells but not in cells overexpressing HDAC1. Pull-down experiments showed that acetylation of UBF augments the interaction with Pol I. Consistent with acetylation of UBF being important for association of PAF53 and recruitment of Pol I, the level of Pol I associated with rDNA and pre-rRNA synthesis were reduced in cells overexpressing HDAC1. The results suggest that acetylation and deacetylation of UBF regulate rRNA synthesis during cell cycle progression.

Acetylation↗

PubMed2006

The chromatin remodelling complex WSTF-SNF2h interacts with nuclear myosin 1 and has a role in RNA polymerase I transcription.

Nuclear actin and myosin 1 (NM1) are key regulators of gene transcription. Here, we show by biochemical fractionation of nuclear extracts, protein-protein interaction studies and chromatin immunoprecipitation assays that NM1 is part of a multiprotein complex that contains WICH, a chromatin remodelling complex containing WSTF (Williams syndrome transcription factor) and SNF2h. NM1, WSTF and SNF2h were found to be associated with RNA polymerase I (Pol I) and ribosomal RNA genes (rDNA). RNA interference-mediated knockdown of NM1 and WSTF reduced pre-rRNA synthesis in vivo, and antibodies to WSTF inhibited Pol I transcription on pre-assembled chromatin templates but not on naked DNA. The results indicate that NM1 cooperates with WICH to facilitate transcription on chromatin.

Adenosine Triphosphatases↗

PubMed2006

Actin and myosin as transcription factors.

The proteins actin and myosin have a firm place in the muscles, where they are responsible for contraction. Although recent investigations have shown that they are found in the nucleus, it has been unclear as to what they are doing there. The discovery of actin as a component of the transcription apparatus, chromatin-remodeling complexes, as well as RNA processing machines, implies important roles for actin in the readout of genetic information. Actin is associated with all three nuclear RNA polymerases and acts in concert with nuclear myosin 1 (NM1) to drive transcription. Actin-NMI interactions are involved in the transition of the initiation complex into the elongation complex, presumably by triggering a structural change of the transcription apparatus or by generating force that supports RNA polymerase movement.

Actins↗

PubMed2005

Cellular stress and nucleolar function.

All organisms sense and respond to conditions that stress their homeostatic mechanisms. Here we review current studies showing that the nucleolus, long regarded as a mere ribosome producing factory, plays a key role in monitoring and responding to cellular stress. After exposure to extra- or intracellular stress, cells rapidly down-regulate the synthesis of ribosomal RNA. Impairment of nucleolar function in response to stress is accompanied by perturbation of nucleolar structure, cell cycle arrest and stabilization of p53. The nucleolar target for down-regulation of rDNA transcription is TIF-IA, an essential transcription factor that modulates the activity of RNA polymerase I (Pol I). Upon stress, TIF-IA is phosphorylated by c-Jun N-terminal kinase 2 (JNK2). Phosphorylation prevents TIF-IA from interaction with Pol I, thereby impairing transcription complex formation and rRNA synthesis. Furthermore, stress-induced inactivation of TIF-IA is accompanied by translocation of TIF-IA from the nucleolus to the nucleoplasm. These findings, together with other data showing stress-induced release of nucleolar proteins to carry out other regulatory functions, reinforce the growing realization that nucleoli orchestrate the chain of events the cell uses to properly respond to stress signals.

Animals↗

PubMed2005

The PHD finger/bromodomain of NoRC interacts with acetylated histone H4K16 and is sufficient for rDNA silencing.

The SNF2h-containing chromatin-remodeling complex NoRC is responsible for silencing a fraction of mammalian rRNA genes (rDNA). NoRC silences transcription by establishing heterochromatic features-including DNA methylation, hypoacetylation of histone H4, and methylation of H3K9-at the rDNA promoter []. We have investigated the mechanism of NoRC-mediated rDNA silencing and show that binding of the bromodomain of TIP5, the large subunit of NoRC, to acetylated nucleosomes is a prerequisite for NoRC function. A point mutation within the bromodomain impairs the association of NoRC with chromatin, prevents heterochromatin formation, and abolishes transcriptional repression. Moreover, the association of NoRC with chromatin requires acetylation of histone H4 at lysine 16 (acH4K16), and binding to acH4K16 is required for subsequent deacetylation of H4K5, H4K8, and H4K12, indicating that acetylation of H4K16 plays an active role in NoRC-mediated heterochromatin formation. The bromodomain cooperates with an adjacent PHD finger to recruit HDAC1, DNMT1, DNMT3, and SNF2h to rDNA. If specifically targeted to the rDNA promoter, the PHD finger/bromodomain is capable of establishing heterochromatic features and rDNA silencing. Thus, the PHD finger/bromodomain represents an autonomous unit that binds to acH4K16 and coordinates the chain of events that establish the repressed state of rDNA.

Acetylation↗

PubMed2005

Genetic inactivation of the transcription factor TIF-IA leads to nucleolar disruption, cell cycle arrest, and p53-mediated apoptosis.

Growth-dependent regulation of rRNA synthesis is mediated by TIF-IA, a basal transcription initiation factor for RNA polymerase I. We inactivated the murine TIF-IA gene by homologous recombination in mice and embryonic fibroblasts (MEFs). TIF-IA-/- embryos die before or at embryonic day 9.5 (E9.5), displaying retardation of growth and development. In MEFs, Cre-mediated depletion of TIF-IA leads to disruption of nucleoli, cell cycle arrest, upregulation of p53, and induction of apoptosis. Elevated levels of p53 after TIF-IA depletion are due to increased binding of ribosomal proteins, such as L11, to MDM2 and decreased interaction of MDM2 with p53 and p19(ARF). RNAi-induced loss of p53 overcomes proliferation arrest and apoptosis in response to TIF-IA ablation. The striking correlation between perturbation of nucleolar function, elevated levels of p53, and induction of cell suicide supports the view that the nucleolus is a stress sensor that regulates p53 activity.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription.

The c-Myc oncoprotein regulates transcription of genes that are associated with cell growth, proliferation and apoptosis. c-Myc levels are modulated by ubiquitin/proteasome-mediated degradation. Proteasome inhibition leads to c-Myc accumulation within nucleoli, indicating that c-Myc might have a nucleolar function. Here we show that the proteins c-Myc and Max interact in nucleoli and are associated with ribosomal DNA. This association is increased upon activation of quiescent cells and is followed by recruitment of the Myc cofactor TRRAP, enhanced histone acetylation, recruitment of RNA polymerase I (Pol I), and activation of rDNA transcription. Using small interfering RNAs (siRNAs) against c-Myc and an inhibitor of Myc-Max interactions, we demonstrate that c-Myc is required for activating rDNA transcription in response to mitogenic signals. Furthermore, using the ligand-activated MycER (ER, oestrogen receptor) system, we show that c-Myc can activate Pol I transcription in the absence of Pol II transcription. These results suggest that c-Myc coordinates the activity of all three nuclear RNA polymerases, and thereby plays a key role in regulating ribosome biogenesis and cell growth.

Animals↗

PubMed2005

Epigenetic mechanism of rRNA gene silencing: temporal order of NoRC-mediated histone modification, chromatin remodeling, and DNA methylation.

Epigenetic control mechanisms silence about half of the rRNA genes in eukaryotes. Previous studies have demonstrated that recruitment of NoRC, a SNF2h-containing remodeling complex, silences rRNA gene transcription. NoRC mediates histone H4 deacetylation, histone H3-Lys9 dimethylation, and de novo DNA methylation, thus establishing heterochromatic features at the rRNA gene promoter. Here we show that inhibition of any of these activities alleviates NoRC-dependent silencing, indicating that these processes are intimately linked. We have studied the temporal order of epigenetic events at the rRNA gene promoter during gene silencing and demonstrate that recruitment of NoRC by TTF-I is a prerequisite for the deacetylation of histone H4 and the dimethylation of histone H3-Lys9. Inhibition of histone deacetylation prevents DNA methylation, while inhibition of DNA methylation does not affect histone modification. Importantly, ATP-dependent chromatin remodeling is required for methylation of a specific CpG dinucleotide within the upstream control element of the rRNA gene promoter, and this modification impairs preinitiation complex formation. The results of this study reveal a clear hierarchy of epigenetic events that control de novo DNA methylation and lead to silencing of RNA genes.

Acetylation↗

PubMed2004

The chromatin remodeling complex NoRC controls replication timing of rRNA genes.

The ATP-dependent chromatin remodeling complex NoRC silences a fraction of mammalian ribosomal RNA genes (rDNA) by establishing heterochromatic structures at the rDNA promoter. Here we show that NoRC also plays a role in replication timing of rDNA. rDNA is replicated in a biphasic manner, active genes ( approximately 60%) replicating early and silent ones ( approximately 40%) replicating late in S-phase. The chromatin structure that marks active and silent rDNA repeats is propagated during cell division. To examine the function of NoRC in epigenetic inheritance and replication timing, we have monitored the chromatin structure, transcriptional activity and replication timing of rDNA in a cell line that moderately overexpresses NoRC. NoRC is exclusively associated with late-replicating rDNA arrays. Overexpression of NoRC silences rDNA transcription, reduces the size and number of nucleoli, impairs cell proliferation and resets replication timing from early to late. The results demonstrate that NoRC is an important determinant of replication timing and epigenetic marks are heritably maintained through DNA replication.

Adenosine Triphosphate↗

PubMed2004

Nuclear actin and myosin I are required for RNA polymerase I transcription.

The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.

Actins↗

PubMed2004

The chromatin remodeling complex NoRC and TTF-I cooperate in the regulation of the mammalian rRNA genes in vivo.

The transcription termination factor (TTF)-I is a multifunctional nucleolar protein that terminates ribosomal gene transcription, mediates replication fork arrest and regulates RNA polymerase I transcription on chromatin. TTF-I plays a dual role in rDNA regulation, being involved in both activation and silencing of rDNA transcription. The N-terminal part of TTF-I contains a negative regulatory domain (NRD) that inhibits DNA binding. Here we show that interactions between the NRD and the C-terminal part of TTF-I mask the DNA-binding domain of TTF-I. However, interaction with TIP5, a subunit of the nucleolar chromatin remodeling complex, NoRC, recovers DNA-binding activity. We have mapped the protein domains that mediate the interaction between TTF-I and TIP5. The association of TIP5 with the NRD facilitates DNA binding of TTF-I and leads to the recruitment of NoRC to the rDNA promoter. Thus, TTF-I and NoRC act in concert to silence rDNA transcription.

Animals↗

PubMed2004

mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability.

In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. Here, we show that the mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Se 44 (S44) and hyperphosphorylation of Se 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target formTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of RNA synthesis.

3T3 Cells↗

PubMed2004

Role of pescadillo and upstream binding factor in the proliferation and differentiation of murine myeloid cells.

Pescadillo (PES1) and the upstream binding factor (UBF1) play a role in ribosome biogenesis, which regulates cell size, an important component of cell proliferation. We have investigated the effects of PES1 and UBF1 on the growth and differentiation of cell lines derived from 32D cells, an interleukin-3 (IL-3)-dependent murine myeloid cell line. Parental 32D cells and 32D IGF-IR cells (expressing increased levels of the type 1 insulin-like growth factor I [IGF-I] receptor [IGF-IR]) do not express insulin receptor substrate 1 (IRS-1) or IRS-2. 32D IGF-IR cells differentiate when the cells are shifted from IL-3 to IGF-I. Ectopic expression of IRS-1 inhibits differentiation and transforms 32D IGF-IR cells into a tumor-forming cell line. We found that PES1 and UBF1 increased cell size and/or altered the cell cycle distribution of 32D-derived cells but failed to make them IL-3 independent. PES1 and UBF1 also failed to inhibit the differentiation program initiated by the activation of the IGF-IR, which is blocked by IRS-1. 32D IGF-IR cells expressing PES1 or UBF1 differentiate into granulocytes like their parental cells. In contrast, PES1 and UBF1 can transform mouse embryo fibroblasts that have high levels of endogenous IRS-1 and are not prone to differentiation. Our results provide a model for one of the theories of myeloid leukemia, in which both a stimulus of proliferation and a block of differentiation are required for leukemia development.

Animals↗