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Ingrid Grummt

Publications and source records attributed to Ingrid Grummt.

31 records · Page 2Linked to original sources
PubMed2004

Recruitment of the nucleolar remodeling complex NoRC establishes ribosomal DNA silencing in chromatin.

The rRNA gene cluster consists of multiple transcription units. Half of these are active, while the other half are transcriptionally inactive. Previously, in vivo studies have demonstrated that silencing of ribosomal DNA (rDNA) is mediated by the chromatin remodeling NoRC (nucleolar remodeling complex). To explore the mechanisms underlying NoRC-directed silencing of rDNA transcription, we investigated the effect of recombinant NoRC on RNA polymerase I transcription on reconstituted chromatin templates. We show that NoRC interacts with the transcription terminator factor (TTF-I), and this interaction is required both for the binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome, thereby repressing RNA polymerase I transcription. This NoRC-directed rDNA repression requires the N terminus of histone H4. Repression is effective before preinitiation complex formation and as such is unable to exert an effect upon activated rDNA genes. Furthermore, the early steps of rDNA repression do not depend on DNA and histone modifications. These results reveal an important role for TTF-I in recruiting NoRC to rDNA and an active role for NoRC in the establishment of rDNA silencing.

Acetylation↗
PubMed2003

ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth.

Phosphorylation of transcription factors by mitogen-activated protein kinase (MAPK) cascades links cell signaling with the control of gene expression. Here we show that growth factors induce rRNA synthesis by activating MAPK-dependent signaling cascades that target the RNA polymerase I-specific transcription initiation factor TIF-IA. Activation of TIF-IA and ribosomal gene transcription is sensitive to PD98059, indicating that TIF-IA is targeted by MAPK in vivo. Phosphopeptide mapping and mutational analysis reveals two serine residues (S633 and S649) that are phosphorylated by ERK and RSK kinases. Replacement of S649 by alanine inactivates TIF-IA, inhibits pre-rRNA synthesis, and retards cell growth. The results provide a link between growth factor signaling, ribosome production, and cell growth, and may have a major impact on the mechanism of cell transformation.

3T3 Cells↗
PubMed2003

Epigenetic silencing of RNA polymerase I transcription.

The genes that encode ribosomal RNA exist in two distinct types of chromatin--an 'open' conformation that is permissive to transcription and a 'closed' conformation that is transcriptionally refractive. Recent studies have provided insights into the molecular mechanisms that silence either entire nucleolus organizer regions (NORs) in genetic hybrids or individual rRNA genes within a NOR. An emerging theme from these studies is that epigenetic mechanisms operating at the level of DNA methylation and histone modifications alter the chromatin structure and control the ratio of active and inactive rRNA genes.

Animals↗
PubMed2002

A kinetic framework for a mammalian RNA polymerase in vivo.

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.

Animals↗
PubMed2002

Multiple interactions between RNA polymerase I, TIF-IA and TAF(I) subunits regulate preinitiation complex assembly at the ribosomal gene promoter.

In mammals, growth-dependent regulation of rRNA synthesis is brought about by the transcription initiation factor TIF-IA. TIF-IA is associated with a fraction of the TBP-containing factor TIF-IB/SL1 and the initiation-competent form of RNA polymerase I (Pol I). We investigated the mechanisms that down-regulate cellular pre-rRNA synthesis and demonstrate that nutrient starvation, density arrest and protein synthesis inhibitors inactivate TIF-IA and impair the association of TIF-IA with Pol I. Moreover, we used a panel of TIF-IA deletion mutants to map the domains that mediate the interaction of TIF-IA with Pol I and TIF-IB/SL1. We found that amino acids 512-609 interact with two subunits of Pol I, RPA43 and PAF67, whereas a short, conserved motif (LARAK, amino acids 411-415) is required for the association of TIF-IA with TAF(I)95 and TAF(I)68. The results uncover an interphase for essential protein-protein interactions that facilitate Pol I preinitiation complex formation.

Amino Acid Sequence↗
PubMed2002

The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription.

Epigenetic control mechanisms silence about half of the ribosomal RNA (rRNA) genes in metabolically active cells. In exploring the mechanism by which the active or silent state of rRNA genes is inherited, we found that NoRC, a nucleolar remodeling complex containing Snf2h (also called Smarca5, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily a, member 5), represses rDNA transcription. NoRC mediates rDNA silencing by recruiting DNA methyltransferase and histone deacetylase activity to the rDNA promoter, thus establishing structural characteristics of heterochromatin such as DNA methylation, histone hypoacetylation and methylation of the Lys9 residue of histone H3. These results indicate that active and inactive rRNA genes can be demarcated by their associated proteins, and link chromatin remodeling to DNA methylation and specific histone modifications.

3T3 Cells↗
PubMed2002

The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase I transcription.

Mammalian chromatin remodeling complexes are involved in both activation and repression of transcription. Here, we show that NoRC, a SNF2h- containing nucleolar chromatin remodeling complex, represses ribosomal gene transcription. NoRC-mediated rDNA silencing was alleviated by trichostatin A, indicating that histone deacetylation is causally involved in silencing. Chromatin immunoprecipitation experiments demonstrate that overexpression of TIP5, the large subunit of NoRC, mediates deacetylation of nucleosomes in the vicinity of the rDNA promoter. Protein-protein interaction assays reveal association of TIP5 with the histone deacetylase HDAC1 in vivo and in vitro. Deletion of the C-terminal PHD finger and bromodomain abolishes the interaction of TIP5 and HDAC1, and abrogates transcriptional repression. The results suggest that NoRC silences the rDNA locus by targeting the SIN3 corepressor complex to the rDNA promoter, thereby establishing a repressed chromatin structure.

3T3 Cells↗
PubMed2002

TFIIH plays an essential role in RNA polymerase I transcription.

TFIIH is a multisubunit protein complex that plays an essential role in nucleotide excision repair and transcription of protein-coding genes. Here, we report that TFIIH is also required for ribosomal RNA synthesis in vivo and in vitro. In yeast, pre-rRNA synthesis is impaired in TFIIH ts strains. In a mouse, part of cellular TFIIH is localized within the nucleolus and is associated with subpopulations of both RNA polymerase I and the basal factor TIF-IB. Transcription systems lacking TFIIH are inactive and exogenous TFIIH restores transcriptional activity. TFIIH is required for productive but not abortive rDNA transcription, implying a postinitiation role in transcription. The results provide a molecular link between RNA polymerase I transcription and transcription-coupled repair of active ribosomal RNA genes.

Animals↗
PubMed2002

Identification of protein phosphorylation sites by combination of elastase digestion, immobilized metal affinity chromatography, and quadrupole-time of flight tandem mass spectrometry.

Using the combination of in-gel elastase digestion, immobilized metal affinity chromatography and high resolution electrospray tandem mass spectrometry, the phosphorylation sites of two phosphoproteins were determined. Complete coverage of all phosphorylation sites (Ser10, Ser139, Thr197, Ser338) of the model phosphoprotein protein kinase A C(alpha)-subunit could be achieved by this strategy in the low picomole range. In addition, three previously unknown phosphorylation sites of the human transcription initiation factor TIF-IA (Ser44, Ser170, Ser172) were determined in this way. Both phosphoproteins could be identified in a protein database on the basis of their elastase generated phosphopeptides alone. The data of seven phosphopeptides were used for identification of protein kinase A, and those of two phosphopeptides for TIF-IA, respectively. The accurate mass data of the electrospray mass spectra recorded at high resolution are extremely useful for sequencing of the elastase generated phosphopeptides and for protein identification by database searching.

Chromatography, Affinity↗
PubMed2002

CSB is a component of RNA pol I transcription.

Mutation in the CSB gene results in the human Cockayne's syndrome (CS). Here, we provide evidence that CSB is found not only in the nucleoplasm but also in the nucleolus within a complex (CSB IP/150) that contains RNA pol I, TFIIH, and XPG and promotes efficient rRNA synthesis. CSB is active in in vitro RNA pol I transcription and restores rRNA synthesis when transfected in CSB-deficient cells. We also show that mutations in CSB, as well as in XPB and XPD genes, all of which confer CS, disturb the RNA pol I/TFIIH interaction within the CSB IP/150. In addition to revealing an unanticipated function for CSB in rRNA synthesis, we show that the fragility of this complex could be one factor contributing to the CS phenotype.

Blotting, Western↗
PubMed2002

Mechanism of inhibition of RNA polymerase I transcription by DNA-dependent protein kinase.

DNA-dependent protein kinase represses RNA polymerase I (Pol I) transcription in vitro. To investigate the mechanism underlying transcriptional repression, we compared Pol I transcription in extracts from cells that either contain or lack the catalytic subunit of DNA-PK (DNA-PKcs). ATP-dependent repression of Pol I transcription was observed in extracts from DNA-PKcs-containing but not -deficient cells, required templates with free DNA ends, and was overcome by exogenous SL1, the factor that nucleates initiation complex formation. Order-of-addition experiments demonstrate that DNA-PKcs does not inactivate component(s) of the Poll transcription machinery. Instead, phosphorylated Ku protein competes with SL1 for binding to the rDNA promoter and, as a consequence, prevents initiation complex formation. The results reveal a novel mechanism of transcriptional regulation by DNA-PK. Once targeted to DNA, autophosphorylated Ku may displace positive- or negative-acting factors from their target sites, thereby repressing or activating transcription in a gene-specific manner.

Antigens, Nuclear↗
PubMed2002

Autoantibodies to NOR 90/hUBF: longterm clinical and serological followup in a patient with limited systemic sclerosis suggests an antigen driven immune response.

We describe the clinical and serological followup of a 9-year-old girl with anti-nucleolar organizing region 90/human upstream-binding factor (anti-NOR 90/hUBF) who had features of systemic sclerosis over a period of 17 years, from childhood into adulthood. We review the associations of anti-UBF autoantibodies, and provide evidence that anti-NOR 90/UBF immune response is antigen driven.

Autoantibodies↗