A cytological approach to the ordering of events in gene activation using the Sgs-4 locus of Drosophila melanogaster.

The polytene chromosomes of Drosophila strains that differ in the synthesis of the major salivary gland glue protein sgs-4 were examined by indirect immunofluorescence using antisera to several nonhistone chromosomal proteins. The Oregon-R X chromosome, which produces sgs-4 messenger RNA, showed a strong fluorescent band at locus 3C11-12 when stained with anti-RNA polymerase II, whereas the null mutant Berkeley 1 failed to exhibit fluorescence at that locus. The presence of another antigen (Band 2), normally associated with developmentally active loci, was clearly evident at locus 3C11-12 of both transcriptionally competent and null strains, indicating that the association of Band 2 antigen with the chromatin is an event independent of RNA polymerase II binding. Antibodies directed against Drosophila topoisomerase I stained 3C11-12 in the Sgs-4+ (wild-type) strain brightly, but gave significantly less staining in the null strain. This indicates that the high concentrations of topoisomerase I seen at active loci are closely associated with the transcriptional event. In some of these analyses, we have made use of flies heterozygous for the wild-type and null alleles in order to make internally controlled comparisons. The results suggest that this type of analysis will enable conclusions to be drawn concerning the interdependence and order of action of chromosomal proteins involved in developmental gene activation.

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Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

Journal of Biological Chemistry ◽

10.1074/jbc.m009634200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 12266-12273 ◽

Cited By ~ 31

Author(s):

Wenxiang Wei ◽

Dorjbal Dorjsuren ◽

Yong Lin ◽

Weiping Qin ◽

Takahiro Nomura ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Middle Part ◽

Wild Type ◽

Binding Region ◽

Pol Ii ◽

Transcription Factor Iif ◽

B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0591 ◽

2014 ◽

Vol 25 (12) ◽

pp. 1916-1924 ◽

Cited By ~ 3

Author(s):

David Öling ◽

Rehan Masoom ◽

Kristian Kvint

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Life Span ◽

Rna Polymerase Ii ◽

Ribosomal Dna ◽

Genetic Evidence ◽

Wild Type ◽

Replicative Life Span ◽

Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

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Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation

Molecular and Cellular Biology ◽

10.1128/mcb.7.12.4308-4316.1987 ◽

1987 ◽

Vol 7 (12) ◽

pp. 4308-4316

Author(s):

E Egyházi ◽

E Durban

Keyword(s):

Salivary Gland ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Immunoglobulin G ◽

Topoisomerase I ◽

Chironomus Tentans ◽

Balbiani Ring ◽

Gland Cells ◽

Salivary Gland Cells ◽

Transcriptional Process

Purified anti-topoisomerase I immunoglobulin G (IgG) was microinjected into nuclei of Chironomus tentans salivary gland cells, and the effect on DNA transcription was investigated. Synthesis of nucleolar preribosomal 38S RNA by RNA polymerase I and of chromosomal Balbiani ring RNA by RNA polymerase II was inhibited by about 80%. The inhibitory action of anti-topoisomerase I IgG could be reversed by the addition of exogenous topoisomerase I. Anti-topoisomerase I IgG had less effect on RNA polymerase II-promoted activity of other less efficiently transcribing heterogeneous nuclear RNA genes. The pattern of inhibition of growing nascent Balbiani ring chains indicated that the transcriptional process was interrupted at the level of chain elongation. The highly decondensed state of active Balbiani ring chromatin, however, remained unaffected after injection of topoisomerase I antibodies. These data are consistent with the interpretation that topoisomerase I is an essential component in the transcriptional process but not in the maintenance of the decondensed state of active chromatin.

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CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

Molecular and Cellular Biology ◽

10.1128/mcb.01993-06 ◽

2007 ◽

Vol 27 (5) ◽

pp. 1631-1648 ◽

Cited By ~ 104

Author(s):

Igor Chernukhin ◽

Shaharum Shamsuddin ◽

Sung Yun Kang ◽

Rosita Bergström ◽

Yoo-Wook Kwon ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Activation ◽

Ctcf Binding ◽

Pol Ii ◽

Genome Wide ◽

Target Sites ◽

On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

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Rpb7 Can Interact with RNA Polymerase II and Support Transcription during Some Stresses Independently of Rpb4

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2672 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2672-2680 ◽

Cited By ~ 48

Author(s):

Ayelet Sheffer ◽

Mazal Varon ◽

Mordechai Choder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Stress Conditions ◽

Cold Sensitivity ◽

Starvation Period ◽

Wild Type ◽

Pol Ii ◽

Growth Phenotype ◽

Mild Temperature

ABSTRACT Rpb4 and Rpb7 are two yeast RNA polymerase II (Pol II) subunits whose mechanistic roles have recently started to be deciphered. Although previous data suggest that Rpb7 can stably interact with Pol II only as a heterodimer with Rpb4, RPB7 is essential for viability, whereas RPB4 is essential only during some stress conditions. To resolve this discrepancy and to gain a better understanding of the mode of action of Rpb4, we took advantage of the inability of cells lacking RPB4 (rpb4Δ, containing Pol IIΔ4) to grow above 30°C and screened for genes whose overexpression could suppress this defect. We thus discovered that overexpression of RPB7 could suppress the inability ofrpb4Δ cells to grow at 34°C (a relatively mild temperature stress) but not at higher temperatures. Overexpression ofRPB7 could also partially suppress the cold sensitivity ofrpb4Δ strains and fully suppress their inability to survive a long starvation period (stationary phase). Notably, however, overexpression of RPB4 could not override the requirement for RPB7. Consistent with the growth phenotype, overexpression of RPB7 could suppress the transcriptional defect characteristic of rpb4Δ cells during the mild, but not during a more severe, heat shock. We also demonstrated, through two reciprocal coimmunoprecipitation experiments, a stable interaction of the overproduced Rpb7 with Pol IIΔ4. Nevertheless, fewer Rpb7 molecules interacted with Pol IIΔ4 than with wild-type Pol II. Thus, a major role of Rpb4 is to augment the interaction of Rpb7 with Pol II. We suggest that Pol IIΔ4 contains a small amount of Rpb7 that is sufficient to support transcription only under nonstress conditions. When RPB7 is overexpressed, more Rpb7 assembles with Pol IIΔ4, enough to permit appropriate transcription also under some stress conditions.

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Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011665 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3990-4000 ◽

Cited By ~ 3

Author(s):

Sandeep Singh ◽

Karol Szlachta ◽

Arkadi Manukyan ◽

Heather M. Raimer ◽

Manikarna Dinda ◽

...

Keyword(s):

Transcriptional Regulation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Topoisomerase I ◽

Cell Types ◽

Dna Breaks ◽

Transcription Start ◽

Pol Ii ◽

Transcription Start Sites

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

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Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.586 ◽

1987 ◽

Vol 7 (2) ◽

pp. 586-594 ◽

Cited By ~ 60

Author(s):

M S Bartolomei ◽

J L Corden

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Cell Line ◽

Resistance Mutation ◽

Wild Type Mouse ◽

Mutant Form ◽

Wild Type ◽

Gene Encoding ◽

Base Pair Fragment ◽

Alpha Amanitin

RNA polymerase II is inhibited by the mushroom toxin alpha-amanitin. A mouse BALB/c 3T3 cell line was selected for resistance to alpha-amanitin and characterized in detail. This cell line, designated A21, was heterozygous, possessing both amanitin-sensitive and -resistant forms of RNA polymerase II; the mutant form was 500 times more resistant to alpha-amanitin than the sensitive form. By using the wild-type mouse RNA polymerase II largest subunit (RPII215) gene (J.A. Ahearn, M.S. Bartolomei, M. L. West, and J. L. Corden, submitted for publication) as the probe, RPII215 genes were isolated from an A21 genomic DNA library. The mutant allele was identified by its ability to transfer amanitin resistance in a transfection assay. Genomic reconstructions between mutant and wild-type alleles localized the mutation to a 450-base-pair fragment that included parts of exons 14 and 15. This fragment was sequenced and compared with the wild-type sequence; a single AT-to-GC transition was detected at nucleotide 6819, corresponding to an asparagine-to-aspartate substitution at amino acid 793 of the predicted protein sequence. Knowledge of the position of the A21 mutation should facilitate the study of the mechanism of alpha-amanitin resistance. Furthermore, the A21 gene will be useful for studying the phenotype of site-directed mutations in the RPII215 gene.

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The effects of camptothecin on RNA polymerase II transcription: Roles of DNA topoisomerase I

Biochimie ◽

10.1016/j.biochi.2007.01.001 ◽

2007 ◽

Vol 89 (4) ◽

pp. 482-489 ◽

Cited By ~ 35

Author(s):

Giovanni Capranico ◽

Francesca Ferri ◽

Maria Vittoria Fogli ◽

Alessandra Russo ◽

Luca Lotito ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Topoisomerase I ◽

Dna Topoisomerase I ◽

Dna Topoisomerase ◽

Rna Polymerase Ii Transcription

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Assembly and Functional Organization of the Nucleolus: Ultrastructural Analysis ofSaccharomyces cerevisiaeMutants

Molecular Biology of the Cell ◽

10.1091/mbc.11.6.2175 ◽

2000 ◽

Vol 11 (6) ◽

pp. 2175-2189 ◽

Cited By ~ 38

Author(s):

Stéphanie Trumtel ◽

Isabelle Léger-Silvestre ◽

Pierre-Emmanuel Gleizes ◽

Frédéric Teulières ◽

Nicole Gas

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Functional Organization ◽

Ultrastructural Localization ◽

Rna Polymerase I ◽

Wild Type ◽

Rdna Transcription ◽

Nucleolar Structure ◽

Fibrillar Component ◽

Polymerase I

Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization. Although morphologically different, the mininucleoli observed in cells transcribing rDNA with RNA polymerase II also contain a fibrillar subregion of analogous function, in addition to a dense core of unknown nature. Upon repression of rDNA transcription in this strain or in an RNA polymerase I thermosensitive mutant, the nucleolar structure falls apart (in a reversible manner), and nucleolar constituents partially relocate to the nucleoplasm, indicating that rRNA is a primary determinant for the assembly of the nucleolus.

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Histone H3 Lysine 9 Methylation and HP1γ Are Associated with Transcription Elongation through Mammalian Chromatin.

Blood ◽

10.1182/blood.v106.11.1734.1734 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1734-1734 ◽

Cited By ~ 1

Author(s):

Christopher R. Vakoc ◽

Sean A. Mandat ◽

Ben A. Olenschock ◽

Gerd A. Blobel

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Histone Modifications ◽

Transcription Elongation ◽

Gene Activation ◽

Histone H3 ◽

H3k9 Methylation ◽

Erythroid Cells ◽

Cellular Memory ◽

Active Genes

Abstract Epigenetic regulation of gene expression plays a fundamental role during tissue specification and cellular memory. Cells that are committed to a given lineage, for example during hematopoiesis, “remember” their phenotype throughout successive rounds of cell division, reflecting alterations in chromatin structure at genes that are permanently activated or silenced. Cellular memory is anchored in specific sets of histone modifications, which together form the basis for the histone code. This is illustrated in the methylation of histone molecules: while methylation of histone H3 on lysines 4, 36, and 79 is linked with gene activation, methylation of H3 on lysines 9 and 27 and histone H4 on lysine 20 is associated with transcriptionally silent heterochromatin and repressed genes within euchromatin. Not surprisingly, dysregulation of histone methylation contributes to human diseases such as leukemias. Here we examined the methylation of histone molecules during gene activation and repression triggered by the hematopoietic transcription factor GATA-1. Surprisingly, we found that during activation by GATA-1 in erythroid cells, the levels of H3K9 di- and tri-methylation increase dramatically at all examined GATA-1-stimulated genes, including alpha- and beta-globin, AHSP, Band 3 and Glycophorin A. In contrast, at all GATA-1-repressed genes examined (GATA-2, c-kit, and c-myc) these marks are rapidly lost. Peaks of H3K9 methylation were observed in the transcribed portion of genes with lower signals at the promoter regions. Heterochromatin Protein 1γ (HP1γ), a protein containing a chromo-domain that recognizes H3K9 methylation, is also present in the transcribed region of all active genes examined. We extended these analyses to include numerous genes in diverse cell types (primary erythroid cells, primary T-lymphoid cells, epithelial cells and fibroblast) and consistently found a tight correlation between H3K9 methylation and gene activity, highlighting the general nature of our findings. Both the presence of HP1γ and H3K9 methylation at active genes are dependent upon transcription elongation by RNA polymerase II. Finally, HP1γ is in a physical complex with the elongating form of RNA polymerase II. Together, our results show that H3K9 methylation and HP1γ not only function in repressive chromatin, but play a novel and unexpected role during transcription activation. These results further elucidate new combinations of histone modifications that distinguish between repressed and actively transcribing chromatin.

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