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C S Pikaard

Publications and source records attributed to C S Pikaard.

At least 19 recordsLinked to original sources
PubMed2001

Restricted chromosomal silencing in nucleolar dominance.

Failure of one parent's chromosomes to organize nucleoli in an interspecific hybrid is an epigenetic phenomenon known as nucleolar dominance. Selective gene silencing on a scale of millions of bp is known to be involved, but the full extent to which nucleolus organizer region (NOR)-bearing chromosomes are inactivated beyond the NORs is unknown. Aided by genome sequence data for Arabidopsis thaliana, we have mapped the extent of nucleolar dominance-induced silencing in Arabidopsis suecica, the allotetraploid hybrid of A. thaliana and Arabidopsis arenosa. Using a sensitive reverse transcription PCR assay, we show that the four A. thaliana NORs, each approximately 4 Mbp in size, are approximately 99.5% silenced in A. suecica vegetative leaves, whereas the NORs inherited from A. arenosa remain fully active. The two A. thaliana NORs, NOR2 and NOR4, abut the telomeres on chromosomes 2 and 4, thus there are no genes distal to the NORs. The three protein-coding genes nearest NOR4 on its centromere-proximal side, the closest of which is only 3.1 kb from rRNA gene sequences, are shown to be transcribed in the hybrid despite the silencing of the adjacent approximately 4-Mbp NOR. These data argue against hypotheses in which NOR inactivation is attributed to the spread of silencing from adjacent chromosomal regions, but favor models in which NORs or rRNA genes are the targets of regulation.

Arabidopsis↗
PubMed2001

RNA polymerase I holoenzyme-promoter complexes include an associated CK2-like protein kinase.

In eukaryotes, RNA polymerase I (pol I) transcribes the tandemly repeated genes that encode the precursor of 18S, 5.8S and 25S ribosomal RNAs. In plants and animals, the pol I enzyme can be purified in a holoenzyme form that is self-sufficient for promoter binding and accurate, promoter-dependent transcription in a cell-free system. In this report, we show that a casein kinase 2 (CK2)-like protein kinase co-purifies with pol I holoenzyme activity purified from broccoli (Brassica oleracea). Using an immobilized template assay, we show that the CK2-like activity is part of the protein-DNA complex that results upon binding of the holoenzyme to the rRNA gene promoter. The CK2 activity phosphorylates a similar set of holoenzyme proteins both before and after promoter binding. These data provide further evidence that pol I holoenzyme activity can be attributed to a single, multi-protein complex self-sufficient for promoter association and accurate, promoter-dependent transcription.

Brassica↗
PubMed2000

RNA polymerase I holoenzyme-promoter interactions.

In plants and animals, RNA polymerase I (pol I) can be purified in a form that is self-sufficient for accurate rRNA gene promoter-dependent transcription and that has biochemical properties suggestive of a single complex, or holoenzyme. In this study, we examined the promoter binding properties of a highly purified Brassica pol I holoenzyme activity. DNase I footprinting revealed protection of the core promoter region from approximately -30 to +20, in good agreement with the boundaries of the minimal promoter defined by deletion analyses (-33 to +6). Using conventional polyacrylamide electrophoretic mobility shift assays (EMSA), protein-DNA complexes were mostly excluded from the gel. However, agarose EMSA revealed promoter-specific binding activity that co-purified with promoter-dependent transcription activity. Titration, time-course, and competition experiments revealed the formation or dissociation of a single protein-DNA complex. This protein-DNA complex could be labeled by incorporation of radioactive ribonucleotides into RNA in the presence of alpha-amanitin, suggesting that the polymerase I enzyme is part of the complex. Collectively, these results suggest that transcriptionally competent pol I holoenzymes can associate with rRNA gene promoters in a single DNA binding event.

Brassica↗
PubMed2000

The epigenetics of nucleolar dominance.

Epigenetic phenomena are heritable, alternative states of gene activity that are not explained by mutation, changes in gene sequence or normal developmental regulation. Among the earliest examples was nucleolar dominance, a common phenomenon in interspecific hybrids in which only ribosomal RNA (rRNA) genes inherited from one parent are transcribed. Only active rRNA genes initiate formation of a nucleolus, hence the name for the phenomenon. As in other epigenetic phenomena, chromatin modifications enforce selective gene silencing in nucleolar dominance. However, the mechanisms that discriminate between parental sets of rRNA genes are unclear. Possibilities include sequence differences that affect transcription factor affinities. Other evidence suggests that chromosomal context is more important than rRNA gene sequences, implying control on a larger scale.

Cell Nucleolus↗
PubMed2000

Nucleolar dominance: uniparental gene silencing on a multi-megabase scale in genetic hybrids.

Nucleolar dominance is a phenomenon in hybrids or allopolyploids in which nucleoli form on chromosomes inherited from only one of the two parents. The molecular basis for nucleolar dominance is the transcription by RNA polymerase I of only one parental set of ribosomal RNA genes (rRNA genes). These rRNA genes are clustered by the hundreds, or thousands, of copies, often spanning tens of millions of basepairs of chromosomal DNA at loci known as nucleolus organizer regions (NORs). Enforcement of nucleolar dominance appears to be accomplished by selectively silencing one set of rRNA genes via chemical modifications of chromatin. However, the mechanisms responsible for initially discriminating among the parental sets of rRNA genes and establishing nucleolar dominance remain unclear. Possibilities include mechanisms that act on each rRNA gene or mechanisms that affect whole NORs or even larger chromosomal domains. This review provides a historical perspective of nucleolar dominance research, explores the most popular hypotheses and their shortcomings, and offers some speculations concerning alternative hypotheses to be considered.

Chimera↗
PubMed1999

RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: Tests of transcription factor involvement in nucleolar dominance.

In interspecific hybrids or allopolyploids, often one parental set of ribosomal RNA genes is transcribed and the other is silent, an epigenetic phenomenon known as nucleolar dominance. Silencing is enforced by cytosine methylation and histone deacetylation, but the initial discrimination mechanism is unknown. One hypothesis is that a species-specific transcription factor is inactivated, thereby silencing one set of rRNA genes. Another is that dominant rRNA genes have higher binding affinities for limiting transcription factors. A third suggests that selective methylation of underdominant rRNA genes blocks transcription factor binding. We tested these hypotheses using Brassica napus (canola), an allotetraploid derived from B. rapa and B. oleracea in which only B. rapa rRNA genes are transcribed. B. oleracea and B. rapa rRNA genes were active when transfected into protoplasts of the other species, which argues against the species-specific transcription factor model. B. oleracea and B. rapa rRNA genes also competed equally for the pol I transcription machinery in vitro and in vivo. Cytosine methylation had no effect on rRNA gene transcription in vitro, which suggests that transcription factor binding was unimpaired. These data are inconsistent with the prevailing models and point to discrimination mechanisms that are likely to act at a chromosomal level.

Binding, Competitive↗
PubMed1999

Histone acetyltransferase and protein kinase activities copurify with a putative Xenopus RNA polymerase I holoenzyme self-sufficient for promoter-dependent transcription.

Mounting evidence suggests that eukaryotic RNA polymerases preassociate with multiple transcription factors in the absence of DNA, forming RNA polymerase holoenzyme complexes. We have purified an apparent RNA polymerase I (Pol I) holoenzyme from Xenopus laevis cells by sequential chromatography on five columns: DEAE-Sepharose, Biorex 70, Sephacryl S300, Mono Q, and DNA-cellulose. Single fractions from every column programmed accurate promoter-dependent transcription. Upon gel filtration chromatography, the Pol I holoenzyme elutes at a position overlapping the peak of Blue Dextran, suggesting a molecular mass in the range of approximately 2 MDa. Consistent with its large mass, Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels reveal approximately 55 proteins in fractions purified to near homogeneity. Western blotting shows that TATA-binding protein precisely copurifies with holoenzyme activity, whereas the abundant Pol I transactivator upstream binding factor does not. Also copurifying with the holoenzyme are casein kinase II and a histone acetyltransferase activity with a substrate preference for histone H3. These results extend to Pol I the suggestion that signal transduction and chromatin-modifying activities are associated with eukaryotic RNA polymerases.

Acetyltransferases↗
PubMed1998

Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids.

Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural Arabidopsis suecica, an allotetraploid (amphidiploid) hybrid of Arabidopsis thaliana and Cardaminopsis arenosa, the A. thaliana rRNA genes are repressed. Interestingly, A. thaliana rRNA gene silencing is variable in synthetic Arabidopsis suecica F1 hybrids. Two generations are needed for A. thaliana rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic A. suecica to tetraploid A. thaliana yielded progeny with active A. thaliana rRNA genes and, in some cases, silenced C. arenosa rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that A. thaliana and C. arenosa rRNA genes can be expressed transiently in the other species. Transfected A. thaliana genes are also active in A. suecica protoplasts in which chromosomal A. thaliana genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.

Arabidopsis↗
PubMed1997

Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro.

RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inflorescence that supports promoter-dependent RNA pol I transcription in vitro. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain approximately 30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.

Arabidopsis↗
PubMed1997

Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance.

Nucleolar dominance is an epigenetic phenomenon that describes nucleolus formation around rRNA genes inherited from only one progenitor of an interspecific hybrid or allopolyploid. The phenomenon is widespread, occurring in plants, insects, amphibians, and mammals, yet its molecular basis remains unclear. We have demonstrated nucleolar dominance in three allotetraploids of the plant genus Brassica. In Brassica napus, accurately initiated pre-rRNA transcripts from one progenitor, Brassica rapa are detected readily, whereas transcripts from the approximately 3000 rRNA genes inherited from the other progenitor, Brassica oleracea, are undetectable. Nuclear run-on confirmed that dominance is controlled at the level of transcription. Growth of B. napus seedlings on 5-aza-2'-deoxycytidine to inhibit cytosine methylation caused the normally silent, under-dominant B. oleracea rRNA genes to become expressed to high levels. The histone deacetylase inhibitors sodium butyrate and trichostatin A also derepressed silent rRNA genes. These results reveal an enforcement mechanism for nucleolar dominance in which DNA methylation and histone modifications combine to regulate rRNA gene loci spanning tens of megabase pairs of DNA.

Azacitidine↗
PubMed1997

Transcriptional analysis of nucleolar dominance in polyploid plants: biased expression/silencing of progenitor rRNA genes is developmentally regulated in Brassica.

Nucleolar dominance is an epigenetic phenomenon that describes the formation of nucleoli around rRNA genes inherited from only one parent in the progeny of an interspecific hybrid. Despite numerous cytogenetic studies, little is known about nucleolar dominance at the level of rRNA gene expression in plants. We used S1 nuclease protection and primer extension assays to define nucleolar dominance at a molecular level in the plant genus Brassica. rRNA transcription start sites were mapped in three diploids and in three allotetraploids (amphidiploids) and one allohexaploid species derived from these diploid progenitors. rRNA transcripts of only one progenitor were detected in vegetative tissues of each polyploid. Dominance was independent of maternal effect, ploidy, or rRNA gene dosage. Natural and newly synthesized amphidiploids yielded the same results, arguing against substantial evolutionary effects. The hypothesis that nucleolar dominance in plants is correlated with physical characteristics of rRNA gene intergenic spacers is not supported in Brassica. Furthermore, in Brassica napus, rRNA genes silenced in vegetative tissues were found to be expressed in all floral organs, including sepals and petals, arguing against the hypothesis that passage through meiosis is needed to reactivate suppressed genes. Instead, the transition of inflorescence to floral meristem appears to be a developmental stage when silenced genes can be derepressed.

Base Sequence↗
PubMed1997

Cytokinin induction of RNA polymerase I transcription in Arabidopsis thaliana.

RNA polymerase I (pol I) transcribes the repeated genes that encode the precursor of 17-18, 5.8, and 25-28 S ribosomal RNA (rRNA). Pol I transcription is up-regulated in growing cells and down-regulated in quiescent cells, presumably reflecting the demand for ribosomes and protein synthesis. However, the signal transduction pathways responsible for pol I regulation are poorly understood. We tested the effects of exogenously applied plant hormones on promoter-dependent rRNA transcription in Arabidopsis thaliana. Gibberellic acid, abscisic acid, auxin, and ethylene had no detectable effect on rRNA transcription, but kinetin (a cytokinin) stimulated rRNA transcription within 1 h of treatment. Increased steady-state levels of accurately initiated rRNA transcripts, detected by S1 nuclease protection, were paralleled by increased levels of nascent rRNA transcripts in isolated nuclei. Therefore, the primary effect of cytokinin appears to be at the level of transcription initiation rather than rRNA stability. Pol I accounts for approximately 34% of total nuclear transcription in untreated plants and approximately 60% following cytokinin treatment. The specific responsiveness of pol I transcription to kinetin suggests that cytokinins may act as general regulators of protein synthetic capacity and growth status in plant cells.

Adenine↗
PubMed1997

Nucleosome binding by the polymerase I transactivator upstream binding factor displaces linker histone H1.

Upstream binding factor (UBF) is a vertebrate RNA polymerase I transcription factor that can bend and wrap DNA. To investigate UBF's likely role as an architectural protein of rRNA genes organized in chromatin, we tested UBF's ability to bind rRNA gene enhancers assembled into nucleosome cores (DNA plus core histones) and nucleosomes (DNA plus core histones plus histone H1). UBF bound with low affinity to nucleosome cores formed with enhancer DNA probes of 162 bp. However, on nucleosome cores which contained approximately 60 bp of additional linker DNA, UBF bound with high affinity similar to its binding to naked DNA, forming a ternary DNA-core histone-UBF complex. UBF could be stripped from ternary complexes with competitor DNA to liberate nucleosome cores, rather than free DNA, suggesting that UBF binding to nucleosome cores does not displace the core histones H2A, H2B, H3, and H4. DNase I, micrococcal nuclease, and exonuclease III footprinting suggests that UBF and histone H1 interact with DNA on both sides flanking the histone octamer. Footprinting shows that UBF outcompetes histone H1 for binding to a nucleosome core and will displace, if not dissociate, H1 from its binding site on a preassembled nucleosome. These data suggest that UBF may act to prevent or reverse the assembly of transcriptionally inactive chromatin structures catalyzed by linker histone binding.

Animals↗
PubMed1996

Species-specificity of rRNA gene transcription in plants manifested as a switch in RNA polymerase specificity.

Rapid evolution of ribosomal RNA (rRNA) gene promoters often prevents their recognition in a foreign species. Unlike animal systems, we show that foreign plant rRNA gene promoters are recognized in an alien species, but tend to program transcription by a different polymerase. In plants, RNA polymerase I transcripts initiate at a TATATA element (+1 is underlined) important for promoter strength and start-site selection. However, transcripts initiate from +32 following transfection of a tomato promoter into Arabidopsis. The rRNA gene promoter of a more closely related species, Brassica oleracea, programs both +1 and +29 transcription. A point mutation at +2 improving the identity between the Brassica and Arabidopsis promoters increases +1 transcription, indicating a role for the initiator element in species-specificity. Brassica +29 transcripts can be translated to express a luciferase reporter gene, implicating RNA polymerase II. TATA mutations that disrupt TATA-binding protein (TBP) interactions inhibit +29 transcription and luciferase expression. Co-expressed TBP proteins bearing compensatory mutations restore +29 transcription and luciferase activity, suggesting a direct TBP-TATA interaction. Importantly, +1 transcription is unaffected by the TATA mutations, suggesting that in the context of pol I recognition, the TATA-containing initiator element serves a function other than TBP binding.

Arabidopsis↗
PubMed1996

RFLP and physical mapping with an rDNA-specific endonuclease reveals that nucleolus organizer regions of Arabidopsis thaliana adjoin the telomeres on chromosomes 2 and 4.

Ribosomal RNA genes are organized in tandem arrays called nucleolus organizer regions (NORs). In a prior study, RFLP mapping on pulsed-field gels placed NOR2 at the northern tip of Arabidopsis thaliana chromosome 2. New polymorphisms have allowed the other NOR, NOR4, to be mapped to the northern tip of chromosome 4. To map NOR-associated loci, rDNA-specific cleavage by I-Ppol, an endonuclease with a 15 nucleotide recognition sequence involved in rDNA-homing of a mobile, self-splicing Group I intron in Physarum was exploited. I-Ppol digestion of A. thaliana genomic DNA liberated two telomere-containing fragments no larger than 13 kbp, and telomere polymorphisms identified using I-Ppol cosegregated with NOR2 and NOR4. Restriction mapping suggested that telomere-proximal rRNA genes are oriented with their 5' ends nearest the chromosome ends and their 3' ends nearest the centromere. This orientation was confirmed using the polymerase chain reaction to clone one of the telomere-rDNA junctions, most likely the junction on chromosome 4. The telomeric repeats join the terminal rRNA gene downstream of its promoter, suggesting that this first gene is inactive. Subtelomeric repetitive DNAs are absent at the telomere-rDNA junction. Localization of NOR2, NOR4 and their associated telomeres, TEL2N and TEL4N, respectively, provides end points for the genetic and physical maps of chromosomes 2 and 4.

Arabidopsis↗
PubMed1996

Two-dimensional RFLP analyses reveal megabase-sized clusters of rRNA gene variants in Arabidopsis thaliana, suggesting local spreading of variants as the mode for gene homogenization during concerted evolution.

Eukaryotic genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNA (rRNA genes or rDNA) are virtually identical within a species, yet they evolve rapidly between species, a phenomenon known as concerted evolution. The mechanisms by which sequence homogenization and fixation of new rRNA gene variants occurs within a genome are not clear. In diploid Arabidopsis thaliana, approximately 1500 rRNA genes are tandemly arrayed at two nucleolus organizer regions, one on chromosome 2 (NOR2), the other on chromosome 4 (NOR4). This paper shows that NOR2 and NOR4 are similar in size, each spanning approximately 3.5-4.0 Mbp. Using two-dimensional mapping techniques involving a combination of pulsed-field and conventional gel electrophoresis, the distributions of four distinct rRNA gene variants at NOR2 and NOR4 have been determined. rRNA genes at NOR4 are homogeneous with respect to a HindIII site occurring once per gene. In contrast, fewer than 10% of the rRNA genes at NOR2 are HindIII-bearing variants. A single intergenic spacer length is found among rRNA genes at NOR2 but three classes of spacer length variants are present at NOR4. The NOR4 variants are not intermingled with one another; instead, they are highly clustered over distances as large as 1.5 Mbp. These data suggest that in the concerted evolution of rRNA genes, homogenization is a consequence of local spreading of new rRNA gene variants.

Arabidopsis↗
PubMed1995

The minimal ribosomal RNA gene promoter of Arabidopsis thaliana includes a critical element at the transcription initiation site.

The genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNAs are transcribed in the nucleolus by RNA polymerase I. Unlike rRNA gene promoters in animals which differ substantially across species boundaries, plant rRNA gene promoters share sequence similarity for several nucleotides upstream and downstream of the transcription start site (+ 1). The conserved sequence consists of a near-consensus TATA box, a critical promoter element of most genes transcribed by RNA polymerase II and certain genes transcribed by RNA polymerase III, followed by a cluster of four to six guanosines. Using transient expression of cloned promoter deletions in Arabidopsis thaliana protoplasts, it is shown that the 5' boundary of the Arabidopsis rRNA gene promoter is located between -55 and -33 and the 3' promoter boundary is approximately +6. The critical role of the TATA element at the initiation site was demonstrated by altering this region using tandem point mutations within constructs containing essentially complete intergenic spacer sequences from -2590 to +6. The initiation site mutations either abolished transcription or dramatically reduced transcript abundance relative to the wild-type promoter. In some mutants, transcription initiation was shifted to a new site, suggesting a role for the TATA-like initiator region in both start site selection and promoter strength. It is suggested that minimal rRNA gene promoters might be similar in design to minimal promoters of protein-encoding genes, many of which utilize an initiator element, or INR, as an important promoter element located directly at the transcription start site.

Arabidopsis↗