Arnold Kristjuhan

Extraction of genomic DNA from yeasts for PCR-based applications

We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.

Oligomerization of p53 is necessary to inhibit its transcriptional transactivation property at high protein concentration

We have previously shown that transactivation by tumor suppressor protein p53 can be inhibited in vivo at elevated protein concentrations. In this study we characterize the structural requirements of this function. We show that oligomerization domain of p53 is involved in loss of transactivation at high protein concentrations: mutants not able to oligomerize are neither able to suppress transactivation, although these transactivating properties can be untouched.

Acetylation of H3 K56 Is Required for RNA Polymerase II Transcript Elongation through Heterochromatin in Yeast

ABSTRACT In Saccharomyces cerevisiae SIR proteins mediate transcriptional silencing, forming heterochromatin structures at repressed loci. Although recruitment of transcription initiation factors can occur even to promoters packed in heterochromatin, it is unclear whether heterochromatin inhibits RNA polymerase II (RNAPII) transcript elongation. To clarify this issue, we recruited SIR proteins to the coding region of an inducible gene and characterized the effects of the heterochromatic structure on transcription. Surprisingly, RNAPII is fully competent for transcription initiation and elongation at the locus, leading to significant loss of heterochromatin proteins from the region. A search for auxiliary factors required for transcript elongation through the heterochromatic locus revealed that two proteins involved in histone H3 lysine 56 acetylation, Rtt109 and Asf1, are needed for efficient transcript elongation by RNAPII. The efficiency of transcription through heterochromatin is also impaired in a strain carrying the K56R mutation in histone H3. Our results show that H3 K56 modification is required for efficient transcription of heterochromatic locus by RNAPII, and we propose that transcription-coupled incorporation of H3 acetylated K56 (acK56) into chromatin is needed for efficient opening of heterochromatic loci for transcription.

Chromatin-dependent and -independent regulation of DNA replication origin activation in budding yeast.

To elucidate the role of the chromatin environment in the regulation of replication origin activation, autonomously replicating sequences were inserted into identical locations in the budding yeast genome and their activation times in S phase determined. Chromatin‐dependent origins adopt to the firing time of the surrounding locus. In contrast, the origins containing two binding sites for Forkhead transcription factors are activated early in the S phase regardless of their location in the genome. Our results also show that genuinely late‐replicating parts of the genome can be converted into early‐replicating loci by insertion of a chromatin‐independent early replication origin, ARS607, whereas insertion of two Forkhead‐binding sites is not sufficient for conversion.

p53 protein accumulation in addition to the transactivation activity is required for p53-dependent cell cycle arrest after treatment of cells with camptothecin.

In this study we characterize the connections between p53-dependent G1 cell cycle arrest, transcriptional activation of the protein and the increase of its intracellular steady-state concentration. Several cell lines expressing wild-type p53 protein were treated with increasing concentrations of DNA-damaging drug camptothecin. Lower doses of the drug caused transcriptional activation of p53, but no accumulation of the protein was detected. Only after a certain threshold dose of camptothecin does the amount of the protein rapidly increase and reach its plateau levels. The threshold dose was different for different cell lines, but the general non-linear profile was similar. Increase of p53 level was accompanied by additional transcriptional activation of some p53 target genes (i.e. waf1), but not the others (mdm2). We demonstrate here that transcriptional activation of p53 after the treatment of camptothecin is not sufficient to cause p53-dependent G1 cell cycle arrest. The latter is observable only after the inrease of steady-state level of p53. Low drug concentrations, although accompanied by transcriptional activation of p53, do not cause either p53 protein accumulation nor cell cycle arrest at G1. We propose a model for p53 acting as a part of cellular sensor system detecting the severity of DNA damage.

Relicensing of transcriptionally inactivated replication origins in budding yeast.

DNA replication origins are licensed in early G1 phase of the cell cycle where the origin recognition complex (ORC) recruits the minichromosome maintenance (MCM) helicase to origins. These pre-replicative complexes (pre-RCs) remain inactive until replication is initiated in the S phase. However, transcriptional activity in the regions of origins can eliminate their functionality by displacing the components of pre-RC from DNA. We analyzed genome-wide data of mRNA and cryptic unstable transcripts in the context of locations of replication origins in yeast genome and found that at least one-third of the origins are transcribed and therefore might be inactivated by transcription. When investigating the fate of transcriptionally inactivated origins, we found that replication origins were repetitively licensed in G1 to reestablish their functionality after transcription. We propose that reloading of pre-RC components in G1 might be utilized for the maintenance of sufficient number of competent origins for efficient initiation of DNA replication in S phase.

Distribution and Maintenance of Histone H3 Lysine 36 Trimethylation in Transcribed Locus

Post-translational modifications of core histones play an important role in the epigenetic regulation of chromatin dynamics and gene expression. In Saccharomyces cerevisiae methylation marks at K4, K36, and K79 of histone H3 are associated with gene transcription. Although Set2-mediated H3K36 methylation is enriched throughout the coding region of active genes and prevents aberrant transcriptional initiation within coding sequences, it is not known if transcription of one locus impacts the methylation pattern of neighbouring areas and for how long H3K36 methylation is maintained after transcription termination. Our results demonstrate that H3K36 methylation is restricted to the transcribed sequence only and the modification does not spread to adjacent loci downstream from transcription termination site. We also show that H3K36 trimethylation mark persists in the locus for at least 60 minutes after transcription inhibition, suggesting a short epigenetic memory for recently occurred transcriptional activity. Our results indicate that both replication-dependent exchange of nucleosomes and the activity of histone demethylases Rph1, Jhd1 and Gis1 contribute to the turnover of H3K36 methylation upon shut-down of transcription.

Transcriptional repression of the Ahr gene by LHCGR signaling in preovulatory granulosa cells is controlled by chromatin accessibility.

Recent advances in establishing the role of the aryl hydrocarbon receptor (Ahr) in normophysiology have discovered its fundamental role, amongst others, in female reproduction. Considering previous studies suggesting the hormonal modulation of Ahr, we aimed to investigate whether in murine granulosa cells (GCs) the gonadotropins regulate Ahr expression and how this is mechanistically implemented. We found that the FSH-like substance--pregnant mare serum gonadotropin--led to stimulation of Ahr expression. More importantly hCG produced relatively rapid reduction of Ahr mRNA in GCs of preovulatory follicles. We show for the first time that LHCGR signaling in regulating the Ahr message involves protein kinase A pathway and is attributable to decreased transcription rate. Finally, we found that Ahr promoter accessibility was decreased by hCG, implicating chromatin remodeling in Ahr gene regulation by LH.

Uniform Distribution of Elongating RNA Polymerase II Complexes in Transcribed Gene Locus

The intensity of gene transcription is generally reflected by the level of RNA polymerase II (RNAPII) recruitment to the gene. However, genome-wide studies of polymerase occupancy indicate that RNAPII distribution varies among genes. In some loci more polymerases are found in the 5′ region, whereas in other loci, in the 3′ region of the gene. We studied the distribution of elongating RNAPII complexes at highly transcribed GAL-VPS13 locus in Saccharomyces cerevisiae and found that in the cell population the amount of polymerases gradually decreased toward the 3′ end of the gene. However, the conventional chromatin immunoprecipitation assay averages the signal from the cell population, and no data on single cell level can be gathered. To study the spacing of elongating polymerases on single chromosomes, we used a sequential chromatin immunoprecipitation assay for the detection of multiple RNAPII complexes on the same DNA fragment. Our results demonstrate uniform distribution of elongating polymerases throughout all regions of the GAL-VPS13 gene.

Recruitment of Fkh1 to replication origins requires precisely positioned Fkh1/2 binding sites and concurrent assembly of the pre-replicative complex

In budding yeast, activation of many DNA replication origins is regulated by their chromatin environment, whereas others fire in early S phase regardless of their chromosomal location. Several location-independent origins contain at least two divergently oriented binding sites for Forkhead (Fkh) transcription factors in close proximity to their ARS consensus sequence. To explore whether recruitment of Forkhead proteins to replication origins is dependent on the spatial arrangement of Fkh1/2 binding sites, we changed the spacing and orientation of the sites in early replication origins ARS305 and ARS607. We followed recruitment of the Fkh1 protein to origins by chromatin immunoprecipitation and tested the ability of these origins to fire in early S phase. Our results demonstrate that precise spatial and directional arrangement of Fkh1/2 sites is crucial for efficient binding of the Fkh1 protein and for early firing of the origins. We also show that recruitment of Fkh1 to the origins depends on formation of the pre-replicative complex (pre-RC) and loading of the Mcm2-7 helicase, indicating that the origins are regulated by cooperative action of Fkh1 and the pre-RC. These results reveal that DNA binding of Forkhead factors does not depend merely on the presence of its binding sites but on their precise arrangement and is strongly influenced by other protein complexes in the vicinity.