An optimization framework for unsupervised identification of rare copy number variation from SNP array data

Background

Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with active and inactive genes. There is growing evidence that genes that respond to environmental or developmental signals may possess distinct chromatin marks. Using a T cell model and both genome-wide and gene-focused approaches, we examined the chromatin characteristics of genes that respond to T cell activation.

Results

To facilitate comparison of genes with similar basal expression levels, we used expression-profiling data to bin genes according to their basal expression levels. We found that inducible genes in the lower basal expression bins, especially rapidly induced primary response genes, were more likely than their non-responsive counterparts to display the histone modifications of active genes, have RNA polymerase II (Pol II) at their promoters and show evidence of ongoing basal elongation. There was little or no evidence for the presence of active chromatin marks in the absence of promoter Pol II on these inducible genes. In addition, we identified a subgroup of genes with active promoter chromatin marks and promoter Pol II but no evidence of elongation. Following T cell activation, we find little evidence for a major shift in the active chromatin signature around inducible gene promoters but many genes recruit more Pol II and show increased evidence of elongation.

Conclusions

These results suggest that the majority of inducible genes are primed for activation by having an active chromatin signature and promoter Pol II with or without ongoing elongation.     

Phylogeny of parasitic parabasalia and free-living relatives inferred from conventional markers vs. Rpb1, a single-copy gene

Background

Parabasalia are single-celled eukaryotes (protists) that are mainly comprised of endosymbionts of termites and wood roaches, intestinal commensals, human or veterinary parasites, and free-living species. Phylogenetic comparisons of parabasalids are typically based upon morphological characters and 18S ribosomal RNA gene sequence data (rDNA), while biochemical or molecular studies of parabasalids are limited to a few axenically cultivable parasites. These previous analyses and other studies based on PCR amplification of duplicated protein-coding genes are unable to fully resolve the evolutionary relationships of parabasalids. As a result, genetic studies of Parabasalia lag behind other organisms.

Principal Findings

Comparing parabasalid EF1α, α-tubulin, enolase and MDH protein-coding genes with information from the Trichomonas vaginalis genome reveals difficulty in resolving the history of species or isolates apart from duplicated genes. A conserved single-copy gene encodes the largest subunit of RNA polymerase II (Rpb1) in T. vaginalis and other eukaryotes. Here we directly sequenced Rpb1 degenerate PCR products from 10 parabasalid genera, including several T. vaginalis isolates and avian isolates, and compared these data by phylogenetic analyses. Rpb1 genes from parabasalids, diplomonads, Parabodo, Diplonema and Percolomonas were all intronless, unlike intron-rich homologs in Naegleria, Jakoba and Malawimonas.

Conclusions/Significance

The phylogeny of Rpb1 from parasitic and free-living parabasalids, and conserved Rpb1 insertions, support Trichomonadea, Tritrichomonadea, and Hypotrichomonadea as monophyletic groups. These results are consistent with prior analyses of rDNA and GAPDH sequences and ultrastructural data. The Rpb1 phylogenetic tree also resolves species- and isolate-level relationships. These findings, together with the relative ease of Rpb1 isolation, make it an attractive tool for evaluating more extensive relationships within Parabasalia.     

RNA polymerase II degradation in response to rapamycin is not mediated through ubiquitylation

In Saccharomyces cerevisiae, the immunosuppressor rapamycin engenders the degradation of excessive RNA polymerase II leading to growth arrest but the regulation of this process is not known yet. Here, we show that this mechanism is dependent on the peptidyl prolyl cis/trans isomerase Rrd1. Strikingly this degradation is independent of RNA polymerase II polyubiquitylation and does not require the elongation factor Elc1. Our data reveal that there are at least two alternative pathways to degrade RNA polymerase II that depend on different type of stresses.     

A simple solubilization method for loosely and tightly chromatin-bound RNA polymerase II from rat liver nuclei

Rat liver chromatin-bound RNA polymerase II could be differentially solubilized into two distinct populations, loosely and tightly bound enzymes, by a simple method. By this method the recovery of the solubilized enzyme from the chromatin fraction could be increased considerably as compared with the procedure of Yu (1). The two chromatin-bound enzymes had different properties:

1. Loosely bound enzyme was easily extractable from chromatin with relatively mild ionic condition (0.5 M NaCl); the tightly bound enzyme had to be solubilized by more drastic conditions such as sonication or nuclease treatment.
2. Loosely bound enzyme could not efficiently transcribe the chromatin template, but the tightly bound enzyme was active toward the same template. The latter enzyme is involved in the tight complex with the RNA synthesis activating factors.
3. Cycloheximide treatment in vivo suggests that the two enzymes have different turn-over rates.

Therefore, with this simple solubilization method the functionally different two chromatin-bound RNA polymerase II activities can be estimated.