Transcription and FACT facilitate the restoration of replication-coupled chromatin assembly defects.

Genome duplication occurs through the coordinated action of DNA replication and nucleosome assembly at replication forks. Defective nucleosome assembly causes DNA lesions by fork breakage that need to be repaired. In addition, it causes a loss of chromatin integrity. These chromatin alterations can be restored, even though the mechanisms are unknown. Here, we show that the process of chromatin restoration can deal with highly severe chromatin defects induced by the absence of the chaperones CAF1 and Rtt106 or a strong reduction in the pool of available histones, and that this process can be followed by analyzing the topoisomer distribution of the 2µ plasmid. Using this assay, we demonstrate that chromatin restoration is slow and independent of checkpoint activation, whereas it requires the action of transcription and the FACT complex. Therefore, cells are able to "repair" not only DNA lesions but also chromatin alterations associated with defective nucleosome assembly.

Human prefoldin modulates co-transcriptional pre-mRNA splicing.

Prefoldin is a heterohexameric complex conserved from archaea to humans that plays a cochaperone role during the co-translational folding of actin and tubulin monomers. Additional functions of prefoldin have been described, including a positive contribution to transcription elongation and chromatin dynamics in yeast. Here we show that prefoldin perturbations provoked transcriptional alterations across the human genome. Severe pre-mRNA splicing defects were also detected, particularly after serum stimulation. We found impairment of co-transcriptional splicing during transcription elongation, which explains why the induction of long genes with a high number of introns was affected the most. We detected genome-wide prefoldin binding to transcribed genes and found that it correlated with the negative impact of prefoldin depletion on gene expression. Lack of prefoldin caused global decrease in Ser2 and Ser5 phosphorylation of the RNA polymerase II carboxy-terminal domain. It also reduced the recruitment of the CTD kinase CDK9 to transcribed genes, and the association of splicing factors PRP19 and U2AF65 to chromatin, which is known to depend on CTD phosphorylation. Altogether the reported results indicate that human prefoldin is able to act locally on the genome to modulate gene expression by influencing phosphorylation of elongating RNA polymerase II, and thereby regulating co-transcriptional splicing.

CYP3A5 is unlikely to mediate anticancer drug resistance in hepatocellular carcinoma.

Recently, it was published that CYP3A5 contributes to chemotherapeutic drug resistance in a wide range of solid tumors, including hepatocellular carcinoma. However, CYP3A5 is highly polymorphic and 90% of Caucasians are homozygous for the loss-of-function allele CYP3A5*3. Here, we evaluate the relationship between CYP3A5 genotype and expression level of both CYP3A5 transcripts and protein in biopsies from 19 pairs of liver tumors and corresponding peritumoral tissue. We find that CYP3A5 transcript levels are reduced compared with peritumoral controls. Moreover, we do not detect CYP3A5 protein in homozygous CYP3A5*3 carriers and no relative increase of CYP3A5 in tumoral tissue of CYP3A5*1 carriers. We conclude that anticancer drug resistance is unlikely to be caused by increased CYP3A5 expression.