DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae.

To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

A new marker set that identifies fetal cells in maternal circulation with high specificity.

OBJECTIVE: Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking. METHOD: Blood samples from 78 normal pregnancies were used for testing the marker-set CD105 and CD141 for fetal cell enrichment. Fetal cell candidates were subsequently stained by a cocktail of cytokeratin antibodies, and the gender of the fetal cells was explored by fluorescence in situ hybridization (FISH) of the X and Y chromosomes. RESULTS: Fetal cell candidates could be detected in 91% of the samples, and in 85% of the samples, it was possible to obtain X and Y chromosomal FISH results for gender determination. The concordance between gender determined by FISH on fetal cells in maternal blood and gender found at birth reached 100% if three or more fetal cells with FISH signals could be found in a sample. CONCLUSION: The marker set identifies fetal cells with specificity high enough to make cell-based noninvasive prenatal diagnosis realistic.

Characterization of fetal cells from the maternal circulation by microarray gene expression analysis--could the extravillous trophoblasts be a target for future cell-based non-invasive prenatal diagnosis?

INTRODUCTION: Circulating fetal cells in maternal blood provide a tool for risk-free, non-invasive prenatal diagnosis. However, fetal cells in the maternal circulation are scarce, and to effectively isolate enough of them for reliable diagnostics, it is crucial to know which fetal cell type(s) should be targeted. MATERIALS AND METHODS: Fetal cells were enriched from maternal blood by magnetic-activated cell sorting using the endothelial cell marker CD105 and identified by XY fluorescence in situ hybridization. Expression pattern was compared between fetal cells and maternal blood cells using stem cell microarray analysis. RESULTS: 39 genes were identified as candidates for unique fetal cell markers. More than half of these are genes known to be expressed in the placenta, especially in extravillous trophoblasts (EVTs). Immunohistochemical staining of placental tissue confirmed CD105 staining in EVTs and 76% of fetal cells enriched by CD105 were found to be cytokeratin-positive. DISCUSSION: The unique combination of mesodermal (CD105) and ectodermal (cytokeratin) markers in EVTs could be a potential marker set for cell enrichment of this cell type in maternal blood and could be the basis for future cell-based non-invasive prenatal diagnosis.

Identification of circulating fetal cell markers by microarray analysis.

OBJECTIVE: Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester. METHOD: Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem cell microarray analysis. RESULTS: Twenty-eight genes were identified as fetal cell marker candidates. CONCLUSION: Of the 28 fetal marker candidate genes, five coded for proteins, which are located on the outer surface of the cell membrane and not expressed in blood. The protein product of these five genes, MMP14, MCAM, KCNQ4, CLDN6, and F3, may be used as markers for fetal cell enrichment.

The QTK loop is essential for the communication between the N-terminal atpase domain and the central cleavage--ligation region in human topoisomerase IIalpha.

We have characterized a human topoisomerase IIalpha enzyme with a deletion of the conserved QTK loop, which extends from the transducer domain to the ATP-binding pocket in the GHKL domain. The loop has been suggested to play a role for interdomain communication in type II topoisomerases. The mutant enzyme performs only very low levels of strand passage, although it is able to cleave and ligate DNA as well as close the N-terminal clamp. Cleavage is nearly unaffected by ATP and ATP analogues relative to the wild-type enzyme. Although the enzyme is able to close the clamp, the clamp has altered characteristics, allowing trapping of DNA also in the absence of an ATP analogue. The enzyme furthermore retains intrinsic levels of ATPase activity, but the activity is not stimulated by DNA. Our observations demonstrate that the QTK loop is an important player for the interdomain communication in human topoisomerase IIalpha. First, the loop seems to play a role in keeping the N-terminal clamp in an open conformation when no nucleotide is present. Once the nucleotide binds, it facilitates clamp closure, although it is not essential for this event. The QTK loop, in contrast, is essential for the DNA-stimulated ATPase activity of human topoisomerase IIalpha.

Bimodal recognition of DNA geometry by human topoisomerase II alpha: preferential relaxation of positively supercoiled DNA requires elements in the C-terminal domain.

Human topoisomerase IIalpha, but not topoisomerase IIbeta, can sense the geometry of DNA during relaxation and removes positive supercoils >10-fold faster than it does negative superhelical twists. In contrast, both isoforms maintain lower levels of DNA cleavage intermediates with positively supercoiled substrates. Since topoisomerase IIalpha and IIbeta differ primarily in their C-terminal domains (CTD), this portion of the protein may play a role in sensing DNA geometry. Therefore, to more fully assess the importance of the topoisomerase IIalpha CTD in the recognition of DNA topology, hTop2alphaDelta1175, a mutant human enzyme that lacks its CTD, was examined. The mutant enzyme relaxed negative and positive supercoils at similar rates but still maintained lower levels of cleavage complexes with positively supercoiled DNA. Furthermore, when the CTD of topoisomerase IIbeta was replaced with that of the alpha isoform, the resulting enzyme preferentially relaxed positively supercoiled substrates. In contrast, a chimeric topoisomerase IIalpha that carried the CTD of the beta isoform lost its ability to recognize the geometry of DNA supercoils during relaxation. These findings demonstrate that human topoisomerase IIalpha recognizes DNA geometry in a bimodal fashion, with the ability to preferentially relax positive DNA supercoils residing in the CTD. Finally, results with a series of human topoisomerase IIalpha mutants suggest that clusters of positively charged amino acid residues in the CTD are required for the enzyme to distinguish supercoil geometry during DNA relaxation and that deletion of even the most C-terminal cluster abrogates this recognition.