Heterochromatin is important for gene regulation and chromosome structure, but the genes that are occupied by heterochromatin proteins in the mammalian genome are largely unknown. We have adapted the DamID method to systematically identify target genes of the heterochromatin proteins HP1 and SUV39H1 in human and mouse cells. Unexpectedly, we found that CBX1 (formerly HP1␤) and SUV39H1 bind to genes encoding KRAB domain containing zinc finger (KRAB-ZNF) transcriptional repressors. These genes constitute one of the largest gene families and are organized in clusters in the human genome. Preference of CBX1 for this gene family was observed in both human and mouse cells. High-resolution mapping on human chromosome 19 revealed that CBX1 coats large domains 0.1-4 Mb in size, which coincide with the position of KRAB-ZNF gene clusters. These domains show an intricate CBX1 binding pattern: While CBX1 is globally elevated throughout the domains, it is absent from the promoters and binds more strongly to the 3Ј ends of KRAB-ZNF genes. KRAB-ZNF domains contain large numbers of LINE elements, which may contribute to CBX1 recruitment. These results uncover a surprising link between heterochromatin and a large family of regulatory genes in mammals. We suggest a role for heterochromatin in the evolution of the KRAB-ZNF gene family.

In an attempt to identify subtypes of breast cancer and pinpoint patterns of cell cycle regulatory defects associated with clinical behaviour, proliferation and other transformation associated events, a multitude of cell cycle regulatory proteins were analysed in a material of 113 primary breast cancers. Increased proliferation was observed in two different scenarios; (1) with high cyclin D1 and elevated retinoblastoma protein (pRb) phosphorylation, (cyclin D1 high tumours) or (2) with high cyclin E protein but low cyclin D1 and lack of corresponding pRb phosphorylation (cyclin E high tumours) indicative of an interrupted pRb pathway. Characteristic for cyclin E high tumours were further defects in p53, p27 and bcl-2, while c-erbB2 overexpression and c-myc amplification was found in both cyclin D1high and E high tumours. Using transfected cell lines overexpressing cyclin E, cyclin E high and D1 high tumours were mimicked and the cyclin D1 high cell line normalized the cyclin E kinase activity by an induction and redirection of p21 and p27 to the cyclin E complex whereas cyclin E high cell lines obtained increased kinase activity without redirection of inhibitors. Based on differences in genetic aberrations as well as function of the pRb node we therefore propose a model in which cyclin D1 high and cyclin E high tumours represent two alternative mechanisms to inactivate the pRb pathway and thereby achieve unrestrained growth in the tumorogenesis of breast cancer. Oncogene (2002Oncogene ( ) 21, 4680 -4690. doi:10.1038 Keywords: breast cancer; cell cycle; cyclins; cdkinhibitors; pRb pathway; proliferation IntroductionThe G1/S transition in normal cells is a thoroughly controlled checkpoint where the important decision to initiate DNA-replication or not is taken (Draetta, 1994;Weinberg, 1995). As has been obvious the last couple of years, aberrations in G1/S regulatory proteins are common in various tumours and aberrant expression of cyclin E and D1, downregulation of p16 and p27 as well as mutation of the retinoblastoma gene (Rb) has frequently been observed in several cancers and it can be hypothesized that G1/S defects might be obligatory in tumour development (Landberg and Roos, 1997;Sandhu and Slingerland, 2000).Cyclin D1 links mitogenic signals to cell cycle progression through increased phosphorylation of pRb (Lukas et al., 1996). Amplification of the encoding cyclin D1 gene, CCND1, resulting in high cyclin D1 protein content, has been observed in a significant numbers of breast cancers and together with impaired cyclin D1 protein degradation potentially cause unbalanced phosphorylation of pRb (Sherr, 1996;Russell et al., 1999). The relation between cyclin D1 and in vivo pRb phosphorylation in primary breast cancer has nevertheless not been investigated. Overexpression of cyclin D1 has been associated with ERpositivity (Michalides et al., 1996;van Diest et al., 1997), while the relation to proliferation and survival has been without consensus (Jares et al., 1997;Barnes and Gillett, 1998). Nevertheless, simila...

BackgroundDiagnosis of leptospirosis by the microscopic agglutination test (MAT) or by culture is confined to specialized laboratories. Although ELISA techniques are more common, they still require laboratory facilities. Rapid Diagnostic Tests (RDTs) can be used for easy point-of-care diagnosis. This study aims to evaluate the diagnostic performance of the RDTs LeptoTek Dri Dot, LeptoTek Lateral Flow, and Leptocheck-WB, prospectively.MethodologyDuring 2001 to 2012, one or two of the RDTs at the same time have been applied prior to routine diagnostics (MAT, ELISA and culture) on serum specimens from participants sent in for leptospirosis diagnosis. The case definition was based on MAT, ELISA and culture results. Participants not fulfilling the case definition were considered not to have leptospirosis. The diagnostic accuracy was determined based on the 1st submitted sample and paired samples, either in an overall analysis or stratified according to days post onset of illness.ResultsThe overall sensitivity and specificity for the LeptoTek Dri Dot was 75% respectively 96%, for the LeptoTek Lateral Flow 78% respectively 95%, and for the Leptocheck-WB 78% respectively 98%. Based on the 1st submitted sample the sensitivity was low (51% for LeptoTek Dri Dot, 69% for LeptoTek Lateral Flow, and 55% for Leptocheck-WB), but substantially increased when the results of paired samples were combined, although accompanied by a lower specificity (82% respectively 91% for LeptoTek Dri Dot, 86% respectively 84% for LeptoTek Lateral Flow, and 80% respectively 93% for Leptocheck-WB).ConclusionsAll three tests present antibody tests contributing to the diagnosis of leptospirosis, thus supporting clinical suspicion and contributing to awareness. Since the overall sensitivity of the tested RDTs did not exceed 80%, one should be cautious to rely only on an RDT result, and confirmation by reference tests is strongly recommended.

The RH-MLPA genotyping assay is a fast, easy, and reliable method to determine almost all clinically relevant RHD and RHCE variant alleles, RHD zygosity, and RHD+/RHD- chimeras in blood donors, blood recipients, and pregnant women.