CTCF (CCCTC-binding factor) binds sites around the mouse ␤-globin locus that spatially cluster in the erythroid cell nucleus. We show that both conditional deletion of CTCF and targeted disruption of a DNA-binding site destabilize these long-range interactions and cause local loss of histone acetylation and gain of histone methylation, apparently without affecting transcription at the locus. Our data demonstrate that CTCF is directly involved in chromatin architecture and regulates local balance between active and repressive chromatin marks. We postulate that throughout the genome, relative position and stability of CTCF-mediated loops determine their effect on enhancer-promoter interactions, with gene insulation as one possible outcome. Chromatin insulators are DNA sequences that confer autonomous expression on genes by protecting them against inadvertent signals coming from neighboring chromatin. CTCF (CCCTC-binding factor) is the prototype vertebrate protein exhibiting insulator activity that can act as an enhancer blocker or as a barrier against repressive forces from nearby heterochromatin in vitro (Defossez and Gilson 2002; RecillasTarga et al. 2002). In vivo, CTCF binds to the imprinting control region of the H19/insulin-like growth factor (Igf2) locus, where it acts as a methylation-sensitive enhancer blocker (Bell and Felsenfeld 2000;Hark et al. 2000). Moreover, CTCF-binding sites have been foundand its insulator activity has been anticipated-at the imprinting center that determines choice of X inactivation (Chao et al. 2002), at boundaries of domains that escape X inactivation (Filippova et al. 2005), and at sites flanking CTG/CAG repeats at the DM1 locus (Filippova et al. 2001). CTCF was first defined as an insulator protein when it was found to be required for the enhancerblocking activity of a hypersensitive site 5Ј of the chicken ␤-globin locus (5ЈHS4) (Bell et al. 1999). A similar CTCF-dependent insulator site was subsequently found at the 3Ј end of the locus and both sites coincide with erythroid-specific transitions in DNase I sensitivity of chromatin (Saitoh et al. 2000). Such observations suggested that CTCF partitions the genome in physically distinct domains of gene expression. The molecular mechanism underlying CTCF's insulating activity is still unknown.CTCF-binding sites also flank the human and mouse ␤-globin locus (Fig. 1A), which contains a number of developmentally regulated, erythroid-specific ␤-globin genes and an upstream locus control region (LCR) required for high ␤-globin expression levels. In mice, three CTCF-binding sites have been identified upstream (HS-85, HS-62, and HS5) and one downstream (3ЈHS1) of the locus (Farrell et al. 2002;Bulger et al. 2003). Previously, we applied chromosome conformation capture (3C) technology (Dekker et al. 2002) to study long-range DNA interactions between these and other sites in the ␤-globin locus. In erythroid cells, the CTCF-binding sites (including HS-85; see below) were found to participate in spatial interactions between the LCR and th...

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The shape of the genome is thought to play an important part in the coordination of transcription and other DNA-metabolic processes. Chromosome conformation capture (3C) technology allows us to analyze the folding of chromatin in the native cellular state at a resolution beyond that provided by current microscopy techniques. It has been used, for example, to demonstrate that regulatory DNA elements communicate with distant target genes through direct physical interactions that loop out the intervening chromatin fiber. Here we discuss the intricacies of 3C and new 3C-based methods including the 4C, 5C and ChIP-loop assay.

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Expression of the ␤-globin genes proceeds from basal to exceptionally high levels during erythroid differentiation in vivo. High expression is dependent on the locus control region (LCR) and coincides with more frequent LCR-gene contacts. These contacts are established in the context of an active chromatin hub (ACH), a spatial chromatin configuration in which the LCR, together with other regulatory sequences, loops toward the active ␤-globin-like genes. Here, we used recently established I/11 cells as a model system that faithfully recapitulates the in vivo erythroid differentiation program to study the molecular events that accompany and underlie ACH formation. Upon I/11 cell induction, histone modifications changed, the ACH was formed, and the ␤-globin-like genes were transcribed at rates similar to those observed in vivo. The establishment of frequent LCR-gene contacts coincided with a more efficient loading of polymerase onto the ␤-globin promoter. Binding of the transcription factors GATA-1 and EKLF to the locus, although previously shown to be required, was not sufficient for ACH formation. Moreover, we used knock-out mice to show that the erythroid transcription factor p45 NF-E2, which has been implicated in ␤-globin gene regulation, is dispensable for ␤-globin ACH formation.The mammalian ␤-globin gene loci serve as a model system for studying developmental gene regulation. The murine ␤-globin locus contains four ␤-like globin genes that are arranged on the DNA in the order of their developmental expression. Expression of the ␤-like globin genes is restricted to the erythroid lineage. In erythroid progenitor cells, they are expressed at basal transcription levels, comparable with that of most other genes. However, at later stages of erythroid maturation they are expressed at exceptionally high levels. This high transcription rate is dependent on the ␤-globin locus control region (LCR) 2 (1), a cis-regulatory DNA element located upstream of the ␤-like globin genes that contains six erythroid-specific DNase I hypersensitive sites (HSs) (Fig. 1A).Recently developed chromosome conformation capture (3C) technology provides insight into the spatial conformation of the ␤-globin locus. This technique involves quantitative PCR analysis of formaldehyde cross-links made between selected DNA fragments as a measure of their interaction frequency. 3C technology revealed that high expression of the ␤-globin genes at later stages of differentiation coincides with the formation of an active chromatin hub (ACH), a spatial configuration of the locus in which the LCR, together with additional HSs upstream and downstream of the locus, loops toward the active ␤-like globin genes (2). At early stages of erythroid differentiation a smaller chromatin hub (CH), is present. It is composed of contacts between the outer HSs of the locus and part of the LCR but does not contain the genes (3). The ␤-globin genes switch interaction with the ACH in relation to their transcriptional activity during development. The embryonic genes contact...

The erythrocyte sedimentation rate (ESR) is still a widely used parameter for acute phase inflammation. Recently, new methods based on direct undiluted measurement of ESR in a standard EDTA tube have been developed. We evaluated the analytic performance of one of these new methods, the Ves-Matic Cube 200 (Diesse Diagnostica Senese, Siena, Italy), and compared it with several established Westergren-based diluted methods. The Ves-Matic Cube 200 showed a poor correlation (r = 0.83) with the International Council for Standardization in Haematology Westergren reference method, mainly caused by a considerable negative bias at low ESR levels. Moreover, a random bias was found at higher ESR levels that correlated with hematocrit levels, suggesting a differential influence of packed cell volume on the Ves-Matic Cube 200 results compared with Westergren results. We conclude that the Ves-Matic Cube 200 method is not interchangeable with Westergren-based diluted methods and generates ESR results that are too deviant to be clinically acceptable.

The aim of our study was to assess the fetal RBC count in maternal blood during uncomplicated pregnancies from 26 weeks onward. We used a flow cytometric method specifically designed for use in a routine hematology analyzer. Pregnant women were recruited through midwives. The participating laboratories used the FMH QuikQuant method (Trillium Diagnostics, Brewer, ME) in a CELL-DYN Sapphire hematology analyzer (Abbott Diagnostics, Santa Clara, CA). The method is based on a monoclonal antibody to hemoglobin F. Flow cytometric data were analyzed by 2 independent observers. The 95th percentile reference range was estimated according to Clinical and Laboratory Standards Institute guidelines. A total of 236 samples were statistically analyzed. Gestational ages ranged from 21.6 to 41 weeks (mean, 32.0 weeks), and the fetal RBC count in maternal blood ranged from 0.00% to 0.50% (median, 0.025%). The fetal RBC count in maternal blood shows no correlation with gestational age. The established reference range during normal pregnancy is less than 0.125%.

Background Alinity hq (Abbott) is a new high-throughput hematology analyzer that exclusively employs optical principles for detecting and enumerating blood cells. It reports 29 parameters, including a six-part white blood cell (WBC) differential. The aim of this multicenter study was to evaluate the analytical and clinical performance of the Alinity hq. Methods Complete blood count (CBC) results and morphological flagging were compared to that of CELL-DYN Sapphire (Abbott) and 2 × 200-cell manual differential results, on 1473 whole-blood samples from a well-defined patient population from three different clinical laboratories in the Netherlands. In addition, within-run and within-laboratory precision, linearity, limit of quantitation, carryover and sample stability were assessed. External quality assessment samples were also evaluated. Results Data analysis demonstrated strong concordance of Alinity hq results with those of CELL-DYN Sapphire for all CBC parameters, except for basophil granulocytes. Alinity hq WBC differential showed high level of agreement with manual differential results and exhibited a better agreement with manual basophil results than CELL-DYN Sapphire. The sensitivity of the Alinity hq Blast flag was 57.6%, equal to the 57.6% sensitivity of the CELL-DYN Sapphire’s Blast Alert. When considering samples with ≥5% blasts, the sensitivity of the Alinity hq Blast flag was 70.0%. Analytical performance of Alinity hq was shown to be consistent with state-of-the-art (SOTA) performance characteristics. Conclusions Alinity hq CBC measurands demonstrated good overall agreement with results obtained with CELL-DYN Sapphire, as well as manual WBC differential. The analytical and clinical performance characteristics of Alinity hq make it well suited for clinical laboratories.