During erythroid differentiation, beta-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for beta-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of beta-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of beta-globin transcription in committed cells before terminal differentiation.

In order to create an extended map of chromatin features within a mammalian multigene locus, we have determined the extent of nuclease sensitivity and the pattern of histone modifications associated with the mouse ␤-globin genes in adult erythroid tissue. We show that the nuclease-sensitive domain encompasses the ␤-globin genes along with several flanking olfactory receptor genes that are inactive in erythroid cells. We describe enhancer-blocking or boundary elements on either side of the locus that are bound in vivo by the transcription factor CTCF, but we found that they do not coincide with transitions in nuclease sensitivity flanking the locus or with patterns of histone modifications within it. In addition, histone hyperacetylation and dimethylation of histone H3 K4 are not uniform features of the nuclease-sensitive mouse ␤-globin domain but rather define distinct subdomains within it. Our results reveal a complex chromatin landscape for the active ␤-globin locus and illustrate the complexity of broad structural changes that accompany gene activation.

DNA replication in the human ␤-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the ␤-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5HS2-5 in the normal chromosomal context of the human ␤-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5HS2-5 in the classically defined LCR do not control replication in the human ␤-globin locus. Recent studies also show that targeted deletion of 5HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.The eukaryotic genome is divided into independently regulated domains that initiate DNA replication from defined sequences at specific times during S phase. Control mechanisms exist to specify both the sites of replication initiation (origins) and the temporal order of replication throughout the genome. Although great progress has been made in understanding the control of DNA replication in viruses, bacteria, and yeasts, we are only beginning to understand the control of DNA replication in higher eukaryotes. Over a dozen origins have been mapped in metazoans (reviewed in reference 10), and it appears that DNA replication in these organisms, in contrast to prokaryotes and lower eukaryotes, does not depend only on origin-proximal sequences, but is subject to long-distance regulation as well. The human ␤-globin locus is one of several loci in which this long-distance regulation has so far been demonstrated; sequences over 50 kb from the origin of replication are necessary for proper replication initiation and replication timing of the locus. Because of the tools available with which to make genetic modifications and perform functional analyses, the human ␤-globin locus offers an excellent opportunity to dissect possible replication control mechanisms.A single bidirectional origin, located in the vicinity of the ␤-globin gene, is used to replicate the human ␤-globin locus ( Fig. 1) (1, 35). Although this origin is used in both erythroid and nonerythroid cells, replication timing follows patterns of gene activity and chromatin structure: in erythroid cells, the globin genes are transcribed, the locus is gen...

A population sample from people of diverse ethnic origins living in New Zealand serves as a database to test methods for inference of population subdivision. The initial null hypothesis, that the population sample is homogeneous across ethnic groups, is easily rejected by likelihood ratio tests. Beyond this, methods for quantifying subdivision can be based on the probability of drawing alleles identical by descent (FST), probabilities of matching multiple locus genotypes, and occurrence of unique alleles. Population genetic theory makes quantitative predictions about the relation between FST, population sizes, and rates of migration and mutation. Some VNTR loci have mutation rates of 10(-2) per generation, but, contrary to theory, we find no consistent association between the degree of population subdivision and mutation rate. Quantification of population substructure also allows us to relate the magnitudes of genetic distances between ethnic groups in New Zealand to the colonization history of the country. The data suggests that the closest relatives to the Maori are Polynesians, and that no severe genetic bottleneck occurred when the Maori colonized New Zealand. One of the central points of contention regarding the application of VNTR loci in forensics is the appropriate means for estimating match probabilities. Simulations were performed to test the merits of the product rule in the face of subpopulation heterogeneity. Population heterogeneity results in large differences in estimates of multilocus genotype frequencies depending on which subpopulation is used for reference allele frequencies, but, of greater importance for forensic purposes, no five locus genotype had an expected frequency greater than 10(-6). Although this implies that a match with an innocent individual is unlikely, in a large urban area such chance matches are going to occur.

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