Homeotic protein binding sites, origins of replication, and nuclear matrix anchorage sites share the ATTA and ATTTA motifs.

Nuclear matrix organizes the mammalian chromatin into loops. This is achieved by binding of nuclear matrix proteins to characteristic DNA landmarks in introns as well as proximal and distal sites flanking the 5' and 3' ends of genes. Matrix anchorage sites (MARs), origins of replication (ORIs), and homeotic protein binding sites share common DNA sequence motifs. In particular, the ATTA and ATTTA motifs, which constitute the core elements recognized by the homeobox domain from species as divergent as flies and humans, are frequently occurring in the matrix attachment sites of several genes. The human apolipoprotein B 3' MAR and a stretch of the Chinese hamster DHFR gene intron and human HPRT gene intron shown to anchor these genes to the nuclear matrix are mosaics of ATTA and ATTTA motifs. Several origins of replication also share these elements. This observation suggests that homeotic proteins which control the expression level of many genes and pattern formation during development are components of the nuclear matrix. Thus, the nuclear matrix, known as the site of DNA replication, might sculpture the crossroads of the differential activation of origins during development and S-phase and the control of gene expression and pattern formation in embryogenesis.     

Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.     

The Chromatin of Active Genes Is Not in a Permanently Open Conformation

Quantitative measurements of local chromatin accessibility to DNase I in 15-day chicken embryo erythrocyte nuclei have been performed using a range of nuclease concentrations and real-time TaqMan PCR to monitor the loss of short (∼ 80 bp) amplicons. At the β-globin locus, well-established DNase I hypersensitive sites stand out against a background in which actively transcribed gene sequences (e.g., β-adult and β-hatching) are no more sensitive than the nearby constitutive heterochromatin that has previously been shown to form the 30-nm fibre structure. Similar observations were made at the lysozyme locus containing the active Gas41 gene and also at the GAPDH locus. We conclude that active genes are not continuously held in an open ‘beads-on-a-string’ configuration, but adopt a 30-nm-type structure most of the time. This implies that the compact nucleosomal supercoil re-forms in the wake of the polymerase complex.     

Isolation and sequence analysis of a hybrid delta-globin pseudogene from the brown lemur

The β-globin gene cluster of the brown lemur, a prosimian, is very short and contains a single ?-, γ- and β-globin gene, with an additional β-related gene sequence between the γ- and β-globin genes. Brown lemur DNA was cloned into the bacteriophage vector λL47.1 and a recombinant was isolated which contained an 11 × 10³ base insert including the β-globin gene and the additional putative β-globin pseudogene. The nucleotide sequence of this β-related gene was completely determined. A complete gene sequence was found, containing four frameshift mutations sufficient to establish its pseudogene status. The gene was interrupted by two intervening sequences with sizes and locations typical of mammalian β-related globin genes. The pseudogene sequence was compared in detail with human ?-, γ-, δ- and β-globin genes. The beginning of the pseudogene, from the 5′ flanking region to the second exon, was homologous to the corresponding regions of the human ?- and γ-globin genes. In contrast, the second intron, third exon and 3′ flanking region showed a remarkably close homology to the δ-globin, but not β-globin, gene of man. This suggests that the δ-globin gene is not the product of a recent gene duplication, but instead is present in most or all primates. This gene has been silenced on at least two separate occasions in primate evolution (in lemurs and in old world monkeys). In addition, the 5′ end of the lemur ψδ gene appears to have exchanged sequences with an ?- or γ-globin gene, and an analogous exchange with the β-globin gene seems to have occurred recently in the human δ-globin gene. The evolution and function of the δ-globin gene are discussed.     

Nuclear factors regulating erythroid progenitor cell differentiation and gene expression

Nuclear factors are effector molecules that directly regulate proliferation and differentiation of progenitor cells. Gene targeting experiments in mice have demonstrated that several nuclear factors are essential at different stages of erythroid development including cell cycling factors (Rb), and both ubiquitous (c-myb) and erythroid-specific (GA TA-1) DNA binding factors. In addition, DNA binding factors are required to establish the DNase I hypersensitive sites in the human β-globin locus control region (LCR). By 'opening' chromatin in the β-globin locus early in development, the LCR permits correct temporal and spatial expression of the globin genes in the maturing cells of the erythroid lineage.     

The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The β-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant β-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.     

Characterization of the chromatin structure in the upstream region of the chicken alpha-globin gene domain

The distribution of DNase I hypersensitive sites upstream of the chicken -globin gene cluster was studied. A group of hypersensitive sites with a complex pattern of tissue specificity, including erythroid-specific elements, was found at a distance of 11.5–14.5 kb upstream of the gene, the first gene in the cluster. The observations indicate that this area, located upstream of the block of AT-rich sequences and MAR sites (at –8 kb) and upstream of the site of permanent DNA attachment to the nuclear matrix (–3 kb), still belongs to the domain of the -globin genes.     

Restriction enzyme analysis of the β-globin gene in DNA from β°-thalassaemic subjects from Ferrara

The map of restriction sites including and surrounding the δ- and β-globin genes has been established for three Ferrara β°-thalassaemic subjects. The fragments obtained using nine restriction enzymes do not show any differences from normal DNA. Among others, restriction enzymes giving short fragments at the 5′ and 3′ ends of the β-globin structural gene have been employed. The results obtained for the thalassaemic DNA are identical to those for control DNA, thus excluding the presence of extensive deletions in or adjacent to the coding regions of the β-globin gene in Ferrara β°-thalassaemia.     

Characterization of the major regulatory element upstream of the human alpha-globin gene cluster.

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.     

Quantitation of DNase I sensitivity in Xenopus chromatin containing active and inactive globin, albumin and vitellogenin genes.

The disappearance of defined restriction fragments of the beta 1-globin, an albumin and the A1 vitellogenin gene was quantitated after DNase I digestion and expressed by a sensitivity factor defined by a mathematical model. Analysis of naked DNA showed that the gene fragments have similar but not identical sensitivity factors. DNase I digestion of chromatin revealed for the same gene fragments sensitivity factors differing over a much wilder range. This is correlated to the activity of the genes analyzed: the beta 1-globin gene fragment is more sensitive to DNase I in chromatin of erythrocytes compared to hepatocytes whereas the albumin gene fragment is more sensitive to DNase I in chromatin of hepatocytes. The A1 vitellogenin gene has the same DNase I sensitivity in both cell types. Comparing the DNase I sensitivity of the three genes in their inactive state we suggest that different chromatin conformations may exist for inactive genes.     

Genomic analysis of the mouse protamine 1, protamine 2, and transition protein 2 gene cluster reveals hypermethylation in expressing cells

To understand the role of chromatin structure in the expression of the mouse protamine 1, protamine 2, and transition protein 2 genes during spermatogenesis, we have examined the genomic organization of this cluster of ``haploid-specific' genes. As seen in the human genome, protamine 2, transition protein 2, and approximately 2.8 kb of a CpG island, hereafter called CpG island-dTP2, were clustered in a small region. Methylation analyses of this region have demonstrated that i) unlike most other tissue-specific genes, the protamine 1, protamine 2, and transition protein 2 genes were located in a large methylated domain in round spermatids, the cell type where they are transcribed, ii) the protamine 1 gene was only partially methylated in somatic cells and in testes from 7-day-old mice, and iii) the approximately 2 kb upstream and downstream of the CpG island-dTP2 were only partially methylated in somatic tissues. DNase I analysis revealed the presence of at least five strong DNase I hypersensitive sites over the CpG island-dTP2 in somatic tissues, but not in germ cells, and sequence analysis indicated that the CpG island-dTP2 is homologous to a CpG island located approximately 10.6 kb downstream of the human transition protein 2 gene. Although the nature of a CpG island-dTP2 and the function of a CpG island-dTP2-containing somatic tissue-specific DNase I hypersensitive sites in close proximity to the germ cell-specific gene cluster are unclear, the ``open' chromatin structure of the CpG island-dTP2 may be responsible for the partial methylation pattern of the flanking sequences including the transition protein 2 gene in somatic tissues. Received: 6 September 1996 / Accepted: 14 January 1997     

A fusion gene in man: DNA sequence analysis of the abnormal globin gene of Hemoglobin Miyada

An abnormal globin gene from a patient heterozygous for Hemoglobin Miyada was cloned and sequenced. The results indicated that the 5′ flanking region and the 5′ side of the gene were identical to those of a β-globin gene and that the 3′ side was identical to that of a γ-globin gene. The part of the gene identical to a β-globin gene shifted to the part identical to the δ-globin gene somewhere in a homologous sequence region between the third nucleotide of the 17th codon and the second nucleotide of the 22nd codon of these two genes. Thus, results of analysis of the nucleotide sequence support the idea that the abnormal globin gene of Hemoglobin Miyada was generated as a fusion gene by unequal crossing over between a β- and a δ-globin gene.