Phylogenetic footprinting reveals a nuclear protein which binds to silencer sequences in the human gamma and epsilon globin genes.

Tissue- and developmental stage-specific expression of the human beta-like globin genes is regulated by a combination of ubiquitous and erythroid-restricted trans factors that bind to cis elements near each of the five active genes. Additional interactions of these cis and trans factors with sequences located in the far 5' end of the cluster occur by as yet obscure mechanisms. Because of the complexity of this regulatory puzzle, precise identification of the determinants that control hemoglobin switching has proven difficult. Phylogenetic footprinting is an evolutionary approach to this problem which is based on the supposition that the basic mechanisms of switching are conserved throughout mammalian phylogeny. Alignment of the 5' flanking regions of the gamma genes of several species allows the identification of footprints of 100% conserved sequence. We have now tested oligomers spanning 13 such phylogenetic footprints and find that 12 are bound by nuclear proteins. One conserved element located at -1086 from the gamma genes exhibits repressor activity in transient transfection studies. The protein that binds this element, CSBP-1 (conserved sequence-binding protein 1), also binds at three sites within a silencer element upstream from the epsilon globin gene. Further analysis reveals that the CSBP-1 binding activity is identical to that of a recently cloned zinc finger protein that has been shown to act as a repressor in other systems. The binding of CSPB-1 to silencer sequences in the epsilon and gamma globin genes may be important in the stage-specific silencing of these genes.     

An insulator element 3′ to the CFTR gene binds CTCF and reveals an active chromatin hub in primary cells

Regulation of expression of the CFTR gene is poorly understood. Elements within the basal promoter of the gene do not fully explain CFTR expression patterns, suggesting that cis-regulatory elements are located elsewhere, either within the locus or in adjacent chromatin. We previously mapped DNase I hypersensitive sites (DHS) in 400 kb spanning the CFTR locus including a cluster of sites close to the 3′-end of the gene. Here we focus on a DHS at +6.8 kb from the CFTR translation end-point to evaluate its potential role in regulating expression of the gene. This DHS, which encompasses a consensus CTCF-binding site, was evident in primary human epididymis cells that express abundant CFTR mRNA. We show by DNase I footprinting and electophoretic mobility shift assays that the cis-regulatory element within this DHS binds CTCF in vitro. We further demonstrate that the element functions as an enhancer blocker in a well-established in vivo assay, and by using chromatin immunoprecipitation that it recruits CTCF in vivo. Moreover, we reveal that in primary epididymis cells, the +6.8 kb DHS interacts closely with the CFTR promoter, suggesting that the CFTR locus exists in a looped conformation, characteristic of an active chromatin hub.     

Isolation and characterization of a novel human gene (NESH) which encodes a putative signaling molecule similar to e3B1 protein

Using a conventional cloning technique, a novel full-length cDNA was isolated and sequenced from a human placental cDNA library. This cDNA consists of 2129 bp and has a predicted open reading frame encoding 366 amino acids. It possesses a Src homology 3 (SH3) motif, proline-rich region, serine-rich region and no catalytic domain, suggesting that it seems to be a signaling protein most similar to e3B1, an eps8 SH3 binding protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 17q21.3 near the marker D17S1795.     

Sequence and genomic structure of the human adult skeletal muscle sodium channel alpha subunit gene on 17q.

The amino acid sequence of the sodium channel alpha subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the alpha subunit from rat adult skeletal muscle and 70% identity to the alpha subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene.     

Human placenta type V collagens. Evidence for the existence of an alpha 1(V) alpha 2(V) alpha 3(V) collagen molecule

Human type V collagen was purified from placenta and found to contain alpha 1(V), alpha 2(V), and alpha 3(V) chains in varying ratios. Using any of three independent nondenaturing methods (phosphocellulose chromatography, high-performance ion-exchange chromatography on IEX-540 DEAE, and ammonium sulfate precipitation), this preparation could be resolved into two fractions. Analysis of the two fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that one fraction contained alpha 1(V) and alpha 2(V) in a 2:1 ratio and the other contained alpha 1(V), alpha 2(V), and alpha 3(V) in a 1:1:1 ratio. When the crude placental type V collagen was electrophoresed under nondenaturing conditions, two bands were observed, one co-migrating with purified (alpha 1(V]2 alpha 2(V) and the other co-migrating with the fractions containing alpha 1(V), alpha 2(V), and alpha 3(V) chains in a 1:1:1 ratio. Electrophoresis in a second dimension under denaturing conditions confirmed that the fast-migrating band contained (alpha 1(V]2 alpha 2(V) and that the slow-migrating band contained the three chains in equimolar ratio. CD spectra of the two fractions and resistance to trypsin-chymotrypsin digestion confirmed that the two fractions contain triple helical collagen. Thermal denaturations were monitored by the changes in CD signal at 221 nm. The two fractions purified by ammonium sulfate precipitation melted at 39.1 and 36.4 degrees C for the (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) fractions, respectively. Trypsin cleavage of these two native fractions at temperatures near melting produced completely different fragmentation patterns, indicating different partial unwinding sites of the alpha 1(V) and alpha 2(V) chains in the two preparations and thus different molecular assemblies. Our data demonstrate the existence of two different molecular assemblies of type V collagen in human placenta consisting of (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) heterotrimers.     

The genomic organisation and nucleotide sequence of the HLA-SB(DP) alpha gene.

We have isolated a unique fragment of the HLA-DR alpha gene and probed human genomic DNA at low stringency to search for homologous sequences. A minimum of six non-polymorphic cross-hybridizing high molecular weight fragments were found in all DNAs examined. In order to obtain molecular clones of these cross-hybridizing fragments, we constructed lambda and cosmid libraries of human DNA and screened them at low stringency with the HLA-DR alpha gene specific subclone. We have isolated clones corresponding to each of the six fragments and, in this paper, describe those which contain the gene encoding HLA-SB(DP) alpha.     

Human interferon omega (omega) binds to the alpha/beta receptor.

It was proposed that human interferon omega (omega) binds to the interferon alpha/beta receptor but not to the interferon gamma receptor. However, since no studies were performed to provide direct evidence for this hypothesis, we carried out cross-linking experiments and saturation binding assays between a 32P-labeled human interferon-alpha (Hu-IFN-alpha) and unlabeled Hu-IFN-alpha A, -beta, -gamma, and -omega. These assays demonstrated that Hu-IFN-alpha A, -beta, and -omega, but not Hu-IFN-gamma, were able to block binding of 32P-labeled Hu-IFN-alpha A to human cells. These results indicate that Hu-IFN-omega binds to the alpha/beta receptor.     

MDC-9 (ADAM-9/Meltrin gamma) functions as an adhesion molecule by binding the alpha(v)beta(5) integrin

MDC-9 is a widely expressed member of the metalloproteinase/disintegrin/cysteine-rich protein family. The disintegrin domain of MDC-9 lacks an RGD motif but has recently been reported to bind the alpha(6)beta(1) integrin; however, it is unclear whether MDC-9 can bind other integrins. In the present study myeloma cells, but not lymphoblastoid cells, were shown to bind to immobilised, recombinantly expressed MDC-9 disintegrin domain (A9dis). Binding was divalent cation-dependent, being supported by Mn(2+) and Ca(2+). Adhesion of myeloma cells to A9dis was completely inhibited by an antibody to the alpha(v)beta(5) integrin but not by antibodies to other subunits. RGD-containing peptides had no effect on binding, suggesting that MDC-9 interacts with alpha(v)beta(5) in an RGD-independent manner. Flow cytometric analyses demonstrated that myeloma cells, but not lymphoblastoid cells, expressed alpha(v)beta(5) on the cell membrane. These data indicated that the disintegrin domain of MDC-9 can function as an adhesion molecule by interacting with an alpha(v)beta(5) integrin.