Enhancer-dependent transcriptional oscillations in mouse erythroleukemia cells.
(41/3761)
By using recombinase-mediated cassette exchange, a method that allows integration of single copies of different constructs at the same predetermined chromosomal location, several expression cassettes have been integrated at a randomly chosen locus in the genome of mouse erythroleukemia cells. The cassettes studied contain the human beta-globin promoter fused to lacZ coding sequences either alone or linked to DNase I-hypersensitive site HS2, HS3, or HS234 (a large locus control region fragment containing HS2, HS3, and HS4) of the human beta-globin locus control region. Analysis of expression of these cassettes revealed mosaic expression patterns reminiscent of, but clearly different from, position effect variegation. Further investigations demonstrated that these mosaic expression patterns are caused by dynamic activation and inactivation of the transcription unit, resulting in oscillations of expression. These oscillations occur once in every few cell cycles at a rate specific for the enhancer present at the locus. DNase I sensitivity studies revealed that the chromatin is accessible and that DNase-hypersensitive sites were present whether or not the transcription unit is active, suggesting that the oscillations occur between transcriptionally competent and transcriptionally active chromatin conformations, rather than between open and closed chromatin conformations. Treatment of oscillating cells with trichostatin A eliminates the oscillations only after the cells have passed through late G1 or early S, suggesting that these oscillations might be caused by changes in histone acetylation patterns. (+info)
Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression.
(42/3761)
Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases. (+info)
Probing the reactivity of nucleophile residues in human 2,3-diphosphoglycerate/deoxy-hemoglobin complex by aspecific chemical modifications.
(43/3761)
The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues present on the molecular surface of the human 2,3-diphosphoglycerate/deoxy-hemoglobin complex. In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of methyl groups introduced in the modified globin chains. A combined LCESMS-Edman degradation approach for the analysis of the tryptic peptide mixtures yielded to the exact identification of methylation sites together with the quantitative estimation of their degree of modification. The reactivities observed were directly correlated with the pKa and the relative surface accessibility of the nucleophilic residues, calculated from the X-ray crystallographic structure of the protein. The results here described indicate that this methodology can be efficiently used in aspecific modification experiments directed to the molecular characterization of the surface topology in proteins and protein complexes. (+info)
Induced synthesis of immunoglobulin messenger RNA accompanies induction of immunoglobulin production in cultured mouse spleen cells.
(44/3761)
Immunoglobulin kappa type light chain mRNA (Lkappa mRNA) accumulated in parallel with secretion of immunoglobulin M in cultured mouse spleen cells activated by lipopolysaccharide. Actinomycin D suppressed the accumulation of kappa chain mRNA completely without affecting the degradation rate of kappa chain mRNA. The half life of kappa chain mRNA was about 9 h. Available evidence indicates that lipopolysaccharide stimulates de novo synthesis of kappa chain mRNA. The accumulation of kappa chain mRNA was markedly suppressed by inhibitors of DNA or protein synthesis such as hydroxyurea, cytosine arabinoside and cycloheximide. (+info)
A bipartite sequence element associated with matrix/scaffold attachment regions.
(45/3761)
We have identified a MAR/SAR recognition signature (MRS) which is common to a large group of matrix and scaffold attachment regions. The MRS is composed of two degenerate sequences (AATAAYAA and AWWRTAANNWWGNNNC) within close proximity. Analysis of >300 kb of genomic sequence from a variety of eukaryotic organisms shows that the MRS faithfully predicts 80% of MARs and SARs. In each case where we find a MRS, the corresponding DNA region binds specifically to the nuclear scaffold. Although all MRSs are associated with a SAR, not all known SARs and MARs contain a MRS, suggesting that at least two classes exist, one containing a MRS, the other not. Evidence is presented that the two sequence elements of the bipartite MRS occupy a position on the nucleosome near the dyad axis, together creating a putative protein binding site. The identification of a MAR- and SAR-associated DNA element is an important step forward towards understanding the molecular mechanisms of these elements. It will allow: (i) analysis of the genomic location of SARs, e.g. in relationship to genes, based on sequence information alone, rather than on the basis of an elaborate biochemical assay; (ii) identification and analysis of proteins that specifically bind to the MRS. (+info)
The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expression.
(46/3761)
We have studied replication throughout 325 kb of the telomeric region of a human chromosome (16p13.3) and related the findings to various aspects of chromosome structure and function (DNA sequence organization, nuclease-hypersensitive sites, nuclear matrix attachment sites, patterns of methylation and gene expression). The GC-rich isochore lying adjacent to the telomere, which contains the alpha-globin locus and many widely expressed genes, replicates early in the cell cycle regardless of the pattern of gene expression. In subtelomeric DNA, replication occurs later in the cell cycle and the most telomeric region (20 kb) is late replicating. Juxtaposition of early replicating DNA next to the telomere causes it to replicate later in S-phase. Analysis of the timing of replication in chromosomes with deletions, or in transgenes containing various segments of this telomeric region, suggests that there are no critical origins or zones that initiate replication, rather the pattern of replication appears to be related to the underlying chromatin structure which may restrict or facilitate access to multiple, redundant origins. These results contrast with the pattern of replication at the human beta-globin locus and this may similarly reflect the different chromosomal environments containing these gene clusters. (+info)
DNA bend sites in the human beta-globin locus: evidence for a basic and universal structural component of genomic DNA.
(47/3761)
Here we summarize the DNA bend sites in a 66-kb region of the human beta-globin locus. A total of 98 sites were mapped by circular permutation assay along the locus with an average interval of 679.2 +/- 229.6 bp between them. The distribution of the bend sites indicated that although the most frequent distance was about 650-700 bp, there appeared to be preferences at 300-400, 500-550, 800-850, 1,000-1,050, and 1,150-1,200 bp, indicating that these distances are multimers of a 170-bp basic unit. DNA bend sites in the globin-encoding regions indicated that most of their locations relative to the cap sites were conserved during evolution. Insertion of Alu and L1 sequences that occurred at various times and changed the distances of the sites was corrected for the epsilon-, psi beta-, and delta-globin genes. The only exception of the conservation was observed at the duplication junctions of the two gamma-globin genes, which occurred 25-35 MYA. Among the 75 A/A/A (A2N8A2N8A2) sequences found in the 51 bend sites, 59 sequences from 47 sites showed bending profiles by oligonucleotide-based assay. All of these sites were included in the sites predicted by computer analysis based on the distribution of AA and TT dinucleotides. These lines of evidence suggest that these DNA bend sites are one of the basic structural components universally present in genomic DNA. (+info)
An insulator element and condensed chromatin region separate the chicken beta-globin locus from an independently regulated erythroid-specific folate receptor gene.
(48/3761)
We have identified a folate receptor gene upstream of the chicken beta-globin locus and separated from it by a 16 kbp region of silent chromatin. We find that this receptor is expressed only at a stage of erythroid differentiation (CFU-E) preceding the activation of beta-globin genes, consistent with the role of folate receptors in proliferation. This discovery raises the question of how these two loci are regulated during erythropoiesis. Our data suggest that the folate receptor gene and the beta-globin locus are regulated independently. We show that a 3.3 kbp DNA region upstream of the folate receptor gene is sufficient to induce strong expression of a transgene in CFU-E stage cells. We also find that the region between the beta-globin locus and the folate receptor gene is fully methylated and condensed at this stage of differentiation. Its 3' boundary coincides with the 5' beta-globin insulator. We speculate that the 5' beta-globin boundary element might be important for the proper regulation of two adjacent domains activated at two different stages during differentiation. (+info)