Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.
The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions. (+info)
Transcription factors CCAAT/enhancer-binding protein beta and nuclear factor-Y bind to discrete regulatory elements in the very low density lipoprotein receptor promoter.
Expression of the very low density lipoprotein receptor (VLDL-R) is barely detectable in liver, but occurs in adipose tissue, skeletal muscle, heart, and placenta, where it is postulated to supply triglyceride to tissues that utilize fatty acids. To investigate its tissue-specific expression, cell lines were transfected with luciferase reporter gene constructs driven by the 5'-flanking region of the VLDL-R gene. Transcriptional activity of a 4.2-kb promoter fragment was 5-fold higher in BeWo placental cells than in Huh-7 hepatoma cells, consistent with relative endogenous expression of the VLDL-R. By deletion analysis, DNase I protection assays and site-directed mutagenesis, two regulatory elements were essential for maximal promoter activity in BeWo cells: footprint site D (-856 to -830) and an inverted CCAAT box (-703 to -707). Mutation of either element reduced promoter activity by 60% in BeWo cells, but had little effect in Huh-7 cells, suggesting that these elements direct cell-type specific transcription. Electrophoretic mobility-shift assays with BeWo nuclear extracts revealed that the inverted CCAAT box binds transcription factor NF-Y, and site D binds CCAAT/enhancer-binding protein b (C/EBPbeta) and minor amounts of C/EBPalpha and C/EBPdelta. Overexpression of a dominant negative NF-YA vector confirmed involvement of NF-Y in the regulation of the VLDL-receptor gene through the CCAAT box. However overexpression of C/EBP could not stimulate transcription from the VLDL-receptor promoter nor from site D fused to a heterologous promoter, suggesting that the simultaneous binding of an accessory factor(s) may be necessary for C/EBP transactivation via the D site. (+info)
Interactions of heterologous DNA with polyomavirus major structural protein, VP1.
'Empty' polyomavirus pseudocapsids, self-assembled from the major structural protein VP1, bind DNA non-specifically and can deliver it into the nuclei of mammalian cells for expression [Forstova et al. (1995) Hum. Gene Ther. 6, 297-3061. Formation of suitable VP1-DNA complexes appears to be the limiting step in this route of gene delivery. Here, the character of VP1-DNA interactions has been studied in detail. Electron microscopy revealed that VP1 pseudocapsids can create in vitro at least two types of interactions with double-stranded DNA: (i) highly stable complexes, requiring free DNA ends, where the DNA is partially encapsidated; and, (ii) weaker interactions of pseudocapsids with internal parts of the DNA chain. (+info)
In vivo nuclease hypersensitivity studies reveal multiple sites of parental origin-dependent differential chromatin conformation in the 150 kb SNRPN transcription unit.
Human chromosome region 15q11-q13 contains a cluster of oppositely imprinted genes. Loss of the paternal or the maternal alleles by deletion of the region or by uniparental disomy 15 results in Prader-Willi syndrome (PWS) or Angelman syndrome (AS), respectively. Hence, the two phenotypically distinct neurodevelopmental disorders are caused by the lack of products of imprinted genes. Subsets of PWS and AS patients exhibit 'imprinting mutations', such as small microdeletions within the 5' region of the small nuclear ribonucleoprotein polypeptide N ( SNRPN ) transcription unit which affect the transcriptional activity and methylation status of distant imprinted genes throughout 15q11-q13 in cis. To elucidate the mechanism of these long-range effects, we have analyzed the chromatin structure of the 150 kb SNRPN transcription unit for DNase I- and Msp I-hypersensitive sites. By using an in vivo approach on lymphoblastoid cell lines from PWS and AS individuals, we discovered that the SNRPN exon 1 is flanked by prominent hypersensitive sites on the paternal allele, but is completely inaccessible to nucleases on the maternal allele. In contrast, we identified several regions of increased nuclease hypersensitivity on the maternal allele, one of which coincides with the AS minimal microdeletion region and another lies in intron 1 immediately downstream of the paternal-specific hypersensitive sites. At several sites, parental origin-specific nuclease hypersensitivity was found to be correlated with hypermethylation on the allele contributed by the other parent. The differential parental origin-dependent chromatin conformations might govern access of regulatory protein complexes and/or RNAs which could mediate interaction of the region with other genes. (+info)
Ig heavy chain expression and class switching in vitro from an allele lacking the 3' enhancers DNase I-hypersensitive hs3A and hs1,2.
The murine Ig heavy chain (IgH) 3' regulatory region contains four enhancers: hs3A, hs1,2, hs3B, and hs4. Various studies have suggested a role for these enhancers in regulating IgH expression and class switching. Here we assess the role of hs3A and hs1,2 in these processes by exploiting a naturally occurring deletion of these enhancers from the expressed, C57BL/6 allele of the F1 pre-B cell line, 70Z/3. Equivalent mu expression in 70Z/3 and 18-81 (which has an intact 3' region) indicated that hs3A and hs1,2 were not essential for mu expression at the pre-B cell stage. To further examine the role of hs3A and hs1,2 in IgH function at the plasma cell stage, we fused 70Z/3 with the plasmacytoma NSO. Electromobility shift assay analysis of the 70Z/3-NSO hybrids revealed a transcription factor complement conducive to the activation of the 3' enhancers. Despite the lack of enhancers, hs3A and hs1,2, the level of mu RNA and protein in the 70Z/3-NSO fusion hybrids was substantially elevated relative to its pre-B parent and comparable with that observed in a number of mu-producing spleen cell hybridomas. Additionally, ELISAspot assays showed that the 70Z/3-NSO hybrid underwent spontaneous class switching in culture to IgG1 at a frequency comparable with that of most hybridomas. These results indicate that hs3A and hs1,2 are not essential for high levels of IgH expression or for spontaneous class switching in a plasma cell line. (+info)
Transcriptional repression of the cystic fibrosis transmembrane conductance regulator gene, mediated by CCAAT displacement protein/cut homolog, is associated with histone deacetylation.
Human cystic fibrosis transmembrane conductance regulator gene (CFTR) transcription is tightly regulated by nucleotide sequences upstream of the initiator sequences. Our studies of human CFTR transcription focus on identifying transcription factors bound to an inverted CCAAT consensus or "Y-box element." The human homeodomain CCAAT displacement protein/cut homolog (CDP/cut) can bind to the Y-box element through a cut repeat and homeobox. Analysis of stably transfected cell lines with wild-type and mutant human CFTR-directed reporter genes demonstrates that human histone acetyltransferase GCN5 and transcription factor ATF-1 can potentiate CFTR transcription through the Y-box element. We have found 1) that human CDP/cut acts as a repressor of CFTR transcription through the Y-box element by competing for the sites of transactivators hGCN5 and ATF-1; 2) that the ability of CDP/cut to repress activities of hGCN5 and ATF-1 activity is contingent on the amount of CDP/cut expression; 3) that histone acetylation may have a role in the regulation of gene transcription by altering the accessibility of the CFTR Y-box for sequence-specific transcription factors; 4) that trichostatin A, an inhibitor of histone deacetylase activity, activates transcription of CFTR through the Y-box element; 5) that the inhibition of histone deacetylase activity leads to an alteration of local chromatin structure requiring an intact Y-box sequence in CFTR; 6) that immunocomplexes of CDP/cut possess an associated histone deacetylase activity; 7) that the carboxyl region of CDP/cut, responsible for the transcriptional repressor function, interacts with the histone deacetylase, HDAC1. We propose that CFTR transcription may be regulated through interactions with factors directing the modification of chromatin and requires the conservation of the inverted CCAAT (Y-box) element of the CFTR promoter. (+info)
Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene.
The mouse H19 gene is expressed from the maternal chromosome exclusively. A 2-kb region at 2 to 4 kb upstream of H19 is paternally methylated throughout development, and these sequences are necessary for the imprinted expression of both H19 and the 5'-neighboring Igf2 gene. In particular, on the maternal chromosome this element appears to insulate the Igf2 gene from enhancers located downstream of H19. We analyzed the chromatin organization of this element by assaying its sensitivity to nucleases in nuclei. Six DNase I hypersensitive sites (HS sites) were detected on the unmethylated maternal chromosome exclusively, the two most prominent of which mapped 2.25 and 2.75 kb 5' to the H19 transcription initiation site. Five of the maternal HS sites were present in expressing and nonexpressing tissues and in embryonic stem (ES) cells. They seem, therefore, to reflect the maternal origin of the chromosome rather than the expression of H19. A sixth maternal HS site, at 3.45 kb upstream of H19, was detected in ES cells only. The nucleosomal organization of this element was analyzed in tissues and ES cells by micrococcal nuclease digestion. Specifically on the maternal chromosome, an unusual and strong banding pattern was obtained, suggestive of a nonnucleosomal organization. From our studies, it appears that the unusual chromatin organization with the presence of HS sites (maternal chromosome) and DNA methylation (paternal chromosome) in this element are mutually exclusive and reflect alternate epigenetic states. In addition, our data suggest that nonhistone proteins are associated with the maternal chromosome and that these might be involved in its boundary function. (+info)
Hypersensitive site 2 specifies a unique function within the human beta-globin locus control region to stimulate globin gene transcription.
The human beta-globin locus control region (LCR) harbors both strong chromatin opening and enhancer activity when assayed in transgenic mice. To understand the contribution of individual DNase I hypersensitive sites (HS) to the function of the human beta-globin LCR, we have mutated the core elements within the context of a yeast artificial chromosome (YAC) carrying the entire locus and then analyzed the effect of these mutations on the formation of LCR HS elements and expression of the genes in transgenic mice. In the present study, we examined the consequences of two different HS2 mutations. We first generated seven YAC transgenic lines bearing a deletion of the 375-bp core enhancer of HS2. Single-copy HS2 deletion mutants exhibited severely depressed HS site formation and expression of all of the human beta-globin genes at every developmental stage, confirming that HS2 is a vital, integral component of the LCR. We also analyzed four transgenic lines in which the core element of HS2 was replaced by that of HS3 and found that while HS3 is able to restore the chromatin-opening activity of the LCR, it is not able to functionally replace HS2 in mediating high-level globin gene transcription. These results continue to support the hypothesis that HS2, HS3, and HS4 act as a single, integral unit to regulate human globin gene transcription as a holocomplex, but they can also be interpreted to say that formation of a DNase I hypersensitive holocomplex alone is not sufficient for mediating high-level globin gene transcription. We therefore propose that the core elements must productively interact with one another to generate a unique subdomain within the nucleoprotein holocomplex that interacts in a stage-specific manner with individual globin gene promoters. (+info)