scholarly journals A common pattern of DNase-I footprinting throughout the human mtDNA unveils clues for a chromatin-like organization

2017 ◽  
Author(s):  
Amit Blumberg ◽  
Charles G. Danko ◽  
Anshul Kundaje ◽  
Dan Mishmar
Keyword(s):  
Cell Types ◽  
Regulatory Elements ◽  
Dnase I ◽  
Sequence Specificity ◽  
Physiological Importance ◽  
Dnase I Footprinting ◽  
Dose Dependent Fashion ◽  
Dna Organization ◽  
First Time ◽  
Dose Dependent

AbstractHuman mitochondrial DNA (mtDNA) is believed to lack chromatin and histones. Instead, it is coated solely by the transcription factor TFAM, which binds the mtDNA without sequence specificity and packs it into a bacterial-like nucleoid in a dose-dependent fashion. We asked whether mtDNA packaging is more regulated than once thought. As a first step to address this question, we analyzed mtDNA DNase-I-seq experiments in 324 different human cell types and found, for the first time, a pattern of 29 Genomic footprinting (DGF) sites throughout the mtDNA shared by ∼90% of the tested samples. Low SNP density at the DGF sites, and their conservation in mouse DNase-seq experiments, reflect strong selective constraints. Co-localization of the DGFs with known mtDNA regulatory elements and with recently-discovered transcription pausing sites, suggest a role for such DGFs in mtDNA transcription. Altered mtDNA DGF pattern in IL-3 treated CD+34 cells offer first clue to their physiological importance. Taken together, human mtDNA has a conserved and regulated protein-DNA organization, which is likely involved in regulation of mtDNA gene expression.

scholarly journals Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499 ◽  
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

Regulatory elements controlling pituitary-specific expression of the human prolactin gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4690-4700
Author(s):  
B Peers ◽  
M L Voz ◽  
P Monget ◽  
M Mathy-Hartert ◽  
M Berwaer ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Distal Region ◽  
Dnase I ◽  
Proximal Region ◽  
Base Pairs ◽  
Specific Expression ◽  
Positive Region ◽  
Dnase I Footprinting ◽  
Human Prolactin ◽  
Flanking Sequences

We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region.

scholarly journals Analysis of the mouse Dhfr/Rep-3 major promoter region by using linker-scanning and internal deletion mutations and DNase I footprinting.

1990 ◽  
Vol 10 (11) ◽  
pp. 6003-6012 ◽  
Author(s):  
M L Smith ◽  
P J Mitchell ◽  
G F Crouse
Keyword(s):  
Promoter Region ◽  
Transcriptional Control ◽  
Cpg Island ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Dnase I ◽  
Control Mechanisms ◽  
Cpg Dinucleotides ◽  
Dnase I Footprinting ◽  
Chloramphenicol Acetyltransferase Gene

The mouse dihydrofolate reductase (Dhfr) promoter region is buried within a CpG island (a region rich in unmethylated CpG dinucleotides), has a high G+C content, and lacks CAAT and TATA elements. The region contains four 48-bp repeats, each of which contains an Sp1-binding site. Another gene, Rep-3 (formerly designated Rep-1), shares the same general promoter region with Dhfr, being transcribed in the direction opposite that of Dhfr. Both genes appear to be housekeeping genes and are expressed at relatively low levels in all tissues. The 5' termini of the major Dhfr transcripts are separated from the 5' termini of the Rep-3 transcripts by approximately 140 bp. This curious structural arrangement suggested that the two genes might share common regulatory elements. To investigate the promoter sequences driving bidirectional transcription, a series of promoter mutations was constructed. These mutations were assayed by a replicating minigene system and by promoter fusions to the chloramphenicol acetyltransferase gene. Linker-scanning mutations that spanned the four repeats produced a variety of mRNA transcript phenotypes. The effects were primarily quantitative, generally reducing the abundance of transcripts for one or both genes. Some mutations affected Dhfr in a qualitative manner, such as by changing the startpoint of one of the major Dhfr transcripts or changing the relative abundance of the two major Dhfr transcripts. Additionally, protein transcription factors that bind to sequences in the mouse Dhfr/Rep-3 major promoter region, potentially affecting expression of either or both genes, were investigated by DNase I footprinting. The results indicate that multiple protein-DNA interactions occur in this region, reflecting potentially complex transcriptional control mechanisms that might modulate expression of either or both genes under different physiological conditions.

scholarly journals The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds

Development ◽  
1997 ◽  
Vol 124 (19) ◽  
pp. 3797-3804 ◽  
Author(s):  
N.A. Armes ◽  
J.C. Smith
Keyword(s):  
Cell Types ◽  
Type I ◽  
Cell Stage ◽  
Mesodermal Cell ◽  
Threshold Phenomenon ◽  
Dose Dependent Fashion ◽  
High Concentrations ◽  
Dose Dependent ◽  
Activin Receptors ◽  
Constitutively Active

The TGFbeta family member activin induces different mesodermal cell types in a dose-dependent fashion in the Xenopus animal cap assay. High concentrations of activin induce dorsal and anterior cell types such as notochord and muscle, while low concentrations induce ventral and posterior tissues such as mesenchyme and mesothelium. In this paper we investigate whether this threshold phenomenon involves the differential effects of the two type I activin receptors ALK-2 and ALK-4. Injection of RNA encoding constitutively active forms of the receptors (here designated ALK-2* and ALK-4*) reveals that ALK-4* strongly induces the more posterior mesodermal marker Xbra and the dorsoanterior marker goosecoid in animal cap explants. Maximal levels of Xbra expression are attained using lower concentrations of RNA than are required for the strongest activation of goosecoid, and at the highest doses of ALK-4*, levels of Xbra transcription decrease, as is seen with high concentrations of activin. By contrast, the ALK-2* receptor activates Xbra but fails to induce goosecoid to significant levels. Analysis at later stages reveals that ALK-4* signalling induces the formation of a variety of mesodermal derivatives, including dorsal cell types, in a dose-dependent fashion, and that high levels also induce endoderm. By contrast, the ALK-2* receptor induces only ventral mesodermal markers. Consistent with these observations, ALK-4* is capable of inducing a secondary axis when injected into the ventral side of 32-cell stage embryos whilst ALK-2* cannot. Co-injection of RNAs encoding constitutively active forms of both receptors reveals that ventralising signals from ALK-2* antagonise the dorsal mesoderm-inducing signal derived from ALK-4*, suggesting that the two receptors use distinct and interfering signalling pathways. Together, these results show that although ALK-2* and ALK-4* transduce distinct signals, the threshold responses characteristic of activin cannot be due to interactions between these two pathways; rather, thresholds can be established by ALK-4* alone. Furthermore, the effects of ALK-2* signalling are at odds with it behaving as an activin receptor in the early Xenopus embryo.

Attenuation of ANG II actions by adenovirus delivery of AT1 receptor antisense in neurons and SMC

1998 ◽  
Vol 274 (2) ◽  
pp. H719-H727
Author(s):  
Di Lu ◽  
Hong Yang ◽  
Mohan K. Raizada
Keyword(s):  
Thymidine Incorporation ◽  
Cell Types ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Ang Ii ◽  
Gene Therapy Approach ◽  
Viral Particles ◽  
Receptor Mutant ◽  
First Time ◽  
Dose Dependent

Both central and peripheral renin-angiotensin systems (RAS) are important in the development and establishment of hypertension. Thus, introducing genes relevant to RAS into neuronal and vascular smooth muscle (VSM) cells, two major targets for angiotensin (ANG) II action, is a prerequisite in considering a gene therapy approach for the control of ANG-dependent hypertension. In this study, we explored the use of adenoviral (Ad) vector to transfer AT1 receptor antisense cDNA (AT1R-AS) into neuronal and VSM cells with the anticipation of attenuation of ANG II-mediated cellular actions. Incubation of neurons and VSM cells with viral particles containing AT1R-AS (Ad-AT1R-AS) resulted in a robust expression of AT1R-AS in a majority (∼80%) of the cells. The expression was persistent for at least 28 days and was associated with decreases in the immunoreactive AT1 receptor protein and the maximal binding for AT1 receptor in a time- and dose-dependent manner in both cell types. ANG II stimulation of [3H]thymidine incorporation in VSM cells and norepinephrine transporter gene expression in neuronal cells were attenuated by Ad-AT1R-AS infection. Uninfected cells or cells infected with adenovirus particles containing a mutant AT1 receptor sense cDNA showed no effects on either AT1 receptor or on attenuation of ANG II’s cellular affects. These observations show, for the first time, that adenovirus can be used to deliver AT1 receptor mutant sense and antisense cDNAs into two major ANG II target tissues. This consequently influences AT1 receptor-mediated cellular actions of ANG II.

Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

scholarly journals Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

1987 ◽  
Vol 7 (6) ◽  
pp. 2059-2069 ◽  
Author(s):  
B Kemper ◽  
P D Jackson ◽  
G Felsenfeld
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Dnase I ◽  
Specific Factor ◽  
Mobility Shift ◽  
Protein Binding Sites ◽  
Dnase I Footprinting ◽  
Flanking Region

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

ORP-3, a human oxysterol-binding protein gene differentially expressed in hematopoietic cells

Blood ◽  
2001 ◽  
Vol 98 (7) ◽  
pp. 2279-2281 ◽  
Author(s):  
Claudia C. Gregorio-King ◽  
Gregory R. Collier ◽  
Janine S. McMillan ◽  
Caryll M. Waugh ◽  
Janet L. McLeod ◽  
...  
Keyword(s):  
Binding Protein ◽  
Cholesterol Biosynthesis ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Oxysterol Binding Protein ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Dose Dependent Fashion ◽  
High Degree ◽  
Dose Dependent

Using differential display polymerase chain reaction, a gene was identified in CD34+-enriched populations that had with low or absent expression in CD34− populations. The full coding sequence of this transcript was obtained, and the predicted protein has a high degree of homology to oxysterol-binding protein. This gene has been designated OSBP-related protein 3 (ORP-3). Expression of ORP-3 was found to be 3- to 4-fold higher in CD34+ cells than in CD34− cells. Additionally, expression of this gene was 2-fold higher in the more primitive subfraction of hematopoietic cells defined by the CD34+38− phenotype and was down-regulated with the proliferation and differentiation of CD34+ cells. The ORP-3 predicted protein contains an oxysterol-binding domain. Well-characterized proteins expressing this domain bind oxysterols in a dose-dependent fashion. Biologic activities of oxysterols include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, among them hematopoietic cells. Characterization and differential expression of ORP-3 implicates a possible role in the mediation of oxysterol effects on hematopoiesis.

Sequence specificity of the binding of 9-aminoacridine- and amsacrine-4-carboxamides to DNA studied by DNase I footprinting

Biochemistry ◽  
1992 ◽  
Vol 31 (13) ◽  
pp. 3514-3524 ◽  
Author(s):  
C. Bailly ◽  
W. A. Denny ◽  
L. E. Mellor ◽  
L. P. G. Wakelin ◽  
M. J. Waring
Keyword(s):  
Dnase I ◽  
Sequence Specificity ◽  
Dnase I Footprinting

scholarly journals The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture

1978 ◽  
Vol 172 (1) ◽  
pp. 97-107 ◽  
Author(s):  
D K Fast ◽  
R Felix ◽  
C Dowse ◽  
W F Neuman ◽  
H Fleisch
Keyword(s):  
Cell Types ◽  
Detectable Effect ◽  
Skin Cells ◽  
N2 Atmosphere ◽  
Dose Dependent Fashion ◽  
Cartilage Cells ◽  
Ear Cartilage ◽  
Drug Doses ◽  
Dose Dependent

1. The effects of two diphosphonates (compounds containing a P-C-P bond), disodium dichloromethanediphosphonate and disodium 1-hydroxyethane-1,1-diphosphonate, on the metabolism of cultured rat calvaria cells, rabbit ear cartilage cells and rat skin fibroblasts were investigated. 2. The diphosphonates had no effect on the growth of cartilage cells and on the exponential growth of the calvaria cells and the fibroblasts. However, dichloromethanediphosphonate stopped the growth of the calvaria cells and the fibroblasts after the beginning of confluence, whereas the untreated cells were still growing to a certain extent. This inhibition was dose-dependent. After the drug was withdrawn, the cells recovered slowly. 1-Hydroxyethane-1,1-diphosphonate had no detectable effect on the growth of any of the cell types studied. Both diphosphonates decreased the cloning efficiency of calvaria cells and fibroblasts. 3. The K+ content of cartilage, calvaria and skin cells was diminished only by the highest (0.25 mM) concentration of dichloromethanediphosphonate. 4. Radioactive dichloromethanediphosphonate and 1-hydroxyethane-1,1-diphosphonate were taken up linearly with time for at least 48 h by calvaria cells and fibroblasts. The diphosphonate concentration in the cells depended on its concentration in the medium. 5. Both diphosphonates, in a dose-dependent fashion, markedly inhibited glycolysis, dichloromethanediphosphonate being more effective than 1-hydroxyethane-1,1-diphosphonate, at drug doses that had no effect on cell growth or cellular K+ content. Calvaria cells were much more sensitive than cartilage cells. When cartilage cells were cultured in an N2 atmosphere, these effects on glucose and lactate metabolism disappeared. 6. As increased acid production appears to be associated with resorption of bone, this decrease in lactate may explain why diphosphonates are effective inhibitors of bone resorption in vivo.

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