scholarly journals Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution.

1987 ◽  
Vol 7 (1) ◽  
pp. 15-25 ◽  
Author(s):  
J M Gimble ◽  
E E Max
Keyword(s):  
High Resolution ◽  
B Cell ◽  
Chromatin Structure ◽  
Genomic Sequence ◽  
Dimethyl Sulfate ◽  
Dnase I ◽  
Homologous Region ◽  
Immunoglobulin Kappa ◽  
Gene Enhancer

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.

Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution

1987 ◽  
Vol 7 (1) ◽  
pp. 15-25
Author(s):  
J M Gimble ◽  
E E Max
Keyword(s):  
High Resolution ◽  
B Cell ◽  
Chromatin Structure ◽  
Genomic Sequence ◽  
Dimethyl Sulfate ◽  
Dnase I ◽  
Homologous Region ◽  
Immunoglobulin Kappa ◽  
Gene Enhancer

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.

scholarly journals Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of theHPRT Promoter

2001 ◽  
Vol 21 (22) ◽  
pp. 7682-7695 ◽  
Author(s):  
Chien Chen ◽  
Thomas P. Yang
Keyword(s):  
Chromatin Structure ◽  
Transcription Initiation ◽  
Dimethyl Sulfate ◽  
Dnase I ◽  
Minimal Promoter ◽  
Micrococcal Nuclease ◽  
Promoter Regions ◽  
Permeabilized Cells ◽  
Active Allele

ABSTRACT Differential chromatin structure is one of the hallmarks distinguishing active and inactive genes. For the X-linked human hypoxanthine phosphoribosyltransferase gene (HPRT), this difference in chromatin structure is evident in the differential general DNase I sensitivity and hypersensitivity of the promoter regions on active versus inactive X chromosomes. Here we characterize the nucleosomal organization responsible for the differential chromatin structure of the active and inactive HPRT promoters. The micrococcal nuclease digestion pattern of chromatin from the active allele in permeabilized cells reveals an ordered array of translationally positioned nucleosomes in the promoter region except over a 350-bp region that is either nucleosome free or contains structurally altered nucleosomes. This 350-bp region includes the entire minimal promoter and all of the multiple transcription initiation sites of the HPRT gene. It also encompasses all of the transcription factor binding sites identified by either dimethyl sulfate or DNase I in vivo footprinting of the active allele. In contrast, analysis of the inactive HPRT promoter reveals no hypersensitivity to either DNase I or a micrococcal nuclease and no translational positioning of nucleosomes. Although nucleosomes on the inactive promoter are not translationally positioned, high-resolution DNase I cleavage analysis of permeabilized cells indicates that nucleosomes are rotationally positioned over a region of at least 210 bp on the inactive promoter, which coincides with the 350-bp nuclease-hypersensitive region on the active allele, including the entire minimal promoter. This rotational positioning of nucleosomes is not observed on the active promoter. These results suggest a model in which the silencing of the HPRT promoter during X chromosome inactivation involves remodeling a transcriptionally competent, translationally positioned nucleosomal array into a transcriptionally repressed architecture consisting of rotationally but not translationally positioned nucleosomal arrays.

scholarly journals B-cell nuclear proteins binding in vitro to the human immunoglobulin kappa enhancer: localization by exonuclease protection.

1987 ◽  
Vol 7 (5) ◽  
pp. 1815-1822 ◽  
Author(s):  
J M Gimble ◽  
D Levens ◽  
E E Max
Keyword(s):  
Protein Binding ◽  
B Cell ◽  
Base Pair ◽  
Consensus Sequence ◽  
Dimethyl Sulfate ◽  
Simian Virus 40 ◽  
Human Immunoglobulin ◽  
Core Element ◽  
Immunoglobulin Kappa

Proteins capable of interacting with the enhancer of the immunoglobulin kappa gene in vitro have been detected in extracts of nuclei from human B cells and from human, mouse, and rabbit spleens. The experiments, based on an exonuclease protection technique, demonstrate nuclear protein factors binding to a 30- to 35-base-pair domain containing both the simian virus 40 enhancer core element (TTTCCA) and the octamer CAGGTGGC that was previously identified as the consensus sequence for protein-binding sites in the murine immunoglobulin heavy-chain enhancer. This 30- to 35-base-pair domain in the human kappa enhancer is homologous to a site of protein binding detected in the murine kappa enhancer by other investigators using a gel retardation assay. Our results complement in vivo dimethyl sulfate footprinting studies of the human immunoglobulin kappa enhancer which demonstrated B cell-specific changes in guanine reactivity immediately 5' to the consensus octamer. Together, these findings suggest that DNA-binding proteins in B-cell nuclei interact with the 5' portion of the human kappa-gene enhancer. Such proteins could play a role in the B cell-specific transcription of the human immunoglobulin kappa gene.

scholarly journals High-Resolution Mapping of In vivo Genomic Transcription Factor Binding Sites Using In situ DNase I Footprinting and ChIP-seq

DNA Research ◽  
2013 ◽  
Vol 20 (4) ◽  
pp. 325-338 ◽  
Author(s):  
O. Chumsakul ◽  
K. Nakamura ◽  
T. Kurata ◽  
T. Sakamoto ◽  
J. L. Hobman ◽  
...  
Keyword(s):  
Transcription Factor ◽  
High Resolution ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Dnase I ◽  
High Resolution Mapping ◽  
Dnase I Footprinting ◽  
Resolution Mapping

scholarly journals Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

1996 ◽  
Vol 16 (11) ◽  
pp. 6055-6064 ◽  
Author(s):  
Q H Gong ◽  
J C McDowell ◽  
A Dean
Keyword(s):  
Control Region ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Locus Control Region ◽  
Dnase I ◽  
Beta Globin ◽  
Hypersensitive Site

Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free of nucleosomes. The restricted structural change observed upon transcriptional activation may indicate that the LCR need only make a specific contact with the proximal gene promoter to activate transcription.

Interferon induction of gene transcription analyzed by in vivo footprinting

1992 ◽  
Vol 12 (1) ◽  
pp. 1-9
Author(s):  
J Mirkovitch ◽  
T Decker ◽  
J E Darnell
Keyword(s):  
Transcription Initiation ◽  
Dimethyl Sulfate ◽  
Binding Activity ◽  
Dnase I ◽  
Intact Cells ◽  
Whole Cells ◽  
Ifn Alpha ◽  
Transcription Initiation Complex ◽  
In Vivo Footprinting

The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.

scholarly journals FoxA1 Binding Directs Chromatin Structure and the Functional Response of a Glucocorticoid Receptor-Regulated Promoter

2009 ◽  
Vol 29 (20) ◽  
pp. 5413-5425 ◽  
Author(s):  
Sergey Belikov ◽  
Carolina Åstrand ◽  
Örjan Wrange
Keyword(s):  
Transcription Factor ◽  
Glucocorticoid Receptor ◽  
Chromatin Structure ◽  
Partial Agonist ◽  
Dimethyl Sulfate ◽  
Specific Response ◽  
Hormone Regulation ◽  
Context Specific ◽  
In Vivo Footprinting

ABSTRACT Reconstitution of the glucocorticoid receptor (GR)-regulated mouse mammary tumor virus (MMTV) promoter in Xenopus oocytes was used to monitor the effects of different transcription factor contexts. Three constitutively binding factors, nuclear factor 1 (NF1), octamer transcription factor 1 (Oct1), and the Forkhead box A1 (FoxA1), were shown to act in concert, to direct the chromatin structure, and to enhance the GR response. FoxA1 has a dominant effect in the absence of hormone and induces a cluster of DNase I-hypersensitive sites in the segment comprising bp −400 to +25. This FoxA1-mediated chromatin remodeling does not induce MMTV transcription, as opposed to that of the GR. However, the robust FoxA1-dependent chromatin opening has the following drastic functional consequences on the hormone regulation: (i) GR-DNA binding is facilitated, as revealed by dimethyl sulfate in vivo footprinting, leading to increased hormone-induced transcription, and (ii) the GR antagonist RU486 is converted into a partial agonist in the presence of FoxA1 via ligand-independent GR activation. We conclude that FoxA1 mediates a preset chromatin structure and directs a context-specific response of a nuclear receptor. Furthermore, the alternative nucleosome arrangement induced by GR and FoxA1 implies this to be determined by constitutive binding of transcription factors rather than by the DNA sequence itself.

scholarly journals Characterization of the human immunoglobulin kappa gene 3' enhancer: functional importance of three motifs that demonstrate B-cell-specific in vivo footprints.

1992 ◽  
Vol 12 (11) ◽  
pp. 5206-5216 ◽  
Author(s):  
J G Judde ◽  
E E Max
Keyword(s):  
T Cell ◽  
B Cell ◽  
Regulatory Elements ◽  
Human Immunoglobulin ◽  
Site Directed Mutagenesis ◽  
Cooperative Interaction ◽  
Enhancer Element ◽  
Immunoglobulin Kappa ◽  
In Vivo Footprinting

Using a combination of in vivo footprinting and site-directed mutagenesis, we have functionally characterized an enhancer located 12 kb downstream of the human immunoglobulin kappa constant-region gene. The core enhancer region is highly homologous to the murine 3' kappa enhancer. However, in addition to two regulatory elements homologous to the functional motifs of the murine enhancer, we find a third positive regulatory element in the human enhancer. This element is associated with an 11/12-bp direct repeat (DR) that is well conserved in the murine locus but was not recognized as functionally important in the murine enhancer. Mutation of any of the three motifs of the human enhancer decreases its activity to 3 to 20% of the wild-type level, indicating cooperative interaction between these elements. The DR motif does not resemble any known enhancer element and does not appear to function as a transcriptional activator on its own when present in multiple copies. Interestingly, nuclear extracts from both B- and T-cell lines contain factors binding to DR in vitro, but in vivo footprinting shows no evidence of protein-DNA binding in the T-cell line. This finding suggests that an additional regulatory mechanism, such as the effect of chromatin configuration on accessibility, may be involved in the B-cell-restricted activity of the human 3' kappa enhancer.

scholarly journals The Histone Chaperone Asf1p Mediates Global Chromatin Disassemblyin Vivo

2004 ◽  
Vol 279 (50) ◽  
pp. 52069-52074 ◽  
Author(s):  
Melissa W. Adkins ◽  
Jessica K. Tyler
Keyword(s):  
Histone Acetylation ◽  
Chromatin Structure ◽  
Histone H3 ◽  
Dnase I ◽  
Chromatin Assembly ◽  
Eukaryotic Genome ◽  
Micrococcal Nuclease ◽  
Histone Chaperones ◽  
Assembly Factor

The packaging of the eukaryotic genome into chromatin is likely to be mediated by chromatin assembly factors, including histone chaperones. We investigated the function of the histone H3/H4 chaperones anti-silencing function 1 (Asf1p) and chromatin assembly factor 1 (CAF-1)in vivo. Analysis of chromatin structure by accessibility to micrococcal nuclease and DNase I digestion demonstrated that the chromatin from CAF-1 mutant yeast has increased accessibility to these enzymes. In agreement, the supercoiling of the endogenous 2μ plasmid is reduced in yeast lacking CAF-1. These results indicate that CAF-1 mutant yeast globally under-assemble their genome into chromatin, consistent with a role for CAF-1 in chromatin assemblyin vivo. By contrast,asf1mutants globally over-assemble their genome into chromatin, as suggested by decreased accessibility of their chromatin to micrococcal nuclease and DNase I digestion and increased supercoiling of the endogenous 2μ plasmid. Deletion ofASF1causes a striking loss of acetylation on histone H3 lysine 9, but this is not responsible for the altered chromatin structure inasf1mutants. These data indicate that Asf1p may have a global role in chromatin disassembly and an unexpected role in histone acetylationin vivo.

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