DNase I- and micrococcal nuclease-hypersensitive sites in the human apolipoprotein B gene are tissue specific.

We have mapped the DNase I- and micrococcal nuclease-hypersensitive sites present in the 5' end of the human apolipoprotein B (apo-B) gene in nuclei from cells expressing or not expressing the gene. Four DNase I-hypersensitive sites were found in nuclei from liver-derived HepG2 cells and intestine-derived CaCo-2 cells, which express the apo-B gene, but not in HeLa cells, which do not. These sites are located near positions -120, -440, -700, and +760 base pairs relative to the transcriptional start site. Undifferentiated CaCo-2 cells exhibited another site, near position -540. Six micrococcal nuclease-hypersensitive sites were found in nuclei from HepG2 and CaCo-2 cells, but not in HeLa cells or free DNA. These sites are located near positions -120, -390, -530, -700, -850, and +210. HepG2 cells exhibited another site, near position +460. Comparison of the DNA sequence of the 5' flanking regions of the human and mouse apo-B genes revealed a high degree of evolutionary conservation of short stretches of sequences in the immediate vicinity of each of the DNase I- and most of the micrococcal nuclease-hypersensitive sites.

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The mechanism by which the human apolipoprotein B gene reducer operates involves blocking of transcriptional activation by hepatocyte nuclear factor 3.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1534 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1534-1546 ◽

Cited By ~ 36

Author(s):

B Paulweber ◽

F Sandhofer ◽

B Levy-Wilson

Keyword(s):

Binding Site ◽

Transcriptional Activation ◽

Apolipoprotein B ◽

Regulatory Protein ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Apo B ◽

Human Apolipoprotein ◽

Colon Carcinoma Cells ◽

Mediator Protein

Previously, we showed that when a DNA fragment extending from -3067 to -2734 of the human apolipoprotein B (apo-B) gene is inserted immediately upstream of an apo-B promoter segment (-139 to +121), transcription from this promoter is reduced by about 10-fold in cultured colon carcinoma cells (CaCo-2) but not in cultured hepatoma cells (HepG2). We postulated that this reducer operates by a mechanism involving active repression of a transcriptional activator that binds to the segment from -111 to -88 of the apo-B promoter (B. Paulweber and B. Levy-Wilson, J. Biol. Chem. 266:24161-24168 1991). In the current study, the reducer element has been localized to a 24-bp sequence from -2801 to -2778 of the apo-B gene that contains a binding site for the negative regulatory protein ARP-1. Furthermore, we have demonstrated that the transcription factor hepatocyte nuclear factor 3 alpha (HNF-3 alpha) binds to the sequence 5'-TGTTTGCTTTTC-3' from -95 to -106 of the apo-B promoter, to stimulate transcription. Transcriptional activation by HNF-3 is repressed when the reducer sequence is inserted immediately upstream of the HNF-3 binding site, suggesting a mechanism by which the reducer-bound protein blocks the activation promoted by HNF-3. Data from cotransfection experiments in which ARP-1 is overexpressed in the absence of its binding site suggest that ARP-1 interacts either directly or via a mediator protein with proteins recognizing the HNF-3 site and that this interaction is sufficient to repress transcriptional activation by HNF-3. Because transcriptional activation by Sp1 is not affected by the reducer, it is unlikely that the reducer interacts directly with basic components of the transcriptional machinery.

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Characterization of apolipoprotein B from human serum low density lipoprotein in n-dodecyl octaethyleneglycol monoether: an electron microscope study.

The Journal of Cell Biology ◽

10.1083/jcb.87.3.555 ◽

1980 ◽

Vol 87 (3) ◽

pp. 555-561 ◽

Cited By ~ 14

Author(s):

G Zampighi ◽

J A Reynolds ◽

R M Watt

Keyword(s):

Apolipoprotein B ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Serum Lipoprotein ◽

Low Density ◽

Apo B ◽

Shaped Particle ◽

Orderly Fashion ◽

High Degree

We have studied the structure of the totally delipidated polypeptide (apolipoprotein B [apo B]) present in low-density serum lipoprotein in detergent (n-dodecyl octaethyleneglycol monoether) solution by electron microscopy. The protein-detergent complex appears as a rod-shaped particle, 75-80 nm long and 4.5-5.5 nm wide. The volume of this particle is consistent with the previously published composition reported by Watt and Reynolds (1980, Biochemistry 19:1593-1598) of two copies of apo B and five to six equivalent micelles of detergent. The asymmetric particle possesses a high degree of flexibility and a strong tendency to self-associate in an orderly fashion. The extent of this association is pH dependent.

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A postulated phylogenetic tree for the human apolipoprotein B gene: Unpredicted haplotypes are associated with elevated apo B levels

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism ◽

10.1016/0005-2760(93)90136-w ◽

1993 ◽

Vol 1165 (3) ◽

pp. 271-278 ◽

Cited By ~ 13

Author(s):

Alison M. Dunning ◽

Helmut-H. Renges ◽

Anders Hamsten ◽

Philippa Talmud ◽

Steve Humphries

Keyword(s):

Phylogenetic Tree ◽

Apolipoprotein B ◽

Apo B ◽

Human Apolipoprotein ◽

Apolipoprotein B Gene

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Tunicamycin induces ubiquitination and degradation of apolipoprotein B in HepG2 cells

Biochemical Journal ◽

10.1042/bj3530493 ◽

2001 ◽

Vol 353 (3) ◽

pp. 493-501 ◽

Cited By ~ 16

Author(s):

Wei LIAO ◽

Lawrence CHAN

Keyword(s):

Apolipoprotein B ◽

Hepg2 Cells ◽

Proteasome Inhibitor ◽

Plasma Lipoproteins ◽

Apo B ◽

Net Production ◽

Lipoprotein Particles ◽

Essential Component ◽

Intracellular Degradation

Apolipoprotein (apo) B-100 is an essential component of atherogenic plasma lipoproteins. Previous studies have demonstrated that the production of apoB-100 is regulated largely by intracellular degradation at both the co-translational and post-translational levels and that proteasome-mediated and non-proteasome-mediated pathways are involved in this process. ApoB-100 is a glycoprotein. The present study was undertaken to address the question of whether the inhibition of N-linked glycosylation with tunicamycin would interfere with apoB-100 production. We demonstrated that the treatment of HepG2 cells with tunicamycin decreased the net production of apoB-100 by enhancing co-translational degradation of the protein. This effect of tunicamycin was partly prevented by lactacystin, a specific proteasome inhibitor. Because lactacystin only partly reversed the effects of tunicamycin on apoB biogenesis, tunicamycin seemed also to induce apoB co-translational degradation in HepG2 cells by one or more non-proteasomal pathways. Furthermore, tunicamycin increased apoB ubiquitination approx. 4-fold. The proportion of the newly synthesized apoB-100 that was secreted and incorporated into the nascent lipoprotein particles was unaffected by tunicamycin. Thus the tunicamycin-mediated inhibition of N-linked glycosylation interferes with the production of apoB-100 that is mediated by both proteasomal and non-proteasomal pathways.

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DNaseI hypersensitive sites at the 3′ end of the human apolipoprotein B gene

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(90)91371-x ◽

1990 ◽

Vol 171 (1) ◽

pp. 162-168 ◽

Cited By ~ 1

Author(s):

Beatriz Levy-Wilson

Keyword(s):

Apolipoprotein B ◽

Human Apolipoprotein ◽

Hypersensitive Sites ◽

Apolipoprotein B Gene

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Promoter region of interleukin-2 gene undergoes chromatin structure changes and confers inducibility on chloramphenicol acetyltransferase gene during activation of T cells.

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3042 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3042-3049 ◽

Cited By ~ 91

Author(s):

U Siebenlist ◽

D B Durand ◽

P Bressler ◽

N J Holbrook ◽

C A Norris ◽

...

Keyword(s):

T Cells ◽

Chromatin Structure ◽

Promoter Region ◽

Transcriptional Start Site ◽

Interleukin 2 ◽

Chloramphenicol Acetyltransferase ◽

Dnase I ◽

Jurkat Cells ◽

Hypersensitive Sites ◽

Chloramphenicol Acetyltransferase Gene

The chromatin structure of the interleukin-2 (IL-2) gene was probed by DNase I treatment of isolated nuclei. The 5' region of the IL-2 gene contains three regions of hypersensitivity to DNase I. When peripheral blood T cells or Jurkat T cells are stimulated with mitogens, IL-2 message is induced, and the promoter region of the IL-2 gene develops an additional hypersensitive site. This suggests that a DNA sequence close to the transcriptional start site is involved in the transduction of the extracellular signal. Such a conclusion is further supported by DNA transfection experiments. A short segment of DNA, which includes the region of induced hypersensitivity, confers inducibility on the linked chloramphenicol acetyltransferase gene in transiently transfected Jurkat cells. In addition, cells of nonhematopoietic origins exhibit a strikingly different chromatin pattern of IL-2, suggesting a role during differentiation for some of the hypersensitive sites.

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Developmental regulation of DNase I-hypersensitive sites in Dictyostelium discoideum

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1823-1829.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1823-1829

Author(s):

K Ayres ◽

W Neuman ◽

W G Rowekamp ◽

S Chung

Keyword(s):

Dictyostelium Discoideum ◽

Developmental Regulation ◽

Transcriptional Start Site ◽

Dnase I ◽

Start Site ◽

Hypersensitive Site ◽

Stages Of Development ◽

Vegetative Cells ◽

Dnase I Hypersensitive Sites ◽

Hypersensitive Sites

We have studied two regions of Dictyostelium discoideum chromatin and identified several DNase I-hypersensitive sites in these regions. One of these sites is located about 300 to 500 bases upstream of the transcriptional start site of a gene that is expressed at all stages of development. This site is present in both vegetative cells and postaggregation cells. Another hypersensitive site is associated with a gene that is expressed only after the multicellular stage. This site is located about 400 bases upstream of the start site, and it is present only in postaggregation cells. Thus, much like higher eucaryotes, D. discoideum contains DNase I-hypersensitive sites that may be involved in the regulation of the genes with which they are associated.

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The mechanism by which the human apolipoprotein B gene reducer operates involves blocking of transcriptional activation by hepatocyte nuclear factor 3

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1534-1546.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1534-1546

Author(s):

B Paulweber ◽

F Sandhofer ◽

B Levy-Wilson

Keyword(s):

Binding Site ◽

Transcriptional Activation ◽

Apolipoprotein B ◽

Regulatory Protein ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Apo B ◽

Human Apolipoprotein ◽

Colon Carcinoma Cells ◽

Mediator Protein

Previously, we showed that when a DNA fragment extending from -3067 to -2734 of the human apolipoprotein B (apo-B) gene is inserted immediately upstream of an apo-B promoter segment (-139 to +121), transcription from this promoter is reduced by about 10-fold in cultured colon carcinoma cells (CaCo-2) but not in cultured hepatoma cells (HepG2). We postulated that this reducer operates by a mechanism involving active repression of a transcriptional activator that binds to the segment from -111 to -88 of the apo-B promoter (B. Paulweber and B. Levy-Wilson, J. Biol. Chem. 266:24161-24168 1991). In the current study, the reducer element has been localized to a 24-bp sequence from -2801 to -2778 of the apo-B gene that contains a binding site for the negative regulatory protein ARP-1. Furthermore, we have demonstrated that the transcription factor hepatocyte nuclear factor 3 alpha (HNF-3 alpha) binds to the sequence 5'-TGTTTGCTTTTC-3' from -95 to -106 of the apo-B promoter, to stimulate transcription. Transcriptional activation by HNF-3 is repressed when the reducer sequence is inserted immediately upstream of the HNF-3 binding site, suggesting a mechanism by which the reducer-bound protein blocks the activation promoted by HNF-3. Data from cotransfection experiments in which ARP-1 is overexpressed in the absence of its binding site suggest that ARP-1 interacts either directly or via a mediator protein with proteins recognizing the HNF-3 site and that this interaction is sufficient to repress transcriptional activation by HNF-3. Because transcriptional activation by Sp1 is not affected by the reducer, it is unlikely that the reducer interacts directly with basic components of the transcriptional machinery.

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A Highly Sensitive Assay for Quantitation of Apolipoprotein B48 Using an Antibody to Human Apolipoprotein B and Enhanced Chemiluminescence

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456329703400210 ◽

1997 ◽

Vol 34 (2) ◽

pp. 185-189 ◽

Cited By ~ 40

Author(s):

Darrin Smith ◽

Spencer D Proctor ◽

John C L Mamo

Keyword(s):

Apolipoprotein B ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Apo B ◽

Human Apolipoprotein ◽

Enhanced Chemiluminescence ◽

Coomassie Blue ◽

Sds Page ◽

One Step ◽

Apolipoprotein B48

We describe a method for the rapid quantification of serum apolipoprotein B48 using a commercially available anti-apolipoprotein (apo)B antiserum and compare it to analytical sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with coomassie blue R250 staining. The method described here eliminates the need for de-lipidation of samples and only requires a one-step overnight ultracentrifugation. Western-blotting and enhanced chemiluminescence (ECL) visualization of proteins was approximately 10 times more sensitive than coomassie staining and generally took no longer to complete than staining/destaining of SDS-PAGE gels. The sensitivity of the antiserum/ECL technique enabled quantitation of fasting apolipoprotein B48 which could not be resolved by SDS-PAGE and Coomassie staining.

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