Extinction of immunoglobulin gene expression in B cells upon fusion with HeLa cells is preceded by rapid nuclear depletion of essential transcription factors and is accompanied by widespread inactivation of genes expressed in a B cell-specific manner

1997 ◽  
Vol 110 (20) ◽  
pp. 2579-2587 ◽  
Author(s):  
S. Junker ◽  
M. Lamm ◽  
V. Nielsen ◽  
P. Matthias
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Molecular Mechanism ◽  
Gene Transcription ◽  
Hela Cells ◽  
Immunoglobulin Gene ◽  
Nf Kappa B ◽  
Specific Manner

When immunoglobulin (Ig) expressing B cells are fused with non-B cells, Ig expression is rapidly suppressed at the level of transcription, a phenomenon termed extinction. Here we demonstrate that fusion of HeLa cells with either diploid or tetraploid B cells (Daudi) results in widespread extinction of several other B cell-encoded genes that are expressed in a B cell-specific manner. In contrast, expression of B cell-expressed genes that are not dependent on cell-specific controls is unaffected. We show that the molecular mechanism(s) underlying Ig gene extinction can be explained, at least in part, by a lack of transcription factors that are essential for Ig gene transcription. These transcription factors are either not produced due to block of transcription of their respective genes (Oct-2, OBF-1, PU.1), or are rendered inactive posttranslationally (NF-kappa B, E47). By isolating Daudi × HeLa heterokaryons a few hours after fusion, we have studied the initial fate of two B cell-specific transcription factors involved in Ig gene transcription, Oct-2 and NF-kappa B. This report provides the first demonstration that upon fusion with HeLa cells, the nuclear contents of B cell-expressed transcription factors are depleted within a few hours with kinetics that are as fast or faster than that of Ig gene extinction. Thus, the extinguishing mechanism is effective very early after fusion. We suggest that extinction of Ig genes is part of a global mechanism that suppresses the differentiation program foreign to the HeLa phenotype.

scholarly journals A proximal tissue-specific module and a distal negative regulatory module control apolipoprotein(a) gene transcription

2004 ◽  
Vol 379 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Sarita NEGI ◽  
Saurabh K. SINGH ◽  
Nirupma PATI ◽  
Vikas HANDA ◽  
Ruchi CHAUHAN ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Gene Transcription ◽  
Hela Cells ◽  
Hepg2 Cells ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Tissue Specific ◽  
Apolipoprotein A ◽  
Regulatory Module ◽  
Specific Manner

The apo(a) [apolipoprotein(a)] gene is responsible for variations in plasma lipoprotein(a), high levels of which are a risk factor for atherosclerosis and myocardial infarction. The apo(a) promoter stimulates the expression of reporter genes in HepG2 cells, but not in HeLa cells. In the present study, we demonstrate that the 1.4 kb apo(a) promoter comprises two composite regulatory regions: a distal negative regulatory module (positions −1432 to −716) and a proximal tissue-specific module (−716 to −616). The distal negative regulatory module contains two strong negative regulatory regions [polymorphic PNR (pentanucleotide repeat region) and NREβ (negative regulatory element β)], which sandwich the postive regulatory region PREβ (positive regulatory element β). The PNR was shown to bind to transcription factors in a tissue-specific manner, whereas the ubiquitous transcription factors hepatocyte nuclear factor 3α and GATA binding protein 4 bound to NREβ to repress gene transcription. The proximal tissue-specific module contains two regulatory elements: an activating region (PREα) that activates transcription in HepG2 cells, and NREα, which is responsible for repressing the apo(a) gene in HeLa cells. NREα binds to a HeLa-specific repressor. These multiple regulatory elements might work co-operatively to finely regulate apo(a) gene expression. Although the tissue-specific module is required for apo(a) gene activation and repression in a tissue-specific manner, the combinatorial interplay of the distal and proximal regulators might define the complex pathway(s) of apo(a) gene regulation.

Positive and negative regulation of immunoglobulin gene expression by a novel B-cell-specific enhancer element

1991 ◽  
Vol 11 (1) ◽  
pp. 75-83
Author(s):  
J Wang ◽  
M Oketani ◽  
T Watanabe
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Hela Cells ◽  
Simian Virus 40 ◽  
Immunoglobulin Gene ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Octamer Motif

A new B-cell-specific enhancer element has been identified 3' of E4 and the octamerlike motifs in the human immunoglobulin heavy-chain gene enhancer. Tandem copies of this 67-bp MnlI-AluI fragment, when fused to the chloramphenicol acetyltransferase gene driven by the conalbumin promoter, stimulated transcription in B cells but not in Jurkat T cells or HeLa cells. Footprinting analysis revealed that the identical sequence CCGAAACTGAAAAGG, designated E6, was protected by nuclear extracts from B cells, T cells, or HeLa cells. Gel mobility shift assays using a synthetic E6 motif detected a B-cell-specific complex in addition to a ubiquitous band found also in T cells and HeLa cells. In agreement with the results of gel retardation assays, tandem copies of the E6 motif stimulated transcription in ARH77 and Raji cells but not in Jurkat or HeLa cells. Furthermore, a mutant E6 motif lost both in vitro binding activity and in vivo enhancer activity. In striking contrast to the mouse Ig heavy-chain enhancer, in which the octamer motif acts as a B-cell-specific enhancer element, the human enhancer contains an octamerlike sequence with one base substitution which bound octamer-binding proteins with only very low affinity and showed no enhancer activity of its own. Interestingly, the MnlI-AluI fragment could suppress the basal-level activity of the conalbumin promoter in both Jurkat and HeLa cells. Moreover, simian virus 40 enhancer activity was blocked by the MnlI-AluI fragment in HeLa cells but not in B cells. Thus, the novel enhancer element identified in this study is probably a target site for both positive and negative factors.

scholarly journals Positive and negative regulation of immunoglobulin gene expression by a novel B-cell-specific enhancer element.

1991 ◽  
Vol 11 (1) ◽  
pp. 75-83 ◽  
Author(s):  
J Wang ◽  
M Oketani ◽  
T Watanabe
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Hela Cells ◽  
Simian Virus 40 ◽  
Immunoglobulin Gene ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Octamer Motif

A new B-cell-specific enhancer element has been identified 3' of E4 and the octamerlike motifs in the human immunoglobulin heavy-chain gene enhancer. Tandem copies of this 67-bp MnlI-AluI fragment, when fused to the chloramphenicol acetyltransferase gene driven by the conalbumin promoter, stimulated transcription in B cells but not in Jurkat T cells or HeLa cells. Footprinting analysis revealed that the identical sequence CCGAAACTGAAAAGG, designated E6, was protected by nuclear extracts from B cells, T cells, or HeLa cells. Gel mobility shift assays using a synthetic E6 motif detected a B-cell-specific complex in addition to a ubiquitous band found also in T cells and HeLa cells. In agreement with the results of gel retardation assays, tandem copies of the E6 motif stimulated transcription in ARH77 and Raji cells but not in Jurkat or HeLa cells. Furthermore, a mutant E6 motif lost both in vitro binding activity and in vivo enhancer activity. In striking contrast to the mouse Ig heavy-chain enhancer, in which the octamer motif acts as a B-cell-specific enhancer element, the human enhancer contains an octamerlike sequence with one base substitution which bound octamer-binding proteins with only very low affinity and showed no enhancer activity of its own. Interestingly, the MnlI-AluI fragment could suppress the basal-level activity of the conalbumin promoter in both Jurkat and HeLa cells. Moreover, simian virus 40 enhancer activity was blocked by the MnlI-AluI fragment in HeLa cells but not in B cells. Thus, the novel enhancer element identified in this study is probably a target site for both positive and negative factors.

scholarly journals Formation of an Active Tissue-Specific Chromatin Domain Initiated by Epigenetic Marking at the Embryonic Stem Cell Stage

2005 ◽  
Vol 25 (5) ◽  
pp. 1804-1820 ◽  
Author(s):  
Henrietta Szutorisz ◽  
Claudia Canzonetta ◽  
Andrew Georgiou ◽  
Cheok-Man Chow ◽  
László Tora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Chromatin Domain ◽  
Cell Stage ◽  
General Transcription Factors ◽  
Regulated Gene Expression

ABSTRACT The differentiation potential of stem cells is determined by the ability of these cells to establish and maintain developmentally regulated gene expression programs that are specific to different lineages. Although transcriptionally potentiated epigenetic states of genes have been described for haematopoietic progenitors, the developmental stage at which the formation of lineage-specific gene expression domains is initiated remains unclear. In this study, we show that an intergenic cis-acting element in the mouse λ5-VpreB1 locus is marked by histone H3 acetylation and histone H3 lysine 4 methylation at a discrete site in embryonic stem (ES) cells. The epigenetic modifications spread from this site toward the VpreB1 and λ5 genes at later stages of B-cell development, and a large, active chromatin domain is established in pre-B cells when the genes are fully expressed. In early B-cell progenitors, the binding of haematopoietic factor PU.1 coincides with the expansion of the marked region, and the region becomes a center for the recruitment of general transcription factors and RNA polymerase II. In pre-B cells, E2A also binds to the locus, and general transcription factors are distributed across the active domain, including the gene promoters and the intergenic region. These results suggest that localized epigenetic marking is important for establishing the transcriptional competence of the λ5 and VpreB1 genes as early as the pluripotent ES cell stage.

scholarly journals Transcription factors of the alternative NF-κB pathway are required for germinal center B-cell development

2016 ◽  
Vol 113 (32) ◽  
pp. 9063-9068 ◽  
Author(s):  
Nilushi S. De Silva ◽  
Michael M. Anderson ◽  
Amanda Carette ◽  
Kathryn Silva ◽  
Nicole Heise ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Plasma Cells ◽  
Alternative Pathway ◽  
Cell Development ◽  
B Cell Development ◽  
Cell Surface Receptor ◽  
Signaling Cascade

The NF-κB signaling cascade relays external signals essential for B-cell growth and survival. This cascade is frequently hijacked by cancers that arise from the malignant transformation of germinal center (GC) B cells, underscoring the importance of deciphering the function of NF-κB in these cells. The NF-κB signaling cascade is comprised of two branches, the canonical and alternative NF-κB pathways, mediated by distinct transcription factors. The expression and function of the transcription factors of the alternative pathway, RELB and NF-κB2, in late B-cell development is incompletely understood. Using conditional deletion of relb and nfkb2 in GC B cells, we here report that ablation of both RELB and NF-κB2, but not of the single transcription factors, resulted in the collapse of established GCs. RELB/NF-κB2 deficiency in GC B cells was associated with impaired cell-cycle entry and reduced expression of the cell-surface receptor inducible T-cell costimulator ligand that promotes optimal interactions between B and T cells. Analysis of human tonsillar tissue revealed that plasma cells and their precursors in the GC expressed high levels of NF-κB2 relative to surrounding lymphocytes. Accordingly, deletion of nfkb2 in murine GC B cells resulted in a dramatic reduction of antigen-specific antibody-secreting cells, whereas deletion of relb had no effect. These results demonstrate that the transcription factors of the alternative NF-κB pathway control distinct stages of late B-cell development, which may have implications for B-cell malignancies that aberrantly activate this pathway.

scholarly journals Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation.

1991 ◽  
Vol 11 (10) ◽  
pp. 4885-4894 ◽  
Author(s):  
C L Miller ◽  
A L Feldhaus ◽  
J W Rooney ◽  
L D Rhodes ◽  
C H Sibley ◽  
...  
Keyword(s):  
B Cell ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
De Novo ◽  
Interleukin 1 ◽  
Transcriptional Level ◽  
Immunoglobulin Gene ◽  
B Cell Differentiation ◽  
Nf Kappa B ◽  
Specific Expression

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.

scholarly journals Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors.

1995 ◽  
Vol 15 (2) ◽  
pp. 943-953 ◽  
Author(s):  
R I Scheinman ◽  
A Gualberto ◽  
C M Jewell ◽  
J A Cidlowski ◽  
A S Baldwin
Keyword(s):  
Gene Expression ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Glucocorticoid Receptors ◽  
Interleukin 1 ◽  
Direct Interaction ◽  
Significant Loss ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Important Regulator

Glucocorticoids are potent immunosuppressants which work in part by inhibiting cytokine gene transcription. We show here that NF-kappa B, an important regulator of numerous cytokine genes, is functionally inhibited by the synthetic glucocorticoid dexamethasone (DEX). In transfection experiments, DEX treatment in the presence of cotransfected glucocorticoid receptor (GR) inhibits NF-kappa B p65-mediated gene expression and p65 inhibits GR activation of a glucocorticoid response element. Evidence is presented for a direct interaction between GR and the NF-kappa B subunits p65 and p50. In addition, we demonstrate that the ability of p65, p50, and c-rel subunits to bind DNA is inhibited by DEX and GR. In HeLa cells, DEX activation of endogenous GR is sufficient to block tumor necrosis factor alpha or interleukin 1 activation of NF-kappa B at the levels of both DNA binding and transcriptional activation. DEX treatment of HeLa cells also results in a significant loss of nuclear p65 and a slight increase in cytoplasmic p65. These data reveal a second mechanism by which NF-kappa B activity may be regulated by DEX. We also report that RU486 treatment of wild-type GR and DEX treatment of a transactivation mutant of GR each can significantly inhibit p65 activity. In addition, we found that the zinc finger domain of GR is necessary for the inhibition of p65. This domain is also required for GR repression of AP-1. Surprisingly, while both AP-1 and NF-kappa B can be inhibited by activated GR, synergistic NF-kappa B/AP-1 activity is largely unaffected. These data suggest that NF-kappa B, AP-1, and GR interact in a complex regulatory network to modulate gene expression and that cross-coupling of NF-kappa B and GR plays an important role in glucocorticoid-mediated repression of cytokine transcription.

Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation

1994 ◽  
Vol 14 (8) ◽  
pp. 5349-5359
Author(s):  
H C Liou ◽  
W C Sha ◽  
M L Scott ◽  
D Baltimore
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lineage ◽  
B Cell Differentiation ◽  
Nf Kappa B ◽  
Activator Proteins ◽  
I Kappa B Alpha ◽  
Wehi 231 ◽  
B Lineage

The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs.

scholarly journals Identification of an octamer-binding site in the mouse kappa light-chain immunoglobulin enhancer.

1989 ◽  
Vol 9 (10) ◽  
pp. 4239-4247 ◽  
Author(s):  
R A Currie ◽  
R G Roeder
Keyword(s):  
B Cell ◽  
Binding Site ◽  
Light Chain ◽  
Specific Binding ◽  
Specific Factor ◽  
Immunoglobulin Gene ◽  
Kappa Light Chain ◽  
Nf Kappa B ◽  
Light Chain Immunoglobulin ◽  
A Site

A 215-base-pair (bp) region of the mouse MOPC 41 kappa light-chain immunoglobulin gene enhancer has been analyzed for specific binding of lymphoid and nonlymphoid nuclear factors. Mobility shift assays with a series of overlapping DNA fragments have mapped DNA-binding sites for three unique factors. The B-cell-specific (OTF-2) and ubiquitous (OTF-1) octamer-binding transcription factors specifically bound to a site centered about 136 bp 5' of the nuclear factor NF-kappa B site. A third specific factor, NF-kappa E, bound to a site that was about 75 bp 5' of the NF-kappa B site and within a region important for enhancer function. This novel factor was found in both mature B and HeLa cell nuclei. B-cell OTF-2, B-cell OTF-1, and HeLa OTF-1 bound to the kappa enhancer and kappa promoter octamer sites with similar affinities despite a 2-bp difference in the kappa enhancer octamer sequence. However, DNase I footprint analyses indicated that affinity-purified OTF-2 bound both to the enhancer OTF site and, surprisingly, to 80 bp of A + T-rich flanking sequence. Moreover, methylation interference studies demonstrated distinct differences in OTF interactions between the consensus octamer in the kappa promoter and the nonconsensus octamer identified in the enhancer. This novel observation of an OTF-binding site in the kappa enhancer provides a common link with the OTF sites in the promoter-proximal regions of all kappa promoters and thus mirrors the structural arrangement of OTF sites found in the promoters and enhancers of immunoglobulin heavy-chain genes.

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