Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

Tratner, Isabelle; Nahon, Jean‐Louis; Sala‐Trepat, José M.; Venetianer, Aniko

doi:10.1128/mcb.7.5.1856

Cited by 30 publications

(20 citation statements)

References 44 publications

(63 reference statements)

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“…There was no evidence that the -3.5-kb region could function as an enhancer. Previous studies found a liver-specific hypersensitive site 2.8 kb upstream of the rat albumin promoter (Babiss et al 1986;Turcotte et al 1986;Nahon et al 1987;Tratner et al 1987); this site is in a position similar to the site found at -3.5 kb in mice by Pinkert et al (1987) and in this study. Rodent hepatoma cell lines transcribe the al bumin gene at a much lower rate than liver (Clayton et al 1985b); in such cells the gene is weakly hypersensi tive at the promoter, and not at all at the expected -2.8-kb (Babiss et al 1986;Nahon et al 1987) or -3.5-kb (Y. Bergman and K. Zaret, unpubl.…”

Section: Genes and Developmentsupporting

confidence: 64%

The mouse albumin promoter and a distal upstream site are simultaneously DNase I hypersensitive in liver chromatin and bind similar liver-abundant factors in vitro.

Liu¹,

Bergman²,

Zaret³

1988

Genes Dev.

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In this paper we characterize the chromatin structure and nuclear proteins associated with different transcriptional states of the mouse serum albumin gene. We found the albumin gene to be transcribed in liver at rates 1000-fold or greater than in other tissues tested. We discovered seven DNase I hypersensitive sites encompassing the albumin gene only in liver chromatin, with strong hypersensitivity at the promoter and the enhancer, which is over 10 kb upstream. Using a gel retardation assay, we found a liver nuclear protein, or set of proteins, which binds specifically to DNA of a liver-specific hypersensitive site that maps 3.5 kb upstream, between the promoter and enhancer. Footprinting, heat insensitivity, and binding competition experiments indicate that the protein(s) have characteristics similar to a heat-stable, liver-abundant protein that binds to the albumin promoter and other enhancer and promoter sequences. Finally, we asked whether the liver-specific factors that cause DNase I hypersensitivity in vivo are present concurrently at the various sites in chromatin. We devised a simple new method to reveal that in liver, individual albumin genes are hypersensitive simultaneously at the promoter, the enhancer, and the -3.5-kb site. Thus, transcriptionally active albumin genes appear to contain tissue-abundant factors that are present at three widely spaced points in chromatin, yet at the same point in time. Similar factors binding simultaneously to at least two of these sites could create a specific structure in chromatin required for high-level albumin gene transcription.

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Section: Genes and Developmentsupporting

confidence: 64%

The mouse albumin promoter and a distal upstream site are simultaneously DNase I hypersensitive in liver chromatin and bind similar liver-abundant factors in vitro.

Liu¹,

Bergman²,

Zaret³

1988

Genes Dev.

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“…An extensive amount of work, however, has established the paradigm that active genes reside in open chromatin. This has been demonstrated for many endogenous loci (12,17,29,40) and also has basis in a history of cytogenetic evidence (2,24). Therefore, we favor the interpretation that the observed level of expression and rearrangement from the unmethylated transgene represents leak-through rather than bona fide activity.…”

Section: Discussionmentioning

confidence: 82%

The Bulk Chromatin Structure of a Murine Transgene Does Not Vary with Its Transcriptional or DNA Methylation Status

Weng

Engler

Storb

1995

Molecular and Cellular Biology

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The DNA methylation status of HRD, a murine transgene, can be controlled by the genetic background upon which it is carried. We found the transgene to be transcribed in competent tissues only when undermethylated. Chromatin structure over the transgene was assayed by nuclear accessibility with DNase I, MspI, and PstI. While the transgene was up to fivefold more resistant to MspI when methylated than when not methylated, we observed no such difference with DNase I or PstI. We suggest that methyl-CpG-binding proteins are responsible for the difference observed with MspI, but that the chromatin structures are otherwise similarly compacted. Methylation could, therefore, play a regulatory role in gene expression beyond that which can be accomplished by bulk chromatin structure alone.Cytosine methylation occurs over approximately 60% of the CpG dinucleotides in the vertebrate genome (4) and has long been associated with a repression of gene activity (9). The pattern of methylation across the genome is passed to subsequent cell generations by the maintenance methylation activity of the methyltransferase enzyme (5, 18). The importance of DNA methylation in the mammalian genome has been demonstrated by the inability of mice homozygous for a targeted disruption of the methyltransferase gene to complete development (26).The mechanism by which DNA methylation effects gene repression remains unclear. Several lines of evidence suggest that methylation may interfere with gene expression directly. The cytosine analog 5-azacytidine is capable of reactivating silent genes, presumably by demethylating CpGs which reside in regulatory regions (22). Also, certain transcriptional activator proteins whose cognate sequences contain CpGs have been demonstrated to bind only when the CpG is not methylated (31, 41). These cases of methylation-sensitive binding were determined in vitro, however; genomic footprinting of the tyrosine aminotransferase promoter demonstrated that binding of the CREB transcription factor could not be induced by demethylation of its binding site, even though it exhibited methylation-sensitive binding when assayed in vitro (42). Furthermore, some transcription factors bind their targets equally well in vitro, whether methylated or not (19).Other lines of evidence suggest that the repressive effect of methylation is indirect and mediated by chromatin structure. Although in vitro methylation of DNA constructs prior to transient transfection renders them transcriptionally inert, the onset of repression after transfection is delayed and correlates with the formation of chromatin over the methylated DNA (7). Similarly, V(D)J recombination of extrachromosomal substrates is inhibited by in vitro CpG methylation, but only after replication (20). As the acquisition of resistance to exogenous endonucleases also occurs after replication, the inhibition of V(D)J recombination is presumably the result of the formation of chromatin over the extrachromosomal substrate rather than the presence of the methyl moieties. Other work has...

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“…We find that the segment of rat AFP DNA spanning -1.2 to -4 kb from the AFP transcription initiation site is a typical enhancer, sufficient to confer high activity upon the AFP promoter in well-differentiated hepatoma cells. The active components of this enhancer element lie between -2.2 and -4 kb and match the only two DH sites in the 7-kb domain upstream from the AFP promoter in rat liver chromatin (32,42,44). Transgene and transfection assays leave ambiguities on liver-specific functions of the AFP enhancer domain, one reason being that segments of DNA between -4 and -7 kb can substitute for the downstream enhancer segments in transfected hepatocytes (17, 31; our unpublished results) or transgenic livers (22).…”

Section: Discussionmentioning

confidence: 99%

“…A promoter domain spanning '230 nucleotides comprises a chromatin DNase I-hypersensitive (DH) site (32, 42,44) that is selectively suppressed by dexamethasone (44), glucocorticoid receptor recognition sequences, and octamer motifs similar to those present in other genes under developmental and growth control (8). A distal domain (-2 to -4 kilobases [kb] relative to the AFP transcription initiation site) is comprised of a liver-constitutive DH site and a developmental stagespecific DH site (32,42,44). These structural features may suggest a cooperativity between distal regulatory elements * Corresponding author.…”

mentioning

confidence: 99%

Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Guertin¹,

LaRue²,

Bernier³

et al. 1988

Mol. Cell. Biol.

109

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Mutations were introduced in 7 kilobases of 5'-flanking rat ac-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.The a,-fetoprotein (AFP) gene, a member of the albumin gene family, is expressed by fetal or malignant hepatocytes and repressed in normal mature hepatocytes. This forms the basis of a long-exploited model system to study cell differentiation and its impairment in neoplasia (1). Molecular genetics have brought the AFP model to molecular levels of cell differentiation, toward finely discerning how genes respond to or escape from developmental and growth signals (2,40,41). As yet, little is known about how differential regulation is exerted on the tandemly organized AFPalbumin locus and whether the two genes operate under shared elements of control. However, at least one wellcharacterized transcription factor, the glucocorticoid receptor, is known to selectively shut off the AFP gene in the developing liver (4, 21). This hormonal effect reaches into the mechanisms of neoplastic resistance to differentiation, because the AFP gene in malignant cells is generally refractory to glucocorticoids (2).Analyses of rat AFP gene and chromatin structures have identified domains in the 5' region of the locus potentially involved in its liver-specific, developmental stage-dependent, and glucocorticoid-regulated expression. A promoter domain spanning '230 nucleotides comprises a chromatin DNase I-hypersensitive (DH) site (32, 42, 44) that is selectively suppressed by dexamethasone (44), glucocorticoid receptor recognition sequences, and octamer motifs similar to those present in other genes under developmental and growth control (8). A distal domain (-2 to -4 kilobases [kb] relative to the AFP trans...

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Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

Cited by 30 publications

References 44 publications

The mouse albumin promoter and a distal upstream site are simultaneously DNase I hypersensitive in liver chromatin and bind similar liver-abundant factors in vitro.

The mouse albumin promoter and a distal upstream site are simultaneously DNase I hypersensitive in liver chromatin and bind similar liver-abundant factors in vitro.

The Bulk Chromatin Structure of a Murine Transgene Does Not Vary with Its Transcriptional or DNA Methylation Status

Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

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