Biomedical subjects

F M Sladek

Publications and source records attributed to F M Sladek.

25 records · Page 2Linked to original sources

PubMed1992

Murine chromosomal location of four hepatocyte-enriched transcription factors: HNF-3 alpha, HNF-3 beta, HNF-3 gamma, and HNF-4.

The genes for rat hepatocyte nuclear factors 3 and 4 (HNF-3 alpha, HNF-3 beta, HNF-3 gamma, and HNF-4) have been mapped in mouse by analysis of restriction fragment length polymorphisms in interspecific backcross mice. These hepatocyte-enriched transcription factors are positive-acting transcription factors with binding sites in regulatory regions of many genes expressed in hepatocytes. Both HNF-3 alpha, beta, and gamma and HNF-4 are also expressed in intestine. They have recently been implicated as potential participants in endodermal development from early gut cells because of their close homology to Drosophila genes, which themselves are expressed in the developing gut. Despite having similar functional roles and highly conserved DNA binding domains, the three loci from the Hnf-3 family of genes mapped to three different mouse chromosomes, suggesting that the Hnf-3 family has become widely dispersed during evolution and implying the necessity for independent activation of each member of the HNF-3 family.

Animals↗

PubMed1992

Mechanisms of liver-specific gene expression.

Significant advances in the field of hepatocyte-specific gene expression have been made during the past year. Several new transcription factors have been cloned and partially characterized. Analyses of the promoter regions of several factors have also been initiated and Drosophila homologs for two of these factors have been found, opening the way for studies on development.

Animals↗

PubMed1992

Antagonism between apolipoprotein AI regulatory protein 1, Ear3/COUP-TF, and hepatocyte nuclear factor 4 modulates apolipoprotein CIII gene expression in liver and intestinal cells.

Apolipoprotein CIII (apoCIII), a lipid-binding protein involved in the transport of triglycerides and cholesterol in the plasma, is synthesized primarily in the liver and the intestine. A cis-acting regulatory element, C3P, located at -90 to -66 upstream from the apoCIII gene transcriptional start site (+1), is necessary for maximal expression of the apoCIII gene in human hepatoma (HepG2) and intestinal carcinoma (Caco2) cells. This report shows that three members of the steroid receptor superfamily of transcription factors, hepatocyte nuclear factor 4 (HNF-4), apolipoprotein AI regulatory protein 1 (ARP-1), and Ear3/COUP-TF, act at the C3P site. HNF-4 activates apoCIII gene expression in HepG2 and Caco2 cells, while ARP-1 and Ear3/COUP-TF repress its expression in the same cells. HNF-4 activation is abolished by increasing amounts of ARP-1 or Ear3/COUP-TF, and repression by ARP-1 or Ear3/COUP-TF is alleviated by increasing amounts of HNF-4. HNF-4 and ARP-1 bind with similar affinities to the C3P site, suggesting that their opposing transcriptional effects may be mediated by direct competition for DNA binding. HNF-4 and ARP-1 mRNAs are present within the same cells in the liver and intestine, and protein extracts from hepatic tissue, HepG2, and Caco2 cells contain significantly more HNF-4 than ARP-1 or Ear3/COUP-TF binding activities. These findings suggest that the transcription of the apoCIII gene in vivo is dependent, at least in part, upon the intracellular balance of these positive and negative regulatory factors.

Apolipoprotein C-III↗

PubMed1991

The different tissue transcription patterns of genes for HNF-1, C/EBP, HNF-3, and HNF-4, protein factors that govern liver-specific transcription.

The transcription factors that act in hepatocyte-specific gene expression include proteins that are present mainly in liver cells (HNF-1/LFB1, C/EBP, HNF-3, HNF-4) (HNF, hepatocyte nuclear factor; C/EBP, rat enhancer binding protein) and proteins that are widely distributed (AP-1, NF-1, NF-Y/ACF). We show here that the genes encoding each of these liver-enriched factors exhibit different patterns of transcriptional control in different tissues. In addition, there were several instances in which transcription was detected (e.g., for HNF-1) when no mRNA or specific DNA binding protein was found, suggesting the importance of posttranscriptional control in some instances for these factors. These experiments identify C/EBP, HNF-3, and HNF-4, and perhaps also HNF-1, as targets for the study of cascades of transcriptionally controlled transcription factors in differentiated cells.

Base Sequence↗

PubMed1990

Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.

HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds to sites required for the transcription of the genes for transthyretin (TTR), the carrier protein in the serum for vitamin A and thyroid hormone, and for apolipoprotein CIII (apoCIII), a major constituent of chylomicrons and very low-density lipoproteins (VLDL). Synthetic oligonucleotides derived from amino acid sequence of affinity-purified HNF-4 protein (54 kD) were used in the polymerase chain reaction (PCR) to isolate a cDNA clone encoding the protein. HNF-4 is a member of the steroid hormone receptor superfamily with an unusual amino acid in the conserved "knuckle" of the first zinc finger (DGCKG). Studies with in vitro-translated HNF-4 protein show that it binds to its recognition site as a dimer, and cotransfection assays indicate that it activates transcription in a sequence-specific fashion in nonhepatic (HeLa) cells. Northern blot analysis reveals that HNF-4 mRNA is present in kidney and intestine, as well as liver, but is absent in other tissues. DNA-binding and antisera reactivity data suggest that HNF-4 could be identical to liver factor A1 (LF-A1), a DNA-binding activity implicated in the regulation of transcription of the alpha 1-antitrypsin, apolipoprotein A1, and pyruvate kinase genes. The similarity between HNF-4 and other ligand-dependent transcription factors raises the possibility that HNF-4 and the genes it regulates respond to an as yet unidentified ligand.

Amino Acid Sequence↗

PubMed1989

In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I.

Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.

Cross-Linking Reagents↗

PubMed1989

Incision by UvrABC excinuclease is a step in the path to mutagenesis by psoralen crosslinks in Escherichia coli.

4,5',8-Trimethylpsoralen (psoralen) plus near UV light produces interstrand crosslinks and monoadducts in DNA, both of which are mutagenic. In Escherichia coli, crosslinks are incised by UvrABC excinuclease, an event that can lead to homologous recombination and repair. To determine whether UvrABC incision of crosslinks is a step in the path to mutagenesis as well as repair, the effect of DNA homologous to a target gene on a plasmid was determined. pSV2-gpt DNA was treated with psoralen and transformed into a pair of hosts: one was gpt+, the other was delta (gpt-lac)5. The DNA was extracted and transformed into a tester strain [delta (gpt-lac)5] in which Gpt- mutations in the plasmid were scored. The results show that psoralen-induced mutations were reduced to background levels by the presence of the gpt+ homolog in the host chromosome. delta gpt hosts that were constitutively induced for the SOS response yielded point mutations, whereas noninduced hosts yielded almost exclusively large deletions. Since crosslinks were estimated to be responsible for most of the mutations observed, we conclude that the premutagenic lesion of psoralen crosslinks is recombinagenic and therefore very likely to be the product of UvrABC incision.

Cross-Linking Reagents↗