Isolation and characterization of a protein fraction that binds to enhancer core sequences in intracisternal A-particle long terminal repeats

The U3 region of mouse intracisternal A-particle (IAP) long terminal repeats (LTRs) contains several nuclear protein-binding domains. Two of these contain sequences with homology to the SV40 enhancer core. We refer to these two domains as Enh1 and Enh2. The Enh2 domain is an important determinant of promoter activity in vivo. We report here the isolation of nuclear fractions from human 293 and mouse MOPC-315 cells which interact with Enh1 and Enh2. Purification was achieved via DNA-affinity chromatography on a multimerized oligonucleotide representing the Enh2 region from the LTR of the mouse genomic IAP element, MIA14. Glycerol gradient sedimentation suggested a native Mr of approximately 80-100 for the binding component(s) in both crude and affinity-purified fractions. UV cross-linking showed that the binding activity involved two polypeptides within this size range. The affinity-isolated fraction from each cell line was highly purified, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in vitro binding analysis. Exonuclease III footprinting showed that the two polypeptides interacted preferentially with the Enh1 and Enh2 domains within a 139-base pair segment from the MIA14 LTR. The polypeptides interacted in a sequence-specific manner with oligonucleotides representing these domains within the IAP LTR and with oligonucleotides containing the enhancer core sequence from SV40 and polyoma virus. Equilibrium binding studies indicated that the apparent dissociation constants for the polypeptides binding to the enhancer core sequence from MIA14, SV40, and polyoma virus were similar. Therefore, this affinity-purified fraction may represent a novel enhancer core-binding component which is distinct from the previously characterized rat CCAAT/enhancer-binding protein, C/EBP.     

Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from Lubrol extract of sea urchin egg membranes. The partially purified protein possessed two polypeptides of 39 and 37 kDa; the 39 kDa polypeptide was specifically ADP-ribosylated by IAP and the 37 kDa protein cross-reacted with the antibody prepared against purified beta gamma-subunits of alpha beta gamma-heterotrimeric IAP substrates from rat brain. Incubation of this sea urchin IAP substrate with a non-hydrolyzable GTP analogue resulted in a reduction of the apparent molecular mass on a column of gel filtration as had been the case with purified rat brain IAP substrates, suggesting that the sea urchin IAP substrate was also a heterooligomer dissociable into two polypeptides in the presence of GTP analogues. Thus, the 39 and 37 kDa polypeptides of the sea urchin IAP substrate correspond to the alpha- and beta-subunits, respectively, of mammalian IAP substrates which are involved in the coupling between membrane receptor and effector systems.     

In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR.

We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo.     

Regulation of intracisternal A particles in mouse teratocarcinoma cells: involvement of DNA methylation in transcriptional control

The methylation state of Intracisternal A Particle (IAP) genes in mouse teratocarcinoma cell lines has been investigated as an approach to study the regulation of the expression of these particles. Treatment of the cells with the methylation inhibitor 5-azacytidine induces the production of the particles in all the cells; the induction is particularly striking in an embryonal carcinoma cell line which is normally devoid of IAPs. The induction is accompanied by decrease in DNA methylation as demonstrated by using the methylation sensitive isoschizomer enzymes MspI and HpaII. Hypomethylation of the IAP genes correlates with accumulation of IAP, specific polyadenylated RNA reinforcing the hypothesis that methylation plays an important role in the control of IAP expression.     

Regulation of intracisternal A particles in mouse teratocarcinoma cells: involvement of DNA methylation in transcriptional control

The methylation state of Intracisternal A Particle (IAP) genes in mouse teratocarcinoma cell lines has been investigated as an approach to study the regulation of the expression of these particles. Treatment of the cells with the methylation inhibitor 5-azacytidine induces the production of the particles in all the cells; the induction is particularly striking in an embryonal carcinoma cell line which is normally devoid of IAPs. The induction is accompanied by decrease in DNA methylation as demonstrated by using the methylation sensitive isoschizomer enzymes MspI and HpaII. Hypomethylation of the IAP genes correlates with accumulation of IAP, specific polyadenylated RNA reinforcing the hypothesis that methylation plays an important role in the control of IAP expression.     

Comparison of the sequence organization of related retrovirus-like multigene families in three evolutionarily distant rodent genomes.

Sequences related to mouse intracisternal A-particle (IAP) genes have been isolated from rat and Syrian hamster gene libraries as recombinants in lambda phage. The sequences are moderately reiterated in both these genomes but their sequence organization in the hamster genome is different from that in the rat genome. Restriction analysis and electron microscopy indicate that the Syrian hamster IAP sequences represent a family of relatively homogeneous well-conserved units; in this, they resemble the mouse IAP genes. The rat sequences, in contrast, are heterogeneous. Both the hamster and rat IAP sequences contain regions homologous to mouse IAP genes interspersed with regions of apparent non-homology. The interspersed regions range in size from 0.5-1.0 kilobases (Kb). The regions of homology among the mouse, rat and Syrian hamster IAP sequences have been mapped to a 5-6 Kb internal region on the mouse IAP genes. Mouse IAP long terminal repeat (LTR) sequences were not detected in the rat and Syrian hamster genomes. We used the thermal stability of hybrids between cloned and genomic IAP sequences to measure family homogeneity. Mouse and Syrian hamster IAP sequences are homogeneous by this criterion, but the rat IAP sequences are heterogeneous with a Tm 6 degrees C below the self-hybrid. The contrasting organization of IAP-related elements in the genomes of these rodents indicates that amplification or homogenization of this sequence family has occurred independently and at different periods of time during their evolution.     

Identification of an Xiap-Like Pseudogene on Mouse Chromosome 7

The most thoroughly characterized mammalian IAP is XIAP/BIRC4, which can inhibit caspases 9, 3 and 7, but may also regulate apoptosis through interactions with other proteins such as Smac/DIABLO, HtrA2/Omi, XAF1, TAK1, cIAP1, and cIAP2.High throughput sequencing of the mouse genome revealed the existence of a gene resembling Xiap/Birc4 on mouse chromosome 7. To confirm the existence of this gene, and to determine its functional significance, we performed Southern and Northern blot analysis. This showed the presence of the Xiap-like gene in both wild-type and Xiap gene knock-out mice, but the corresponding mRNA was not detected in any tissues examined by Northern blot. Analysis of the gene sequence in all three possible reading frames predicts that expression of this gene would not give rise to a full-length protein, but only non-functional truncated polypeptides. Because its nucleotide sequence is 92% identical to Xiap, but it has no introns corresponding to those of Xiap, we conclude that Xiap-ps1 is a pseudogene generated by retro-transposition of a spliced Xiap message to chromosome 7.     

A novel phosphatase upregulated in Akp3 knockout mice

Reexamination of the Akp3(-/-) mouse intestine showed that, despite the lack of intestinal alkaline phosphatase (IAP), the Akp3(-/-) gut still had considerable alkaline phosphatase (AP) activity in the duodenum and ileum. This activity is due to the expression of a novel murine Akp6 gene that encodes an IAP isozyme expressed in the gut in a global manner (gIAP) as opposed to duodenum-specific IAP (dIAP) isozyme encoded by the Akp3 gene. Phylogenetically, gIAP is similar to the rat IAP I isozyme. Kinetically, gIAP displays a 5.7-fold reduction in catalytic rate constant (k(cat)) and a 30% drop in K(m), leading to a 4-fold reduction k(cat)/K(m) compared with dIAP, and these changes in enzymatic properties can all be attributed to a crucial R317Q substitution. Western and Northern blot analyses document the expression of Akp6 in the gut, from the duodenum to the ileum, and it is upregulated in the jejunum and ileum of Akp3(-/-) mice. Developmentally, Akp3 expression is turned on during postnatal days 13-15 and exclusively in the duodenum, whereas Akp6 and Akp5 are expressed from birth throughout the gut with enhanced expression at weaning. Posttranslational modifications of gIAP have a pronounced effect on its catalytic properties. Given the low catalytic efficiency of gIAP, its upregulation during fat feeding, its sequence similarity with rat IAP I, and the fact that rat IAP I has been implicated in the upregulation of surfactant-like particles during fat intake, it appears likely that gIAP may have a role in mediating the accelerated fatty acid intake observed in Akp3(-/-) mice fed a high-fat diet.