Reduced DNase I sensitivity of the rearranged c-myc gene in somatic cell hybrids between murine plasmacytoma cells and fibroblasts

In mouse plasmacytoma (MPC) S194, the rearranged c-myc gene was much more sensitive to DNase I digestion than the nonrearranged gene. The sensitivity of the rearranged c-myc was markedly reduced to the same extent as that of the nonrearranged one in hybrids between the MPC cells and the fibroblasts, but not in a hybrid between the MPC and the spleen cells. These results suggest that trans-acting factors in fibroblasts alter the DNase I-sensitive structure of the rearranged c-myc gene.     

Rearranged and germline immunoglobulin kappa genes: different states of DNase I sensitivity of constant kappa genes in immunocompetent and nonimmune cells

The rearrangement of a variable (V) and a constant (C) gene appears to be a necessary prerequisite for immunoglobulin gene expression. Multiple different rearranged kappa genes were found in several mouse myelomas, although these cells produce only one type of kappa chain [Wilson, R., Miller, J., & Storb, U. (1979) Biochemistry 18, 5013--5021]. It is therefore of interest to understand how only one allele within a lymphoid cell becomes expressed, while the other allele remains nonfunctional ("allelic exclusion"). We have studied the chromatin conformation of kappa genes by making use of the preferential digestion of potentially active genes by DNase I described, for example, for globin genes [Weintraub, H., & Groudine, M. (1976) Science (Washington, D.C.) 193, 848--856]. The DNase I sensitivity of kappa genes in myeloma tumors, in a B cell lymphoma, and in liver was determined by hybridization with DNA on Southern blots. It was found that rearranged C kappa genes are DNase I sensitive in myelomas in which several kappa genes are rearranged, regardless of whether the rearranged genes code for the kappa chains synthesized by the cell. Furthermore, the C kappa gene in germline configuration is also DNase I sensitive in a B cell lymphoma; i.e., it is in the same chromatin state as the rearranged C kappa gene which probably codes for the kappa chains produced by the cell. The altered chromatin state appears to be localized: V kappa genes in germline context are not DNase I sensitive in myeloma or B lymphoma cells while C kappa genes present in a kappa gene cluster on the same chromosomes are sensitive. When rearranged, however, the V kappa genes are as sensitive to DNase I as are rearranged C kappa genes. V lambda and C lambda genes are not DNase I sensitive in kappa myelomas. Thus, commitment to kappa gene expression is apparently correlated with a chromatin conformation which confers increased DNase I sensitivity to the DNA in the vicinity of all C kappa genes in the cell. "Allelic exclusion" does not operate on the level of chromatin conformation which can be detected by altered DNase I sensitivity.     

Fibroblast-like transformation and c-myc gene alteration of human hepatocytes induced by novobiocin and butyrate

Topoisomerase II inhibitor novobiocin and deacetylase inhibitor sodium butyrate synergistically transformed Chang liver cells into fibroblast-like cells. In these fibroblast-like cells, the production of type III procollagen was increased and the DNase I hypersensitivity of c-myc gene was reduced. In addition, these changes were associated with an increased acetylation of nuclear proteins, especially those of DNase I sensitive nucleosomes. Therefore, it is suggested that chemical modulation of nuclear proteins by novobiocin and butyrate may be responsible, at least partly, for the alteration of the chromatin structure of c-myc gene and the fibroblast-like transformation of Chang liver cells.     

Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction

The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.     

DNase II and the Chk2 DNA damage pathway form a genetic barrier blocking replication of horizontally transferred DNA

We have previously shown that DNA from dying tumor cells may be transferred to living cells via the uptake of apoptotic bodies and may contribute to tumor progression. DNA encoding H-ras(V12) and c-myc oncogenes may be transferred to the nucleus of the phagocyte but will only integrate and propagate in p53- and p21-deficient mouse embryonic fibroblasts, whereas normal cells are resistant to transformation. Here, we show that this protective mechanism (activation of p53 and p21 after uptake of apoptotic bodies) is dependent on DNA fragmentation, where inhibition of the caspase-activated DNase in the apoptotic cells, in conjunction with genetic ablation of lysosomal DNase II in the phagocytes, completely blocks p53 activation and consequently allows DNA replication of transferred DNA. We, therefore, suggest that there is a causal relationship between DNA degradation during apoptosis and p53 activation. In addition, we could further show that Chk2-/- cells were capable of replicating the hyg(R) gene taken up from engulfed apoptotic cells, suggesting involvement of the DNA damage response. These data show that the phagocytosing cell is sensing the degraded DNA within the apoptotic cell, hence preventing these genes from being replicated, probably through activation of the DNA damage response. We, therefore, hypothesize that DNase II together with the Chk2, p53, and p21 pathway form a genetic barrier blocking the replication of potentially harmful DNA introduced via apoptotic bodies, thereby preventing transformation and malignant development.     

Transformation of quail embryo fibroblasts by a retrovirus carrying a normal human c-myc gene.

We have constructed avian retroviruses expressing the human c-myc oncogene. These viruses morphologically transformed primary quail embryo fibroblasts upon transfection and infection. Transformed cells produced viruses harboring a spliced c-myc gene and contained high levels of p64-67c-myc protein. One of these infectious viruses, vSX-AHM, was molecularly cloned and the nucleotide sequence of the spliced c-myc insert determined. No mutation was found within the c-myc coding sequence of this transforming clone when compared to the normal genomic progenitor. Thus, we concluded that no mutation within the human c-myc gene is required to induce primary avian embryo fibroblast transformation.