Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation

Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2–3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2–3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation

Monoallelic chromatin conformation flanking long-range silenced domains in cancer-derived and normal cells.

Epigenetic inactivation of chromatin plays an important role in determining cell phenotype in both normal and cancer cells, but our knowledge is still incomplete with respect to any potential monoallelic nature of the phenomenon. We have genotyped DNA isolated from chromatin of two colorectal cancer-derived lines and a culture of normal human intestinal epithelial cells (HIEC), which was immunoprecipitated with antibodies to acetylated vs. methylated histone H3K9, and presented the data as B allele frequency differences over multiple single-nucleotide polymorphism (SNP) moving window averages. [B allele is an arbitrary term defined as one of the two alleles at any given SNP, named A and B]. Three different validation tests confirmed that peaks exhibiting differences represented monoallelic domains. These complementary tests confirmed the following: 1) genes in the regions of high B allele frequency difference were expressed monoallelically; 2) in normal cells all five imprinting control regions which carried heterozygous SNPs were characterized by B allele difference peaks; and 3) the haplotypes in the B allele difference peaks were faithfully maintained in the chromatin immunoprecipitated with the respective antibodies. In both samples most of the monoallelic domains were found at the boundaries between regions of open and closed chromatin. With respect to the cancer line, this supports the established concept of conformation spreading, but the results from the normal cells were unexpected. Since these cells were polyclonal, the monoallelic structures were probably not determined by random choice as occurs in X-inactivation, so we propose that epigenetic inactivation in some domains may be heritable and polymorphic in normal human cells

Data from: Monoallelic chromatin conformation flanking long-range silenced domains in cancer-derived and normal cells

Epigenetic inactivation of chromatin plays an important role in determining cell phenotype in both normal and cancer cells, but our knowledge is still incomplete with respect to any potential monoallelic nature of the phenomenon. We have genotyped DNA isolated from chromatin of two colorectal cancer-derived lines and a culture of normal human intestinal epithelial cells (HIEC), which was immunoprecipitated with antibodies to acetylated vs. methylated histone H3K9, and presented the data as B allele frequency differences over multiple single-nucleotide polymorphism (SNP) moving window averages. [B allele is an arbitrary term defined as one of the two alleles at any given SNP, named A and B]. Three different validation tests confirmed that peaks exhibiting differences represented monoallelic domains. These complementary tests confirmed the following: 1) genes in the regions of high B allele frequency difference were expressed monoallelically; 2) in normal cells all five imprinting control regions which carried heterozygous SNPs were characterized by B allele difference peaks; and 3) the haplotypes in the B allele difference peaks were faithfully maintained in the chromatin immunoprecipitated with the respective antibodies. In both samples most of the monoallelic domains were found at the boundaries between regions of open and closed chromatin. With respect to the cancer line, this supports the established concept of conformation spreading, but the results from the normal cells were unexpected. Since these cells were polyclonal, the monoallelic structures were probably not determined by random choice as occurs in X-inactivation, so we propose that epigenetic inactivation in some domains may be heritable and polymorphic in normal human cells

chr12

chr1

Chr8

Chr

chr19

chr1

chr10

chr1

chr22

chr2