Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

Abstract

This study examines the chromatin structure encompassing replication origins in transformed and normal cells. Analysis of the global levels of histone H3 acetylated at K9&14 (open chromatin) and histone H3 trimethylated at K9 (closed chromatin) revealed a higher ratio of open to closed chromatin in the transformed cells. Also, the trithorax and polycomb group proteins, Brg-1 and Bmi-1, respectively, were overexpressed and more abundantly bound to chromatin in the transformed cells. Quantitative comparative analyses of episomal and in situ chromosomal replication origin activity as well as chromatin immunoprecipitation (ChIP) assays, using specific antibodies targeting members of the pre-replication complex (pre-RC) as well as open/closed chromatin markers encompassing both episomal and chromosomal origins, revealed that episomal origins had similar levels of in vivo activity, nascent DNA abundance, pre-RC protein association, and elevated open chromatin structure at the origin in both cell types. In contrast, the chromosomal origins corresponding to 20mer1, 20mer2, and c-myc displayed a 2- to 3-fold higher activity and pre-RC protein abundance as well as higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited similar levels of activity, pre-RC protein abundance, and higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in both cell types. Nucleosomal positioning analysis, using an MNase-Southern blot assay, showed that all the origin regions examined were situated within regions of inconsistently positioned nucleosomes, with the nucleosomes being spaced farther apart from each other prior to the onset of S phase in both cell types. Overall, the results indicate that cellular transformation is associated with differential epigenetic regulation, whereby chromatin structure is more open, rendering replication origins more accessible to initiator proteins, thus allowing increased origin activity.

Ku Protein Levels, Localization and Association to Replication Origins in Different Stages of Breast Tumor Progression

“…In contrast, the activity of the lamin B2 origin was similar in all cell lines (Fig. 3B), in agreement with previous findings 31, 33, 37.…”

“…Also in agreement with previous results, where a high replication origin activity was seen in tumor/transformed cell compared to normal ones 31, 33, 37, 40, the breast metastatic cancer (MCF10AC1a) cells had the highest replication origin activity at the c-myc locus, by comparison to the normal (MCF10A) and hyperplastic (MCF10AT) breast cells (Fig. 3A).…”

“…Prior to assessing the activity of two replication origins used in this study, namely the origins associated with the c-myc 35 and lamin B2 36 loci, we analyzed the copy number of these replication origins and their respective control (non-origin-containing) regions, as previously described 37. Briefly, equal amounts of genomic DNA from each cell line were amplified by quantitative real-time PCR.…”

Profiling of the Chromatin-associated Proteome Identifies HP1BP3 as a Novel Regulator of Cell Cycle Progression

“…Euchromatin is essential for gene transcription and normal cell function between replicative cycles (14 -17), whereas heterochromatin is vital to prevent aberrant DNA access that could disrupt the cell cycle and impede genome transfer into progeny cells (18 -22). Accordingly, imbalance in the chromatin structure-dependent regulatory system de-regulates the cell cycle and promotes carcinogenesis (23,24).…”

Profiling dynamic chromatome reveals HP1BP3 as a key regulator of cell cycle and hypoxia cancer progression

“…Imbalance in the chromatin structure dependent regulatory system causes deregulated cell cycle, responsible for evolution of cancer like diseases [197,198].…”