Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes. J. Cell. Biochem. 67:439–450, 1997. © 1997 Wiley-Liss, Inc.     

Interspersed repetitive and tandemly repetitive sequences are differentially represented in extrachromosomal covalently closed circular DNA of human diploid fibroblasts.

Extrachromosomal covalently closed circular DNA (cccDNA) was isolated from human diploid fibroblasts by alkaline denaturation/renaturation and CsCl-ethidium bromide isopycnic centrifugation. Probing across these gradient fractions showed a higher proportion of cccDNA sequences homologous to the interspersed highly repetitive Alu I and Kpn I sequences than to the human tandemly-repetitive Eco RI (alphoid) DNA. Cloning of these cccDNAs was then carried out following digestion with restriction endonucleases Hind III, Bam HI or Pst I, and ligation into plasmid pBR322. Many isolated recombinant clones were unstable as seen by a high rate of loss over four cycles of antibiotic selection, and frequent plasmid modifications including deletions adjoining the site of insertion. Of 107 cloned sequences which appeared relatively stable, i.e., survived four cycles of antibiotic selection without incurring detectable deletions, 28% and 11% showed homology to Alu I and Kpn I families, respectively, while 4% contained sequences homologous to both. In contrast, less than one percent hybridized to probes for tandemly-repetitive sequences, Eco RI and Satellite III. The average insert size of cloned cccDNA derived from human fibroblasts, 2.52 Kbp, was larger than previously reported for similar clones derived from genetically less stable permanent lines, which may reflect differences in the process of cccDNA generation.     

Human ING1 proteins differentially regulate histone acetylation

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.     

ING1 and 5-Azacytidine Act Synergistically to Block Breast Cancer Cell Growth

Background

Inhibitor of Growth (ING) proteins are epigenetic “readers” that recognize trimethylated lysine 4 of histone H3 (H3K4Me3) and target histone acetyl transferase (HAT) and histone deacetylase (HDAC) complexes to chromatin.

Methods and Principal Findings

Here we asked whether dysregulating two epigenetic pathways with chemical inhibitors showed synergistic effects on breast cancer cell line killing. We also tested whether ING1 could synergize better with chemotherapeutics that target the same epigenetic mechanism such as the HDAC inhibitor LBH589 (Panobinostat) or a different epigenetic mechanism such as 5-azacytidine (5azaC), which inhibits DNA methyl transferases. Simultaneous treatment of breast cancer cell lines with LBH589 and 5azaC did not show significant synergy in killing cells. However, combination treatment of ING1 with either LBH589 or 5azaC did show synergy. The combination of ING1b with 5azaC, which targets two distinct epigenetic mechanisms, was more effective at lower doses and enhanced apoptosis as determined by Annexin V staining and cleavage of caspase 3 and poly-ADP-ribose polymerase (PARP). ING1b plus 5azaC also acted synergistically to increase γH2AX staining indicating significant levels of DNA damage were induced. Adenoviral delivery of ING1b with 5azaC also inhibited cancer cell growth in a murine xenograft model and led to tumor regression when viral concentration was optimized in vivo.

Conclusions

These data show that targeting distinct epigenetic pathways can be more effective in blocking cancer cell line growth than targeting the same pathway with multiple agents, and that using viral delivery of epigenetic regulators can be more effective in synergizing with a chemical agent than using two chemotherapeutic agents. This study also indicates that the ING1 epigenetic regulator may have additional activities in the cell when expressed at high levels.     

Identification of a novel function for the chromatin remodeling protein ING2 in muscle differentiation

The inhibitor of growth (ING) family of zinc-finger plant homeodomain (PHD)-containing chromatin remodeling protein controls gene expression and has been implicated in the regulation of cell proliferation and death. However, the role of ING proteins in cell differentiation remains largely unexplored. Here, we identify an essential function for ING2 in muscle differentiation. We find that knockdown of ING2 by RNA interference (RNAi) blocks the differentiation of C2C12 cells into myotubes, suggesting that ING2 regulates the myogenic differentiation program. We also characterize a mechanism by which ING2 drives muscle differentiation. In structure-function analyses, we find that the leucine zipper motif of ING2 contributes to ING2-dependent muscle differentiation. By contrast, the PHD domain, which recognizes the histone H3K4me3 epigenetic mark, inhibits the ability of ING2 to induce muscle differentiation. We also find that the Sin3A-HDAC1 chromatin remodeling complex, which interacts with ING2, plays a critical role in ING2-dependent muscle differentiation. These findings define a novel function for ING2 in muscle differentiation and bear significant implications for our understanding of the role of the ING protein family in cell differentiation and tumor suppression.