The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.
Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.
Dekker et al. developed the chromosome conformation capture (3C) approach to detect the frequency of interaction between two genomic loci1, and 3C has been used extensively to investigate intra-chromosomal and inter-chromosomal associations between two known DNA regions in mammalian cells2-9. Although newly developed Hi-C methodology can be applied for study of genome-wide DNA association, ACT assay is still an effective technique for study of locus-specific DNA interaction10-11. We have modified this approach to identify unknown DNA regions that are associated with a known DNA region in cultured mouse and human cells (Figure 1). We named this method the associated chromosome trap (ACT) assay, as it provided us a reliable and reproducible method to identify novel unknown DNA partners that associate with a known target DNA region12. A successful 3C assay with appropriate controls is performed before executing the novel aspects of the ACT assay13. In order tofind as many associated DNA regions as possible, it is necessary to use various combinations of first and second restriction enzymes. It is especially important to use restriction enzymes that are insensitive to CpG methylation to perform the first 3C ligation step. Protein binding and DNA methylation can also influence restriction enzyme digestion efficiency and can lead to failure of ligation of the associated DNA regions for certain restriction enzyme digestions. The occurrence of intra- or inter-chromosomal ligation depends on protein-DNA cross-linking and appropriate physical maps of both of the associated DNA regions. Thus, some preliminary experiments are essential to establish a practicable effective formaldehyde concentration and treatment time in the ACT assay. A range of final concentrations of formaldehyde (from1.5% to 2%) have been used in several labs during the 3C portion of the assay7,9. Alternatively, the oligonucleotides used as linkers can be designed to match the cohesive end cut by the second restriction enzyme. Although we found that 18-20 cycles in the first round of PCR and 20-25 cycles in the second round of PCR could provide clear bands, it is necessary to establish the best PCR conditions for each experiment (Figure 2).Intra-molecule annealing between 5′-end and 3′-end complementary adaptor sequence of a DNA strand may occur in PCR, it inhibits adaptor-specific primer annealing with the DNA molecule, and resulted in much lower amplification efficiency in the first several cycles. After the target DNA was amplified for cycles, its amount may be much larger than these nonspecific reactions, and may facilitate the competition of primer annealing to the target DNA molecules. This is also why we may see background amplification, and why the first round PCR product has to be diluted to decrease background amplification.It is important to remove excess primers from the PCR reaction in order to decrease the background in the second round of PCR. As in all PCR-based experiments, it is vital to design primers that are not located in repeat sequence regions, which constitute the majority of human and mouse DNA. Although newly developed Hi-C methodology can be applied for study of genome-wide DNA association, ACT assay is still an effective technique for study of locus-specific DNA interaction.
The authors have nothing to disclose.
We thanks Adelle Murell and Wolf Reik very much for sharing their 3C protocol. This work was supported by the Department of Defense and the Research Service of the Department of Veterans Affairs.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
RPMI1640 medium | Invitrogen | 22400-105 | ||
acrylamide | Invitrogen | 15512-023 | ||
ATP solution, 10mM | Invitrogen | AM8110G | ||
fetal bovine serum | Invitrogen | 16000-044 | ||
penicillin-streptomycin | Invitrogen | 15140-122 | ||
1M Tris pH8.0 | Invitrogen | AM9856 | ||
RNase A | Invitrogen | 12091-039 | ||
SDS | Invitrogen | 15525-017 | ||
urea | Invitrogen | 15505-035 | ||
BamH I | NEB Biolabs | R0136T | ||
Bgl II | NEB Biolabs | R0144M | ||
Dpn II | NEB Biolabs | R0543T | ||
Msp I | NEB Biolabs | R0106S | ||
dNTPs | NEB Biolabs | N0447L | ||
proteinase K | NEB Biolabs | P8102S | ||
T4 DNA ligase | NEB Biolabs | M0202T | ||
37% formaldehyde | Sigma-Aldrich | F8775 | ||
Bis-acrylamide | Sigma-Aldrich | 146072 | ||
dithiothreitol | Sigma-Aldrich | 43815 | ||
glycine | Sigma-Aldrich | 50046 | ||
PMSF | Sigma-Aldrich | 93482 | ||
proteinase inhibitor | Sigma-Aldrich | S8830 | ||
Nonidet P-40 | Roche Applied Science | 11754599001 | ||
KlenTaq1 | Ab peptides | 1001 | ||
dCTP alpha P32 | PerkinElmer | BLU513H250UC | ||
PTC-100 Thermal Cycler | MJ Research | mjptc100 | ||
Power Supply | Bio-Rad | 164-5056 | ||
OmniPAGE Maxi | Aurogene Life Science | VS20D | ||
Typhoon 9400 | GE Healthcare | 63-0055-78 |