Epigenetics is the study of heritable changes in gene function that do not involve changes in DNA sequence. These changes can occur due to the chemical modification of specific genes or gene-associated proteins of an organism. These modifications can affect how genes are expressed and used in cells. For example, methylation of specific regions in gene sequences, such as CpG sites, can make these genes less transcriptionally active. Another example is post-translational modification of histone proteins around which genomic DNA is wound. This histone modification can affect the unwinding of the DNA during transcription which, in turn, can affect the expression of the transcribed genes. Furthermore, chromosome conformation or chromatin compaction can also have an effect on gene expression, such as affecting the accessibility of the template to polymerases or changing the proximity between genes and genomic sequences.
Important chromosomal activities and gene expression have been linked with the structural properties of the chromosome, such as their spatial conformation. Furthermore, the local properties of chromatin fibers have also been shown to influence gene expression. Higher order structures of chromatin, such as 30 nm fibers, chromatin loops and axes, and interchromosomal connections have also been shown to play a role in gene expression and recombination.
Epigenetic mechanisms are essential for normal development and maintenance of the normal gene expression pattern in many organisms including humans. Recent studies suggest that epigenetic alterations may be the key initiating events in some forms of cancers and global changes in the epigenome are a hallmark of cancers. Epigenetic mechanisms that modify chromatin structure can be divided into four main categories: 1) DNA methylation, 2) covalent histone modifications and noncovalent mechanisms such as incorporation of histone variants, 3) nucleosome remodeling and 4) non-coding RNAs which include microRNAs (miRNAs). The role of DNA methylation and histone modifications in cancer initiation and progression is well established; however, the changes in chromatin structure that accompany DNA methylation and histone modifications are less well understood
The analysis of chromosomal conformation has been complicated by technical limitations. For example, analysis by electron microscopy is laborious and cannot be used to view specific loci; fluorescently-labeled DNA binding proteins permit the visualization of specific loci, but only a few loci can be examined simultaneously. Fluorescence in situ hybridization (FISH) analysis can examine multiple loci, but the severe experimental conditions may adversely affect chromosomal organization.
In an attempt to overcome the limitations of visual chromosomal analysis, methods using the polymerase chain reaction (PCR) have been developed. For example, the chromosome conformation capture (3C) method analyzes overall chromosomal spatial organization and physical properties at a higher resolution (see, Dekker et al., Science 295:1306-1311 (2002)). In 3C experiments, genomic DNA (gDNA) and proteins in the chromosomes are fixed in place by cross-linking. The cross-linked gDNA is digested by a restriction enzyme and ligated before being purified for analysis. Physical interactions between genomic loci are identified as specific cross-ligated DNA elements using PCR amplification. As a result of 3C methods, spatial information is converted to quantifiable DNA sequences. However, the wide adaptation of 3C methods has been hindered by the lack of quantitative process controls and cumbersome protocols.
Derivative methods of 3C, such as 4C and 5C, have also been developed. 4C and 5C differ from 3C only in the analysis of the ligation product. In 4C, the ligation product is first amplified by PCR using two outwardly facing primers from the restriction site to create a circular DNA molecule which is then analyzed by microarray technology. In 5C, the ligation products are mixed with special primers designed to anneal at the ends of the restriction fragment. Analysis is carried out either by use of a microarray or by sequencing against a 3C library of ligation products.
However, there are several disadvantages associated with these 3C-based methods. The assays are time consuming and are not precise and sensitive enough for reactions with low digestion or ligation efficiency. These methods also require significant dilution of the DNA sample. As a result, large quantities of the sample may be needed, which may not always be available. In addition, the 3C-based methods listed above suffer from low throughput and are unable to solve the entire spatial arrangement of the chromatin in the nucleus and therefore must focus on capturing interaction partners of a limited number of loci. The present teachings overcome these and other disadvantages and limitations and are useful for capturing interaction partners in the different regions of chromatin in an unbiased fashion.