Figure 2. Genome-wide methods for studying DNA methylation and histone modifications.
In the area of genomic expression, we collaborate with a number of investigators to profile mRNA expression in a variety of lung diseases and immune cell models, using either sequencing or microarray technologies. Within CGEH, one project uses targeted sequencing of mRNA to examine allele-specific expression of a gene found by CGEH investigators to a promoter mutation with a strong genetic risk factor for development of pulmonary fibrosis. Another project studies the transcriptome of clinically relevant pathogens, together with the transcriptional response of their hosts. Other projects couple transcriptional changes with epigenetic modifications to help elucidate regulatory mechanisms.
(A) To study DNA methylation, genomic DNA is treated with methylation sensitive restriction enzymes (a), bisulfate treated to convert methylated cytosines into uracils (b) or methylated DNA is immunoprecipitaded with the anti-methylcytosine antibody (c). Any of these methods can be combined with either hybridization to DNA microarrays or direct sequencing to study DNA methylation on a genomic scale2.
(B) Comprehensive high-throughput arrays for relative methylation (CHARM) have been developed by Andy Feinberg and Rafael Irizarry at Johns Hopkins University to capture CpG loci both in CpG islands that have long been associated with development of cancer and additional more distant CpG motifs (CpG island shores) that have recently been associated with tissue-specific gene expression and cancer development. The top histogram shows distribution of 2.1 million features on the microarray based on their genomic location while the bottom histogram depicts distribution of tissue-specific differentially methylated regions (T-DMRs)1.
(C) CHIP-seq is the method that is currently being used to study histone modifications. Modified chromatin is purified by immunoprecipitation using an antibody that is specific to a particular histone modification (shown in green). DNA library for sequencing is prepared and sequenced. The resulting sequence reads are mapped to a reference genome to obtain genomic coordinates that correspond to the immunoprecipitated fragments2.