LECTURES
BIOINFORMATICS
GENOMICS
TRANSCRIPTOMICS
Transcriptomics is the study of the transcriptome - the all set of RNA transcripts which are produced under specific circumstances in one cell or population of cells - using high throughout methods such as microarray analysis.
EPIGENOMICS
Epigenomcis is the study of the complete set of epigenetic modifications on the genetic material of a cell, known as the epigenome.
{ Epigenetic modifications = genomic modifications that alter gene expression that cannot be attributed to modification
of the primary DNA sequence and that are heritalbe mitotically and meiotically are classified }
## Major two types of epigenemic modifications ##
1) DNA methylation
DNA methylation is the process of by which a methyl group is added to DNA by enzymes DNA methyltransferases (DNMTs) which are responsible for catalyzing this reaction. In eukaryotes, methylation is most commonly found on the carbon 5 position of cytosine residues (5mc) adjacent to guanine. DNA methylation patterns vary greatly between species and even with the same organisms.
2) Histone modification
In eukaryotes, genomic DNA is coiled into protein-DNA complexes called chromatin. Histones, which are the most prevalent type of protein found in chromatin, function to condense the DNA; the net positive charge on histones facilitates their bonding with DNA, which is negatively charged. The basic and repeating units of chromatin, nucleosomes, consist of an octamer of histone proteins. Many different types of histone modification are known, including acetylation, methylation, phosphorylation, ubiquitination etc. The DNA region where histone modification occurs can elicit different effects. Histone modifications regulate gene expression by two mechanisms : by disruption of the contact between nucleosomes and by recruiting chromatin remodeling ATPases.
## Epigenomic methods ##
1) Histone modification assay
The cellular processes of transcription, DNA replication and DNA repair involve the interaction between genomic DNA and nuclear proteins. It had been known that certain regions within chromatin were extremely susceptible to DNase I digestion, which cleaves DNA in a low sequence specificity manner. Such hypersensitive sites were thought to be transcriptionally active regions, as evidenced by their association with RNA polymerase and topoisomerase I and II. It is now known that sensitivity to DNAse I regions correspond to regions of chromatin with loose DNA-histone association. Hypersensitive sites most often represent promoters regions, which require for DNA to be accessible for DNA binding transcriptional machinery to function.
ChIP-Chip and ChIP-Seq
Histone modification was first detected on a genome wide level through the coupling of chromatin immunoprecipitation (ChIP) technology with DNA microarrays, termed ChIP-Chip. However instead of isolating a DNA-binding transcription factor or enhancer protein through chromatin immunoprecipitation, the proteins of interest are the modified histones themselves.
① Histones are cross-linked to DNA in vivo through light chemical treatment.
② The cells are next lysed, allowing for the chromatin to be extracted and fragmented, either by sonication or treatment with a non-specific restriction enzyme.
③ Modification-specific antibodies in turn, are used to immunoprecipitate the DNA-histone complexes.
④ Following immunoprecipitation, the DNA is purified from the histones, amplified via PCR and labeled with a fluorescent tag.
⑤ The final step involves hybridization of labeled DNA, both immunoprecipitated DNA and non-immunoprecipitated onto a microarray containing immobilized gDNA.
⑥ Analysis of the relative signal intensity allows the sites of histone modification to be determined.
2) DNA methylation arrays
Techniques for characterizing primary DNA sequences could not be directly applied to methylation assays. For example, when DNA was amplified in PCR or bacterial cloning techniques, the methylation pattern was not copied and thus the information lost. The DNA hybridization technique used in DNA assays, in which radioactive probes were used to map and identify DNA sequences, could not be used to distinguish between methylated and non-methylated DNA.
PROTEOMICS