Changes

<p><span style="font-size:14px">Epigenomcis is the study of the complete set of <strong>epigenetic modifications</strong> on the genetic material of a cell, known as the epigenome.</span></p> <p><span style="font-size:14px">{ <strong>Epigenetic modifications</strong> = genomic modifications that alter gene expression that cannot be attributed to modification</span></p> <p><span style="font-size:14px">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;of the primary DNA sequence and that are heritalbe mitotically and meiotically are classified }</span></p>

<p>{ <strongspan style="font-size:14px"><u>Epigenetic ##&nbsp;Major two types of epigenemic modifications##</u></strongspan> = genomic modifications that alter gene expression that cannot be attributed to modification</p>

<p>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;of the primary <span style="font-size:14px">1) <strong>DNA sequence and that are heritalbe mitotically and meiotically are classified }methylation</strong></span></p>

<p><uspan style="font-size:14px">DNA methylation is the process of by which a methyl group is added to DNA by enzymes <strong>DNA methyltransferases (DNMTs)</strong>##which are&nbsp;Major two types responsible for catalyzing this reaction. In <u>eukaryotes</u>, methylation is most commonly found on the <u>carbon 5 position of epigenemic modifications ##cytosine residues (5mc)</u> adjacent to guanine. DNA methylation patterns vary greatly between species and even with the same organisms.</span></p>

<p>1<span style="font-size:14px">2) <strong>DNA methylationHistone modification</strong></span></p>

<p><span style="font-size:14px">In eukaryotes, genomic DNA methylation is coiled into protein-DNA complexes called chromatin. Histones, which are the process most prevalent type of by which a methyl group is added protein found in chromatin, function to condense the DNA by enzymes <strong>; the net positive charge on histones facilitates their bonding with DNA methyltransferases (DNMTs)</strong> , 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 areknown, including acetylation, methylation, phosphorylation, ubiquitination etc. The DNA region where histone modification occurs can elicit different effects.&nbsp;responsible for catalyzing this reactionHistone modifications regulate gene expression by two mechanisms : by disruption of the contact between nucleosomes and by recruiting chromatin remodeling ATPases. In <u>eukaryotes</u>, methylation is most commonly found on the <u>carbon 5 position of cytosine residues (5mc)</uspan> adjacent to guanine. DNA methylation patterns vary greatly between species and even with the same organisms.</p>

<p>2) <strongspan style="font-size:14px"><u>## Epigenomic methods ##</u>Histone modification</strongspan></p>

<p>In eukaryotes, genomic DNA is coiled into protein<span style="font-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 size:14px">1) <strong>Histone modification are known, including acetylation, methylation, phosphorylation, ubiquitination etc. The DNA region where histone modification occurs can elicit different effects.&nbsp;Histone modifications regulate gene expression by two mechanisms : by disruption of the contact between nucleosomes and by recruiting chromatin remodeling ATPases.assay</strong></span></p>

<p><uspan style="font-size:14px">## Epigenomic methods ##The cellular processes of transcription,&nbsp;DNA replication&nbsp;and DNA repair&nbsp;involve the interaction between genomic DNA and nuclear proteins. It had been known that certain regions within chromatin were extremely susceptible to DNase I&nbsp;digestion, which cleaves DNA in a low sequence specificity manner. Such hypersensitive sites&nbsp;were thought to be transcriptionally active regions, as evidenced by their association with RNA polymerase&nbsp;and topoisomerase I and&nbsp;II.&nbsp;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.</uspan></p>

<ph4><span style="font-size:14px">1) <strong>Histone modification assayChIP-Chip and ChIP-Seq</strong></pspan></h4>

<p>The cellular processes <span style="font-size:14px">Histone modification was first detected on a genome wide level through the coupling of transcription,&nbsp;DNA replication&nbsp;and DNA repair&nbsp;involve the interaction between genomic DNA and nuclear proteins. It had been known that certain regions within chromatin were extremely susceptible to DNase Iimmunoprecipitation (ChIP)&nbsp;digestion, which cleaves technology with DNA in a low sequence specificity manner. Such hypersensitive sites&nbsp;were thought to be transcriptionally active regionsmicroarrays, as evidenced by their association with RNA polymerase&nbsp;and topoisomerase I and&nbsp;IItermed ChIP-Chip.&nbsp;It is now known that sensitivity to DNAse I regions correspond to regions However instead of chromatin with loose isolating a DNA-histone association. Hypersensitive sites most often represent promoters regionsbinding transcription factor or enhancer protein through chromatin immunoprecipitation, which require for DNA to be accessible for DNA binding transcriptional machinery to functionthe proteins of interest are the modified histones themselves.&nbsp;</span></p>

<h4p><strongspan style="font-size:14px">ChIP-Chip and ChIP①&nbsp;Histones are cross-Seqlinked&nbsp;to DNA in vivo through light chemical treatment.</strongspan></h4p>

<p>Histone modification was first detected on a genome wide level through the coupling of chromatin immunoprecipitation (ChIP)<span style="font-size:14px">②&nbsp;technology with DNA microarraysThe cells are next lysed, allowing for the chromatin to be extracted and fragmented,either by sonication&nbsp;termed ChIP-Chip.&nbsp;However instead of isolating or treatment with a DNAnon-binding transcription factor or enhancer protein through chromatin immunoprecipitation, the proteins of interest are the modified histones themselvesspecific restriction enzyme.&nbsp;</span></p>

<p>①<span style="font-size:14px">③&nbsp;Histones Modification-specific antibodies in turn, are crossused to immunoprecipitate the DNA-linkedhistone complexes.&nbsp;to DNA in vivo through light chemical treatment.</span></p>

<p>②<span style="font-size:14px">④&nbsp;The cells are next lysedFollowing immunoprecipitation, allowing for the chromatin to be extracted DNA is purified from the histones, amplified via PCR and fragmented, either by sonication&nbsp;or treatment labeled with a non-specific restriction enzymefluorescent tag.</span></p>

<p>③<span style="font-size:14px">⑤&nbsp;Modification-specific antibodies in turnThe final step involves hybridization of labeled DNA, are used to immunoprecipitate the both immunoprecipitated DNAand non-histone complexesimmunoprecipitated onto a microarray containing immobilized gDNA.&nbsp;</span></p>

<p>④<span style="font-size:14px">⑥&nbsp;Following immunoprecipitation, Analysis of the DNA is purified from relative signal intensity allows the histones, amplified via PCR and labeled with a fluorescent tagsites of histone modification to be determined.&nbsp;</span></p>

<p>⑤&nbsp;The final step involves hybridization of labeled <span style="font-size:14px">2) <strong>DNA, both immunoprecipitated DNA and non-immunoprecipitated onto a microarray containing immobilized gDNA.methylation arrays</strong></span></p>

<p>⑥<span style="font-size:14px">Techniques for characterizing primary DNA sequences could not be directly applied to methylation assays. For example, when DNA was amplified in PCR&nbsp;Analysis of or bacterial cloning techniques, the relative signal intensity allows methylation pattern was not copied and thus the sites of histone modification to be determinedinformation lost.The DNA hybridization technique&nbsp;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.</span></p>

<p>2) <span style="font-size:14px"><strong>DNA methylation arraysNon genome-wide approaches</strong></span></p>

<p>Techniques for characterizing primary <span style="font-size:14px">The earliest methylation detection assays used methylation modification sensitive restriction endonucleases.&nbsp;Genomic DNA sequences was digested with both methylation-sensitive and insensitive restriction enzymes recognizing the same restriction site. The idea being that whenever the site was methylated, only the methylation insensitive enzyme could not be directly applied cleave at that position. By comparing restriction fragment&nbsp;sizes generated from the methylation-sensitive enzyme to those of the methylation-insensitive enzyme, it was possible to determine the methylation assayspattern of the region. For example, when DNA This analysis step was amplified in done by amplifying the restriction fragments via PCR, separating them through gel electrophoresis&nbsp;and analyzing them via southern blot&nbsp;with probes for the region of interest.&nbsp;or bacterial cloning techniquesDifferent regions of the gene were known to be expressed at different stages of development. Consistent with a role of DNA methylation in gene repression, regions that were associated with high levels of DNA methylation were not actively expressed.</span></p> <p><span style="font-size:14px">This method was limited not suitable for studies on the global methylation pattern , or methylome. Even within specific loci it was not copied fully representative of the true methylation pattern as only those restriction sites with corresponding methylation sensitive and thus insensitive restriction assays could provide useful information. Further complications could arise when incomplete digestion of DNA by restriction enzymes generated false negative results.</span></p> <h5><span style="font-size:14px"><strong>Gemone widei approaches</strong></span></h5> <p><span style="font-size:14px">DNA methylation profiling on a large scale was first made possible through the information lostRestriction Landmark Genome Scanning (RLGS)&nbsp;technique. The Like the locus-specific DNA hybridization methylation assay, the techniqueidentified methylated DNA via its digestion methylation sensitive enzymes. However it was the use of two-dimensional gel electrophoresis&nbsp;that allowed be characterized on a broader scale.&nbsp;used However it was not until the advent of microarray and next generation sequencing technology when truly high resolution and genome-wide DNA methylation became possible. As with RLGS, the endonuclease component is retained in DNA assaysthe method but it is coupled to new technologies. One such approach is the differential methylation hybridization (DMH), in which radioactive probes were one set of genomic DNA is digested with methylation-sensitive restriction enzymes and a parallel set of DNA is not digested. Both sets of DNA are subsequently amplified and each labelled with fluorescent dyes and used in two-colour array hybridization. The level of DNA methylation at a given loci is determined by the relative intensity ratios of the two dyes. Adaptation of next generation sequencing to DNA methylation assay provides several advantages over array hybridization. Sequence-based technology provides higher resolution to map and identify allele specific DNA sequencesmethylation, could not can be used to distinguish between methylated performed on larger genomes, and non-methylated does not require creation of DNAmicroarrays which require adjustments based on CpG density to properly function.</span></p>