PowerPoint PresentationSlide 2Eukaryotic Genomes are Entwined in ChromatinSlide 4Slide 5Slide 6Transcription can be Actively Regulated in 10- and 30-nm Fiber RegionsSlide 8DNA CompactionSlide 10Gene Activity Correlates with Chromatin StateThe Globin ModelActive Globin Gene is Sensitive to DNase I DigestionTranscriptionally Active DNA Regions are Sensitive to Digestion by DNase IIndirect End-Labeling AssayMicrococcal Nuclease Digestion Reveals SpacingNucleosome Spacing Differences Correlate with Gene ActivityHeterochromatin Nucleosome Spacing is More OrderlyChromatin State Varies Across a GenomeHypersensitive Sites can be Detected with either MNase or DNaseThe Two Enzymes Provide Different Information on Nucleosome StructureSlide 22NucleosomeSlide 24Do Nucleosomes Block RNAP II?RNAP Can Displace Histone OctamerHistone TailsSlide 28Histone Code (partial list)‘Code’ is Recognized by Certain ProteinsDifferent PTMs Nucleate Distinct Proteins and Create Different States of ChromatinSlide 32Acetylation vs MethylationK9 Methylation Correlates with Local SilencingK9 vs. K4 MethylationK4 vs K9 MethylationSlide 37Slide 38Slide 39SIR Complex Bind to Unacetylated Histone TailsHeterochromatin Often Initiates at Specific SitesSir2 is an NAD-Dependent Histone DeacetylasePosition Effect Variegation (PEV)Slide 44Slide 45Boundary Elements Restrict Heterochromatin SpreadSlide 47Slide 48Types of Silenced ChromatinMethylated Proteins Leads to Methylated DNADNA Methylation State Correlates with Higher Order PackingSlide 52Slide 53Methylation and CancerSlide 55Marker Genes for Specific CancersBisulphite PCR is Used to Detect Methyl-CUsing the Code Locally1Points for the lecture:Chromatin state correlates with gene activity Histone modifications correlate with chromatin stateDNA modifications correlate with chromatin stateModifications on histones and DNA nucleate specific complexes2Major Modes of Transcriptional Regulation3Eukaryotic Genomes are Entwined in Chromatin45Nucleosome is the Basic Unit of Chromatin67Transcription can be Actively Regulated in 10- and 30-nm Fiber Regions10 nm Fiber 30 nm Fiber89DNA Compaction10Chromatin and Transcription*assays to study chromatin states11Gene Activity Correlates with Chromatin State12The Globin ModelWeintraub and Groudine hypothesized in the ‘70s that changes in chromatin structure occur when globin genes are transcriptionally active. For example, in brain or fibroblast cells chromatin in condensed and globin genes are inactive where as chromatin is open and genes are active in chicken reticulocytes.13Active Globin Gene is Sensitive to DNase I DigestionGlobin Gene is Active in 14-day erythroblast cells and not in MSB cells14Transcriptionally Active DNA Regions are Sensitive to Digestion by DNase I15Indirect End-Labeling AssayAccessibility changes with chromatin state(use either DNase I or Micrococcal nuclease)Carried out after protein removal to delineate sizeSouthern blot to detect specific DNA by ‘indirect end label’16Micrococcal Nuclease Digestion Reveals Spacing17Nucleosome Spacing Differences Correlate with Gene Activity18Heterochromatin Nucleosome Spacing is More OrderlyH2-S (inactive) = heterochromatin39C-X (active) = euchromatinChromatin State Varies Across a Genome1920Hypersensitive Sites can be Detected with either MNase or DNase21The Two Enzymes Provide Different Information on Nucleosome StructureMNase“Spacing”DNase“Location”22How do Nucleosomes Contribute to Gene Regulation?23Nucleosome24Histones can Inhibit Transcription in vitroHelps deposit histones on DNA25Do Nucleosomes Block RNAP II?26RNAP Can Displace Histone Octamer27Histone Tails28Histone Tails Provide Many Sites for PTMs29Histone Code (partial list)30‘Code’ is Recognized by Certain Proteins31Different PTMs Nucleate Distinct Proteins and Create Different States of Chromatin32HyperAcetylation Localizes to Active Promoters33Acetylation vs Methylation34K9 Methylation Correlates with Local Silencing35K9 vs. K4 Methylation36K4 vs K9 Methylation37Types of Chromatin38Major Heterochromatin Sites39Functional Genomic Regions Have Distinct Chromatin Types40SIR Complex Bind to Unacetylated Histone Tails41Heterochromatin Often Initiates at Specific Sites42Sir2 is an NAD-Dependent Histone Deacetylase43Position Effect Variegation (PEV)44Confining Different Chromatin States45General Scheme46Boundary Elements Restrict Heterochromatin Spread47Boundary Elements Serve at least Two Functions48Chromatin and Silencing49Types of Silenced Chromatin50Methylated Proteins Leads to Methylated DNA51DNA Methylation State Correlates with Higher Order Packing52CpG Islands Occur Throughout the Genome53Not All Promoters Correlate with CpG Islands54Methylation and Cancer55Methyl-CpG Islands Can Correlate with Cancer56Marker Genes for Specific Cancers57Bisulphite PCR is Used to Detect Methyl-C58Using the Code
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