CSB349 Lecture 5 Notes

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University of Toronto St. George
Cell and Systems Biology
Alan Moses

CSB349 Lecture 5 – Transcription I: Genome structure and transcription Slide 4 – Model of nucleosome remodelling in a nut shell - When the genome is in a condensed and closed state, there won’t be any transcription - Transcriptional activators bring the closed state to an open state - How does the gene get turned on? Slide 5 – Chromatin remodeling enzymes - Class I o Writing and erasing  making marks on the chromatin - Class II o Reading and doing  reading marks made by Class I and do stuff Slide 6 – Chemical modification of histones within nucleosomes - Histone tails o Flexible parts of the chromatin that do not adopt a crystal structure o Where the class I enzymes will make their marks (e.g., covalent modifications) - There are many types of covalent modifications Slide 7 – Two classes of chromatin remodeling enzymes - There are enzymes that make the histone marks - There are also enzymes that take off the histone marks - Transcription factors, ATP-dependent nucleosomal remodeling enzymes (Class II) and other histone modifying enzymes are recruited once the histone marks are made Slide 8 – Identifying modifications by chromatin immunoprecipitation (ChIP) - This technique allows us to probe the state of the chromatin in the genome - Use a specific antibody that can specifically recognize the acetylated histone tail that we are looking for - Perform an IP to pull down bits of the genome that have those modified histones - Steps o Cross-link the chromatin (e.g., chemical glue that glues all the DNA and protein together) o Isolate the chromatin from cells o Shear the chromatin into pieces (e.g., mechanically) o Add an antibody that is specific against the thing that you are looking for o Perform an IP to pull down that antibody and anything that is stuck to the antibody  In this case, you pull down the pieces of chromatin that have acetylated histones o Reverse the cross-link, so that you end up with DNA o Sequence the DNA by PCR or any other sequencing technique - The DNA that is pulled down tells you where in the genome the acetylated histone was found Slide 9 – Regulation of histone acetylation in yeast - This is illustrating how the Class I enzymes might be recruited - The Class I and Class II enzymes are not one protein, but multi-subunit protein complexes o Typically named after one or two of the subunits - Activator-directed histone hyperacetylation o There is a DNA sequence called UAS that interacts with a protein called DBD o The DBD protein recognizes a specific sequence in the genome and recruits Gcn5, which is a HAT o The HAT modifies all the nucleosomes - Repressor-directed histone deacetylation o Ume6 is bound to a specific sequence in the DNA o Ume6 is recruiting a multi-subunit HDAC to take off the histone marks - Class I enzymes usually don’t recognize DNA themselves, but rely on other proteins to tell them where to go in the genome Slide 10 – Recruitment of remodeling complexes - These proteins use ATP to do some kind of mechanical function to the histones in the chromatin Slide 11 – ATP-dependent chromatin remodeling complexes - Different ATPase subunits are used in different situations - SWI/SNF contains bromo-domain that recognizes acetylated histone tails - CHD contains chromo-domain that recognizes methylated histone tails Slide 12 – Nucleosome remodeling - These mechanical motions of nucleosomes require ATP o Remodeling by loosening up the chromatin o Slide the histones down by pushing them out of the way o Transfer the nucleosome somewhere else Slide 13 – Nucleosomes and transcription in yeast - DNase I hypersensitivity assay o To find which parts the genome are relatively open and which parts of the genome are relatively closed o The DNase I can only digest the open regions of the chromatin o The regions of the chromatin that are closed are not accessible to DNase I - The genes that were highly expressed tended to have more open chromatin - The genes that had low expression tended to have a more protected and closed chromatin - Nucleosome-free regions (e.g., DNase I hypersensitive regions) are associated with transcriptional competence - Drosophila hsp70 o Before heat shock, the chromatin is in a closed conformation o After heat shock, a new DHS region appears  This is the result of remodeling by a HAT complex Slide 14 – Nucleosomes and transcription in yeast - Not only are the nucleosomes more open in front of the actively transcribed genes, but there is a histone variant that is part of histones near the site of active transcription Slide 16 – Model of nucleosome remodeling in yeast - This is the default state (e.g.,
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