CSB349 Lecture 4 Notes

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

CSB349 Lecture 4 – Welcome to the genome III: Genome structure Slide 2 – Organization of DNA in the nucleus  The genetic material inside the nucleus has various distinct structures  The major two structures are the heterochromatin and euchromatin Slide 3 – Organization of DNA in the nucleus  The DNA is not just floating around in the nucleus  There is a large and complicated matrix protein that supports the genome o A network of proteins that is holding the genome together and in a loose conformation  The genome is not fixed; it is flexible but it doesn’t move around completely freely  The genes on the chromosome are not randomly interspersed in the nucleus  Each chromosome occupies a distinct region in the nucleus called chromosome territories  FISH experiment  Fluorescent probes for different chromosomes have been added Slide 4 – Chromosome territories  The chromosome territories are not fixed  It is a tendency for certain chromosomes to be in certain parts of the nucleus  The location of the chromosome in the nucleus important for the expression of all the genes (or certain important genes) on the chromosome o E.g., Many genes of the same related function are organized together and highly transcribed in the nucleolus o E.g., Chromosome territories are important for some co-regulation of different genes  There is a lot of debate and the evidence is not that strong Slide 5 – Chromosome territories are preserved through mitosis  This is evidence that the chromosome territories are actively preserved by the cell through mitosis  They labelled two of the histones (proteins that are always associated with the genome) o One histone is labelled with two different fluorescent colours (CFP, YFP) o Photo-bleaching the YFP marker causes the green fluorescence to disappear  They want to be able to tell apart half of the chromatin from the other half in the nucleus o They want to chromosome territories in a living cell  They allow the cell to go through mitosis o You can still see some of the residual labelling in the top half of the nucleus o The top of the genome before cell division is still at the top of nucleus after cell division o The chromosome territories are maintained in the daughter cells Slide 6 – Techniques to study genome organization and structure  Which parts of the genome are directly contacting other parts of the genome?  The structure of the genome contains loops that bring various parts of the genome together  The idea is to figure out which pieces of the genome are nearby each other  You add a chemical cross-linker to the cell (e.g., chemical glue) o It glues all the molecules that are nearby each other together  You extract the cross-linked chromosome from the cell o The unlinked DNA is digested o The cross-linked parts won’t be digested  The ligase will connect all the free ends of DNA to each other  The cross-link can be reversed o The result is a linear DNA molecule, but it doesn’t correspond to any part of the genome o The linear DNA contains information about what regions of the genome were close to each other in 3D space  Then we can sequence it using PCR primers Slide 7  The genes are close to each other in space (e.g., arrows) even though they are far from each other on the linear sequence  This is the 3C data that supports this structure o Along the diagonal, the genome is essentially linear o When you are off the diagonal, two genes that are far away in the linear sequence of the genome can be close together in 3D space Slide 8 – Organization of heterochromatin  Heterochromatin is something that we don’t understand very much about  Thought to be important for the structure of the genome o Contains satellite repeats o Telomeres and centromeres are heterochromatic  Thought to be highly condensed regions of the genome  Thought that there were few genes and very little transcription in this region, but not true o In Drosophila, about 450 genes are in the heterochromatic regions  Heterochromatin is not always necessarily heterochromatin o Some of the heterochromatin is permanently condensed  Called constitutive heterochromatin o Some of the heterochromatin can sometimes decondense and maybe turn into euchromatin  Called facultative heterochromatin Slide 9 – Organization of euchromatin  Euchromatin makes up most of the genome  There is a nuclear-DNA matrix o Large pieces of the genome are organized along the proteins  The loops of DNA are on the protein scaffolds (e.g., MARs or SARs)  The loops of DNA are made up of other loops of DNA (e.g., 30 nm fiber) Slide 10 – Functional chromatin domains  How can we observe the different structural regions of the genome?  DNase I sensitivity assay o Enzyme can digest DNA efficiently if it is in an
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