Epigenetics in. Saccharomyces cerevisiae. Chapter 4 2/4/14

Transcription

1 Epigenetics in Saccharomyces cerevisiae Chapter 4 2/4/14

2 The budding yeast - Saccharomyces cerevisiae The fission yeast - Schizosaccharomyces pombe The budding yeast, Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are models used to study the formation of heterochromatin. While these organisms have many similarities they also have surprisingly great differences in how they generate heterochromatin. S. pombe is much more similar to mammals. Our focus for the first poster session is Saccharomyces cerevisiae.

3 sir2 is conserved from bacteria to man.

4

5 Saccharomyces cerevisae a budding yeast Eukaryote Very powerful genetics homologous recombination as a tool Mutagens can be used to induce single base mutations or chromosome rearrangements Most yeast genes are in the active chromatin state.

6 Figure 2 and Figure 3 on pages 66 and 67 Figure 2 page 66 Allis

7 heterochromatin euchromatin - active genes, decondensed during interphase heterochromatin - condensed during interphase, packed so tightly that it is nuclease resistant, replicates late in S phase, very repressive with regards to transcription, repression is generalized not promoter specific usually found at centromeres and adjacent to telomeres In S. cerevisiae ONLY at telomeres and silent MAT cassettes. Genetics show that mutations that heterochromatin affect both telomeres and MAT cassettes.

8 Cell cycle repeating pattern of cell growth and division Alternates between interphase and mitosis Interphase period of cell cycle between divisions/cells grow and replicate chromosomes G1 gap phase birth of cell to onset of chromosome replication/cell growth S synthesis phase duplication of DNA G2 gap phase end of chromosome replication to onset of mitosis G0 - post mitotic G2 In Interphase, nucleus is intact Chromosomes are indistinguishable interphase prophase chromosomes condense, replicated centrosomes move apart mitosis is a cycle prometaphase nuclear envelope breaks down microtubules extend metaphase chromosomes aligned on metaphase plate sister chromatids move to opposite poles anaphase S G1 telophase identical nuclei are enclosed in nuclear envelope Fig. 4.7 interphase cytokinesis 4-20

9 Meiosis I Meiosis II Replicate chromosomes once, divide twice..

10 Figure 2 and Figure 3 on pages 66 and 67

11 Silent MAT cassettes

12 There is heterochromatin around HMLalpha and HMRa HML = hidden MAT left HMR = hidden MAT right True wildtype yeast are homothallic. This means that they switch mating type. heterothallic yeast have a mutation in HO endonuclease so that they don t switch. Mutations that interfere with epigenetic MAINTENANCE give rise to expressed silent cassettes and sterility.

13 Telomere Heterochromatin adjacent to telomeres

14 Telomere 3' 5' 3' 5' 5' 3' A B C D A' B' C' D' 3' 5' A B C D A' B' C' D' 3' 5' A B C D A' B' C' D' 3' 5'

15 Telomere

16 solution Telomere

17 Position effect variegation

18 heterochromatin If a chromosome rearrangement moves euchromatin next to heterochromatin you often see the spreading of the heterochromatin. How is this visualized? In general, if it is stochastic it is called Position effect variegation Heritable

19 Tools used to characterize chromatin spreading (PEV) Homologous recombination Ura 3 and ade2 as a way to detect the heterochromatic spreading phenomenon Can map specific locations near the telomere Yeast people call it TPE 5-fluoroorotic acid--->5-fluorouracil Tools allow us to talk in general about how these experiments were done.

20 5-fluoroorotic acid enzyme encoded by ura3 does this: 5-fluoroorotic acid --->5-fluorouracil 5-flurouracil is incorporated into DNA. This kills the cell.

21 yeast/11.html TURN THE GENE OFF

22 ade2 A A. wild type Euchromatic gene ADE2+ B. ade2- tries to make adenine but instead accumulates a red intermediate. C. Sectored colony B C S. cerevisae Gene knockout Heterochromatic gene ade2 ade2;ade2+ transgene

23 pg 69 Allis et al Telomere sir2 sir3 sir4 Heterochromatin is adjacent to the telomeric repeat C1-3A/ TG1-3 about 350 bp in length These are Rap1 binding sites - nonnucleosome cap Sir 2, 3 & 4 form the heterochromatin Sir=Silent Information Regulator

24 Telomere ChromIP used to show what proteins are present Dam methylase accessibility and nuclease sensitivity used to characterize condensation GATC cut by MboI and Sau3A but MboI is Dam methylase sensitive.

25 pg 72 Common feature of heterochromatin is its spreading from a nucleation site Telomeric repeat is recog by Rap1 and maybe yku These t wo proteins can bind Sir4 proteins Sir4 binds Sir2 and Sir3 Sir2 is a HDAC that deacetylates H4K16ac & H3k9ac!!!!! This can then spread through region w/o Rap1 because Sir3 and 4 can bind the deacetylated H4 & H3 tails which helps the spreading. sir2 produces O-acetyl-ADP-ribose NAD --> O-acetyl-ADP-ribose which stimulates Sir3 multimerization and Sir3 binding to Sir4 and Sir2 Looping can be important for spreading see figure 6

26 H4K16 Involved in producing higher order organization of chromatin, above simple nucleosome organization.

27 pg 69 Allis et al Rap1 recruits Sir4 ORC recruits Sir1 Abf1 recruits Sir3 ORC = origin recognition complex

28 What causes the spreading to stop? pg 73 subtelomeric Sas2 is a H4K16 acetylase in Sas2 mutants see extended spreading! occurs on boundary elements: other things that contribute to the boundary are highly expressed genes (trna), other modifications, histone recruiting transcription factors, nuclear pore tethering (probably an area of the nucleus that does not contain the heterochromatin enzymes).

29 Telomere looping Helps to stabilize heterochromatin by providing trans-nucleation sites Pause Accounts for some instances where the pattern is heterochromatin, euchromatin, heterochromatin

30 Real telomeres are more complex Two types of real telomeres exist in yeast XY & X Star and STR are insulators

31 Heterochromatin is compartmentalized in the nucleus During interphase telomeres are at the nuclear periphery Other heterochromatin is found there too (HML and HMR)

32 Heterochromatin is compartmentalized in the nucleus yku and Sir4 do this. Sir4 binds Esc

33 MAT Figure 3 page 67 E&I Silencers

34 Figure 3 page 67

35 How do we tell where the proteins are?

36 The ChIP assay chromatin immunoprecipitation Allows one to determine which proteins and molecules are present on any part of the chromosome. Allows one to detect changes in abundance of proteins and molecules on any part of the chromosome.

37 Chromatin immunoprecipitation = nucleosome with an acetylated histone H4 Chromatin extraction and cross-linking Chromatin shearing (sonication) Chromatin immunoprecipitation Reverse cross-linking DNA purification

38 The entire genome is usually present in the immunoprecipitate but the relative abundance of any given fragment is directly proportional to the abundance of the epitope (acetylation histone in this case). The DNA contained in each immunoprecipitated fragment can be used to identify its position in the genome. Assay the relative abundance of all of the immunoprecipitated fragments. This provides you with a measure of the relative acetylation level of that part of the genome. The relative abundance of each fragment might be determined by PCR followed by Southern blotting Real time PCR hybridization to a tiling array SAGE massively parallel DNA sequencing

39 Wickipedia has a nice and concise write-up. Chromatin Immunoprecipitation (ChIP) Chromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest. This technique gives a picture of the protein-dna interactions that occur inside the nucleus of living cells or tissues. The in vivo nature of this method is in contrast to other approaches traditionally employed to answer the same questions. The principle underpinning this assay is that DNA-binding proteins (including transcription factors) in living cells can be cross-linked to the DNA that they are binding. By using an antibody that is specific to a putative DNA binding protein, one can immunoprecipitate the protein-dna complex out of cellular lysates. The crosslinking is often accomplished by applying formaldehyde to the cells (or tissue), although it is sometimes advantageous to use a more defined and consistent crosslinker such as DTBP. Following crosslinking, the cells are lysed and the DNA is broken into pieces kb in length by sonication. At this point the immunoprecipitation is performed resulting in the purification of protein-dna complexes. The purified protein-dna complexes are then heated to reverse the formaldehyde cross-linking of the protein and DNA complexes, allowing the DNA to be separated from the proteins. The identity and quantity of the DNA fragments isolated can then be determined by PCR. The limitation of performing PCR on the isolated fragments is that one must have an idea which genomic region is being targeted in order to generate the correct PCR primers. This limitation is very easily circumvented simply by cloning the isolated genomic DNA into a plasmid vector and then using primers that are specific to the cloning region of that vector. Alternatively, when one wants to find where the protein binds on a genome-wide scale, a DNA microarray can be used (ChIP-on-chip or ChIP-chip) allowing for the characterization of the cistrome. As well, ChIP- Sequencing has recently emerged as a new technology that can localize protein binding sites in a high-throughput, costeffective fashion.

40 References For MAT: Buhler, M, Gasser, SM (2009) Silent chromatin at the middle and ends: lessons from yeasts. EMBO J, 28: