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Extra Slides. DATA TO SUPPORT STATEMENTS IN PRESENTATION. Outline of talk. General background Introduction to the project Experimental design Experiments and results Conclusions Future experiments. Prior demonstrations of KAP1 complexes. Structure of CSD complex.

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  1. Extra Slides DATA TO SUPPORT STATEMENTS IN PRESENTATION

  2. Outline of talk • General background • Introduction to the project • Experimental design • Experiments and results • Conclusions • Future experiments

  3. Prior demonstrations of KAP1 complexes

  4. Structure of CSD complex

  5. Epigenetic Gene Silencing • Epigenetic effects are those changes in gene function which are heritable through mitosis and/or meiosis and are not due to changes in DNA sequence. • Main types of epigenetic information: • Cytosine DNA Methylation • Genomic impritning • Histone modifications

  6. CSD interacting proteins

  7. Chromatin based epigenetics • Controls chromosome domains and also helps in cell differentiation • X –inactivation (XIST locus. Genomic imprinting, parent-of origin-specific allele silencing) • Developmental reprogramming of cell lineages • Plasticity of stem cells • Implications  human biology and disease, including cancer and aging

  8. Expression Vectors KAP1 BAIT HP1a (BD-HP1a)-(HA-KAP1) Gal4 DBD PREY Gal4 AD ? (AD-?)

  9. HeLa cell cDNA library • Source of human genes (cDNA). • Human cervical carcinoma cell line • Estimated number of Independent Clones: 3.5 x 106 • Average insert (cDNA) size: 2.0 kb • cDNA size range: 0.5 – 4.0 kb HeLa cancer cells

  10. BAITS BD-HP1a (BD-HP1A)-(HA-KAP1) BD-KAP1 (BD-KAP1)-(HA-HP1) (BD-HP1A)-(HA-CAF1p150) PREYS AD-?(HeLa cell cDNA library; Clontech) AD-KIP21 (SETDB1) AD-KIP41 (SETDB1) AD-Mi2b AD-KOX1 AD-Y2H6.2 (POGZ) Hybrid proteins used in this study

  11. Prior demonstrations of KAP1 & HP1 complexes

  12. Histone Code HP1 gene silencing Strahl et al. Nature 2000

  13. Long term Goals • Build complete HP1 network with all its partner proteins and to build a chromatin network database ! • Map both the temporal and Spatial formation of these complexes • Get in depth and complete understanding of gene regulation in cells

  14. Statement of Problem • Studies show that HP1a binds with several proteins and presumably performs different activities (mainly in regulating chromatin structure and function). • But, little is known about what complexes exist in vivo and what controls their formation

  15. Assumptions • The main assumption in this experiment is that there are regulatory factors that help in the formation of HP1 complexes. • Yeast system does not interfere with any of the interactions. (relatively ‘safe’; most of the initial HP1 interactions were done in yeast screens.) • Feasibility of this screen can be demonstrated by showing a ternary complex formation with positive controls.

  16. Limitations • Yeast might inactivate or destroy the foreign (human) proteins. • Some of these human proteins might be toxic to yeast. • Functional homology might be present between yeast proteins and the human proteins. • Presence of false positives or false negatives. • Bait proteins that auto-activate cannot be used. • Cannot directly extrapolate the results to higher eukaryotes without further verification.

  17. Histone H3 tail bound to Chromo Domain of Drosophila HP1a Y : Tyrosine W: Tryptophan E : Glutamine T : Threonine K : Lysine V : Valine N : Asparagine S : Serine A : Alanine Yelow: Me2k9 Red/Orange: Me3k9 Jacobs and Khorasanizadeh (2002), Science express

  18. Number of Transformants = ~ 2 x 106 per ml; For a 5ml final volume of transformed cells number of library colonies screened is around ~107 ! Large enough to cover all clones in the library. (Number of independent clones in the library: 3.5 x 106; clontech ) Mock transformation

  19. HIS3 expression seems to be Leaky on –L/T/H plates • All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1). • BAIT and empty pACT seem to grow on –L/T/H but not –L/T/H/A (observation from mock transformation).

  20. HIS3 titration with competitive inhibitor 3-AT • This assay is to determine the amount of 3-AT needed to reduce noise (growth when there is no interaction) on selection plates. NOTE : 3-AT is 3-AMINO-1,2,4-TRIAZOLE, a competitive inhibiotor of HIS3 gene product. TNC means too numerous to count • All colonies have BAIT in them (pBRIDGE-Gal4HP1a-HA-KAP1).

  21. Cell Lysate prepared for Western blot used for Co-IPs Anti-HA used for IMMUNOPRECIPITATION • Harsh conditions used to obtain cell lysate • Blot demonstrates that the Anti-HA antibody is not as sensitive as expected.

  22. Who's calling the shots? • Some transcription factors have, or recruit proteins that have, histone modification and remodeling activities (Fig. 1). Presumably, gene activation requires at least one such factor that can bind its recognition sequence within 'inactive' chromatin and recruit other factors that collaborate in altering local chromatin structure. These altered regions of chromatin would then expose binding sites for other factors, including the basal transcription machinery3. Histone modifications may also be necessary to allow RNA polymerase to transit across nucleosomal DNA sequences7. • Also, whereas acetylation can be reversed by various histone deacetylases, there are no known histone demethylases. Therefore, once a genomic region is methylated, modified nucleosomes must be replaced rather than altered to remove this epigenetic mark. Further work will be required to understand the mechanisms responsible for the spread of local histone modifications and the impact of these modifications on chromatin structure and transcriptional regulation.

  23. Nature Genetics36, 438 - 440 (2004)

  24. Fig. 3. Model to explain the role of positive and negative factors in heterochromatin and euchromatin. Methylated amino acids in the histone H3 tail are indicated by red lettering, and acetylated residues are shown in blue. The underlying sequence of the satellite repeats promotes the formation of a regular array of stable nucleosomes, which are favoured substrates for methylation at H3 lysine 9 (K9) by SUVAR39H1. Binding of HP1 to long arrays of nucleosomes containing H3 methylated at K9 promotes the formation of the higher-order heterochromatin structure. Transcriptionally active euchromatin is generated by transcription factors binding to clustered recognition sequences resulting in the formation of DNase I hypersensitive sites (HS). The HS generate and maintain the open structure of the euchromatin by promoting H3 K9 acetylation and K4 methylation of neighbouring nucleosomes. They can also act as barriers preventing the spread of heterochromatin into neighbouring euchromatin. Trends Genet. 2002 May;18(5):252-8.

  25. How to identify different classes of interaction?

  26. My Interests • How do cells in different tissues have different functions when they have the same genome? • Is entire human genome expressed? • NO • There is approximately one gene every ~75,000 base pairs. • And only a fraction (~2%)of this part codes for polypeptides. • Global gene expression patterns?

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