Derek Oh & Eric Tarapore Bio Sci D145 2.20.14

1. Derek Oh & Eric TaraporeBio Sci D1452.20.14

2. Chromatin • Complex of proteins and DNA • Two varieties: heterochromatin and euchromatin • Role -package DNA -protect DNA -regulate gene expression

3. Background • Heterochromatins PcG and HP1 identified before paper • PcG: - polycomb-group proteins (PcG) and H3K27 methylation - repressive regulation of developmental genes • HP1: - heterochromatin protein 1 (HP1) and H3K9 methylation -repressive functions

4. What Are They Trying To Learn • Three questions that need answers: • Are there other major types of chromatin? • Is euchromatin organized into distinct domains with distinct protein compositions? • Are there additional types of repressive chromatin that have not been discovered yet?

5. Answered these questions using a Drosophila cell line. • Kc167: • Taken from dorsal closure stage of embryonic development.

6. DamID is the better choice • Protein of interest fused to DNA Adenine Methyltransferase • No crosslinking or antibodies required. • Even transient interactions can be detected. • Researchers used DamID on 53 chromatin proteins.

7. ChIP • Chromatin Immunoprecipitation • Alternative method to DamID

8. Why Not Use ChIP? • Researchers used DamID; essentially giving you the same thing as ChIP. • So why not just use ChIP? • Although it is the most widely used…large scale application is hampered by the availability of specific antibodies. • Results can also depend on choice of crosslinking reagent.

9. Results • First identified five principal chromatin types. • Next analyzed genome coverage, domain number, mRNA expression, length, and other aspects. • Then went on to analyze each of the five chromatin types in detail. • How are DNA Binding Factors (DBFs) affected by the chromatin types

11. Identification of Five Chromatin types • Using the DamID map, in combination with PC analysis and HMM. Five distinct chromatin types were found. • BLACK • BLUE • GREEN • RED • YELLOW

13. BLUE and GREEN chromatin correspond to known chromatin types

14. GREEN chromatin corresponds to classic heterochromatin marked by SU(VAR)3-9. • BLUE chromatin=PcG chromatin. • Validated by using ChiP analysis of H3K9me2 and H3K27me3.

15. BLACK Chromatin • Most abundant (covers 48% of probed genome) • Gene poor, with no transcriptional activity for 66% of genes. However… • Could BLACK chromatin be a type of repressive chromatin? (Yes it can)

16. RED & YELLOW are distinct types of Euchromatin • Trascriptionally active and produces a lot of mRNA. • Both share various chromatin proteins. • But the two types are distinct in several ways.

17. Does Chromatin type guide DBF binding? • Wanted to see if chromatin does affect the ability of DBFs to bind to their similar binding sequences. • Used five DBFs to analyze the binding affinity to the five chromatin types.

19. Conclusions • Integrating 53 protein maps found that the Drosophila genome is packaged into a mosaic of five chromatin types. • These five types differ in a wide range of characteristics (Figure 2). • BLUE and GREEN chromatin has been identified as two well known types of heterochromatin: PcG and HP1 respectively. • BLACK chromatin is a distinct type of repressive chromatin. • While RED and YELLOW are distinct types of trancriptionally active Euchromatin.

20. Further Reading • Schauer, T., Schwalie, P.C., Handley, A., Margulies, C.E., Filcek, P., Ladurner, A.G. 2013. CAST-ChIP Maps Cell-Type-Specific Chromatin States in the Drosophila Central Nervous System. Cell Reports. 5:271-282.