1. RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production

2. What is already known? Without rdp1, heterochromatin is lost
Ago1 and other RITS (RNAi effector complex) proteins bind constitutive heterochromatin at all known loci
Deletion of RNAi components impair silencing at centromeres, initiation of heterochromatin assembly at mat locus
RdRPs thought to convert single stranded to dsRNA, enhancing RNAi effect

3. What are they asking?
Is RdRP (RNA dependent RNA Polymerase) activity responsible for heterochromatin formation, chromosome segregation, centromere silencing?
Is Rdp1 recruited to the centromeres?
Is there a self-inducing pattern where siRNA are generated by the RNAi pathway, then target heterochromatin proteins to homologous sequences. (This should maintain heterochromatin)

4. How did they address these questions?
Model: schizosaccharomyces pombe
ChIP analysis
otr1R::ura4+ construct. Otr1R is not important except that is usually kept as heterochromatin

5. Chromatin Immunoprecipitation
Otr1R
Otr1R
BEAD
Rdp1
Centrifugation pulls down the beads, chemically dissociate
Load result into a gel, run electrophoresis
Stain for reporter gene (ura4)

6. Rdp1 on constitutive heterochromatic domains
ChIP analysis for Rdp1 at the centromeres, telomeres and mat locus (constitutive heterochromatin areas)
WCE= Whole Cell Extract
ChIP analysis of Rdp1 at centromeric repeats with one RITS constituent protein knocked out in each; as well as Dcr1 (involved in RNAi), Clr4 (H3K9 methylation) and Swi6 (heterochromatin formation).

7. What have they shown?
Rdp1 localized at heterochromatin (centromeres, telomeres, mat locus)
Rdp1 localization depends of RITS, Dcr1, and heterochromatin assembly factors

8. S. Pombe
N Crassa
C. Elegans
A. thaliana
expressing ura4 sensitizes cells to FOA
Coomassie Brilliant Blue

9. What have they shown?
Rdp1 localized at heterochromatin (centromeres, telomeres, mat locus)
Rdp1 localization depends of RITS, Dcr1, and heterochromatin assembly factors
Their Rdp1 mutant construct is expressed at the same level, but with the mutation normally silent genes are expressed

10. There is a similar level of Swi6 and H3K9me in the whole cell, but less at the otr1R region and centriole in the mutant; problem with heterochromatin assembly

11. What have they shown?
Rdp1 localized at heterochromatin (centromeres, telomeres, mat locus)
Rdp1 localization depends of RITS, Dcr1, and heterochromatin assembly factors
Their Rdp1 mutant construct is expressed at the same level, but with the mutation normally silent genes are expressed
There is a similar level of Swi6 and H3K9me in the whole cell, but less at the otr1R region and centriole in the mutant; problem with heterochromatin assembly

12. Similar viability with no treatment, but the mutant is more sensitive to TBZ, indicating failure of centromeres
Wild types rarely show “lagging chromosomes”-- which fail to split in anaphase, and don’t get incorporated into the new cells– but about 1/5 of rdp1 mutants
Taz1 is a telomere binding protein, and colocalizes with Swi6 because of its heterochromatin state. This shows problems in telomere clustering (there are too many in the mutants)

13. What have they shown?
Rdp1 localized at heterochromatin (centromeres, telomeres, mat locus)
Rdp1 localization depends of RITS, Dcr1, and heterochromatin assembly factors
Their Rdp1 mutant construct is expressed at the same level, but with the mutation normally silent genes are expressed
There is a similar level of Swi6 and H3K9me in the whole cell, but less at the otr1R region and centriole in the mutant; problem with heterochromatin assembly
Mutation causes mitosis problems, possibly due to failure of heterochromatin assembly

14. Mutation of Rdp1 at D903 abolishes activity in affinity purified samples.
siRNAs were lost in mutants of RdRP activity, reducing it to background levels
Shows that this is exclusively an RNA effect; pattern not affected by DNase

15. What have they shown?
Rdp1 localized at heterochromatin (centromeres, telomeres, mat locus)
Rdp1 localization depends of RITS, Dcr1, and heterochromatin assembly factors
Their Rdp1 mutant construct is expressed at the same level, but with the mutation normally silent genes are expressed
There is a similar level of Swi6 and H3K9me in the whole cell, but less at the otr1R region and centriole in the mutant; problem with heterochromatin assembly
Heterochromatin assembly problem causes mitosis problems
RdRP-affecting mutation removed ability to create siRNA

16. The RITS components, Ago1 Chp1 and Tas3, fail to localize with the mutant strain. In this ChIP, they are not associated with otr1R or the centriole
Rdp1 mutant does not associate with the heterochromatin either

17. What have they shown?
Rdp1 localized at heterochromatin (centromeres, telomeres, mat locus)
Rdp1 localization depends of RITS, Dcr1, and heterochromatin assembly factors
Their Rdp1 mutant construct is expressed at the same level, but with the mutation normally silent genes are expressed
There is a similar level of Swi6 and H3K9me in the whole cell, but less at the otr1R region and centriole in the mutant; problem with heterochromatin assembly
Heterochromatin assembly problem causes mitosis problems
RdRP-affecting mutation removed ability to create siRNA
RdRP-afecting mutation prevents the formation of the RITS complex and association of the Rdp1 to heterochromatin

18. So what is the point?
Rdp1 required for creating dsRNAs that activate Dicer, which in turn amplifies siRNAs
siRNA target heterochromatin in cis, help maintain heterochromatin state: if the heterochromatin opens and expresses, the siRNA will silence it again
siRNA needed for H3K9 methylation, RITS localization

20. STEPS in cycle
Bidirectional transcription of repeat elements creates dsRNA
Dicer cleaves the dsRNA into siRNA
siRNA targets histone methyltransferase and RITS to the repeat element
RITS binds Rdp1, others at heterochromatic loci
Ago1 binds siRNA, uses Rdp1’s RdRP activity to create more dsRNA

21. So what?
Support idea that RNAi maintains heterochromatin in a loop, not a line
Rdp1 converts “aberrant” transcripts into siRNA to resilence the gene
Keeping certain genes off is critical for the differentiation of cells or reactions to the environment