1. Non-coding RNA characterization
Roles in X-chromosome inactivation

2. This paper sought to reveal the mechanisms by which Xist silences the X chromosome.
what elements are important for exclusion of transcription machinery and nuclear reorganization?
what are the temporal and spatial characteristics of Xist-mediated silencing?

3. Is there a relationship between RNA pol II and Xist RNA accumulation?

7. Summary of fig 1
Spatially, transcription machinery excluded from the Xist domain
Repressive epigenetic modifications are associated with the Xist domain
Temporally, exclusion is followed by epigenetic chromatin changes
exlcusion is an early event– differentiation day 1
epigenetic modification is a later event– day 2

8. Paradox:
Prior data shows that X-linked expression falls on day 2-4 of differentiation
(Keohane AM, et al. X-Inactivation and histone H4 acetylation in embryonic stem cells. Dev Biol Dec 15;180(2): )
Resolution:
- Examine lifetime of x-linked RNA transcripts by RNA FISH and RNA pol II IF

12. Fig 2 summary: Exclusion of machinery occurs earlier than repression
- expression persists during differentiation day 1 and 2
- machinery is excluded during the observed expression
- persistence of primary transcripts causes delay in silencing
Jarid1c escapes complete inactivation because it is distal to Xist locus
Depletion of Cot-1 RNA signal follows RNA pol II exclusion
further identifies the temporal relationship between repression and RNA pol II exclusion
Genes at the periphery of Xist domain lag in repression

13. Role of spatial organization in determining txn state
- Xist induced relocalization
- gene position relative to Xist RNA domain
Using 3D reconstruction analysis:
- identify Xist domain via RNA FISH
- identify repressed gene locations via DNA FISH

16. Fig 3 summary:
X-linked genes are re-localized toward the interior of the Xist RNA domain during differentiation
This re-localization is associated with repression of the genes further into the Xist domain

17. Role of Xist A repeats
- ΔA constructs:
- cannot induce silencing
- can coat in cis correctly
- can induce same histone modifications
allows distinction between changes due to Xist silencing or chromatin modifications

20. Fig 5 summary: A-repeat region not necessary for:
exclusion of RNA pol II
repression of Cot-I
formation of silent nuclear compartment
delta-A Xist failed to silence X-linked genes
genes were not re-organized into the silent compartment

21. Conclusions
Xist RNA forms a silent nuclear compartment during female ES differentiation
Txn machinery is rapidly excluded followed by silencing of genes
Exclusion is proceeded by epigenetic silencing modifications
Silencing is correlated to position relative to the Xist domain
Xist domain presents a repressive environment
Xist induces relocalization of genes to the silent Xist RNA domain
Function of A-repeat region of Xist
Not needed for formation of the silent compartment
Not needed for exclusion of RNA pol II
Required for gene re-localization

23. Discussion ? Is their measure of RNA pol II exclusion accurate?
Is fluorescence an accurate enough measure of transcriptional state?
What about the converse experiment express just the A-repeat region…
Experiments using truncations of the delta-A construct….
Does the Xist domain co-localize with the nuclear structure components?
What is the nature of this repressive RNA “domain”?
How do Polycomb Group complexes fit with Xist function?
How does Xist re-organize the X-linked genes into the silent domain?