Non-coding RNA characterization

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Non-coding RNA characterization
Roles in X-chromosome inactivation
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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?
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Is there a relationship between RNA pol II and
Xist RNA accumulation?
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• 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

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• 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.
  1996 Dec 15180(2)618-30.)

Resolution - Examine lifetime of x-linked RNA
transcripts by RNA FISH and RNA pol II IF
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• 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

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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
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• 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

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Role of Xist A repeats - ?A 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
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• 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

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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

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• 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?