RNA meets chromatin

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RNA meets chromatin

• Qi Yao
• 
  2006-10-30

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• This review favors the general view that the
  epigenetic regulation is likely to require
  examination of both RNA and chromatin.
• This review will concentrate on emerging evidence
  that links RNAi-like mechanisms to the regulation
  of TGS through changes in chromatin.
• They proposed that heterochromatin-associated
  proteins may participate in linking RNA and
  chromatin.

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• Discuss Noncoding RNAs chromatin formation
  changes in the following epigenetic progress
• Dosage compensation
• RNAi-mediated heterochromatin assembly and gene
  silencing
• Programmed DNA elimination

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• Experimental models
• Fungi
• Protists
• Plants
• Animals ( Flies , Mammals )

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Main points

• Chromatin dynamics
• RNA intricacies
• Heterochromatin meets RNAi
• Missing links heterochromatin-associated
  proteins that may interact with RNA
• Intriguing links between RNA and chromatin
• Unanswered questions and future directions

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Chromatin dynamics

• Histone modification--serine and threonine
  phosphorylation, lysine acetylation, lysine and
  arginine methylation, lysine ubiquitination and
  sumoylation, and ADP ribosylation
• Euchromatin and heterochromatin--the relationship
  between euchromatin and heterochromatin, in part
  dictated by covalent modifications of histone
  proteins, provides an elegant balance for the
  regulation of epigenetic states, and may have
  much more significance than simply governing gene
  expression.

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RNA intricacies

• PTGS--post-transcriptional gene silencing
• TGS--transcriptional gene silencing
• Dosage compensation a strong link between RNA
  and chromatin (in flies between RNA and active
  histone acetylation marks (rox), while in mammals
  between RNA with repressive histone methylation
  marks (xist) )
• Chromatin and RNA may be intertwined

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Heterochromatin meets RNAi

• RNAi and repetitive elements
• RNAi and centromeres
• RNAi and DNA elimination

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RNAi and repetitive elements

• repetitive elements are often assembled into
  condensed, transcriptionally silent chromatin
  states
• RNAi-like mechanisms are now known to play a
  critical role in mediating heterochromatic gene
  silencing and can prevent the mobilization of
  transposable element
• Furthermore, in an effort to identify endogenous
  targets of RNAi, the sequencing of small RNAs has
  revealed sequences corresponding to endogenous
  transposons and other repetitive sequences in
  Drosophila and plants

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RNAi and centromeres

• Long,noncoding RNAs homologous to the centromeric
  repeats were found to accumulate in the dcr1,
  ago1, and rdp1 mutant cells, but not in wild-type
  cells
• Heterochromatin stabilizes repetitive DNA
  sequences or multiple copies of transposable
  elements at centromeres, telomeres, and other
  regions of the genome
• Using S.pombe, several groups have demonstrated
  that small RNAs play a critical role in
  regulating heterochromatin formation

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RNAi and DNA elimination

• ProtistTetrahymena thermophila
• dramatically rearranges its genome during its
  sexual life cycle.
• siRNAs play a role in DNA elimination and
  heterochromatin formation in this protist, as
  represented by the scan RNA model

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• scanRNAsGermline IESs (internally eliminated
  sequences)
• have been shown to be bidirectionally
  transcribed in micronuclei at a unique stage of
  the sexual pathway and potentially give rise to
  the small RNAs
• These RNAs are associated with Twi1 (an Argonaute
  family member) and are escorted from the parental
  MAC to the new MAC.

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• scanRNA hypothesis
• based upon the finding that Twi1 localizes
• in the cytoplasm early in conjugation,
  followed by its concentration in the parental MAC
  and finally in the new MAC
• propose that small RNAs literally scan the old
  macronuclear genome in order to determine the
  identity of IESs to be eliminated in the new MAC.

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Missing links heterochromatin-associated
proteins that may interact with RNA

• Chromodomain(CD)-containing proteins
• The CDs of the Drosophila DCC
• HP1 and RNA in the maintenance of pericentric
  heterochromatin
• Other chromatin-associated proteins that interact
  with RNA

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Chromodomain(CD)-containing proteins

• Excellent progress has been made in demonstrating
  that the chromodomain is, indeed, a
  proteinprotein interaction module, specifically
  by its ability to bind to methylated histone
  peptides.
• Several reports have suggested that CDs also bind
  to nucleic acids, both RNA and DNA .Here, we
  focus our attention on potential RNA-binding
  properties of the chromodomain, as well as that
  of other chromatin-associated proteins.

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The CDs of the Drosophila DCC

• dosage compensation complex--roX RNAs, MOF and
  MSL.
• MOF MSL
• interact with the X chromosome in an
  RNase-sensitive manner and to bind RNA in vitro
• RNase treatment of Drosophila S2 cells resulted
  in loss of MOF staining on the X chromosome, and
  electromobility shift assays revealed an
  intriguing interaction between MOF and RNA

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HP1 and RNA in the maintenance of pericentric
heterochromatin

• Heterochromatin in mouse pericentromeric regions
  has an RNA component that is required for its
  integrity
• RNase treatment of mouse fibroblasts results in
  delocalization of HP1 from the pericentric
  heterochromatin and prevents detection of the
  normal foci seen by H3 Lys 9 methyl-specific
  antibodies
• HP1 is a hallmark property of constitutive
  heterochromatin and is the effector-binding
  partner of H3 methylated at Lys 9. association of
  HP1 at pericentric regions is dependent not only
  on its histone methyl-lysine-binding ability, but
  also on RNA binding

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Other chromatin-associated proteins that interact
with RNA

• The DDP1 protein of Drosophila contains 15 tandem
  KH domains, which are high-affinity RNA- and
  ssDNAbinding motifs.
• C. elegans homolog of mammalian Polyhomeotic 1,
  sop-2,
• that displays RNA-binding activity

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• Simultaneous chromatin and nucleic acid binding
  may be required in order to regulate gene
  expression appropriately. They envision that this
  may occur in several ways

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• (1) A protein complex may contain a particular
  polypeptide required for chromatin binding and
  another for targeting the complex to a specific
  locus by an RNA-guided interaction (e.g., RITS
  or possibly the DCC in flies)
• (2) A single polypeptide may contain both a
  chromatin-binding domain and an RNA-binding
  domain (HP1)
• (3) a single domain within a single polypeptide
  may perform both of these functions, possibly to
  enhance its binding affinity and specificityfor
  example, a chromodomain

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Intriguing links between RNA and chromatin

• The histone variant macroH2A
• Tudor domains
• PcG proteins
• miRNAs and chromatin

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The histone variant macroH2A

• MacroH2A is an unusually large H2A variant that
  contain an N-terminal H2A region (65 identity
  to core histone H2A) and a large C-terminal
  nonhistone region, the macro domain.
• preferentially concentrated on the inactive X
  chromosome in females.its dependence on the
  expression of the Xist RNA suggests a role in
  maintaining the silenced state of the Xi,
  possibly through an RNA-binding mechanism.
• The wide distribution of this domain suggests a
  conserved and important function.

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Tudor domains

• the tudor gene product of Drosophila contains
  multiple repetitive domains now referred to as
  the tudor domain homeless gene product TSN1, a
  protein that contains five staphylococcal/micrococ
  cal nuclease domains, and is a component of the
  RISC complex involved in RNAi
• Tudor-domaincontaining proteins are also
  implicated in RNA regulation (RNAi, splicing,
  mRNA transport in Drosophila development).
• have dual binding propertiesa specificity for
  nucleic acids and a specificity for appropriately
  modified amino acids (e.g., methyllysine in
  histone peptides). For example, in the case of
  homeless, the tudor domain may bind RNA, and use
  the domain to target other proteins, such as
  modified histones

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PcG proteins

• it remains a formal possibility that RNA may
  regulate PcG targeting through an RNAi-based
  mechanism.
• Evidence
• discovery of genetic interactions between PcG
  mutants and the RNAi machinery in C. elegans and
  Drosophila as well as the aforementioned
  RNA-binding affinity of Polyhomeotic proteins.

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miRNAs and chromatin

• many chromatin-associated proteins also regulate
  development genes (the PcG genes) like miRNAs do.
• It is intriguing to speculate that miRNAs may
  regulate the expression of key chromatin
  regulators.
• a recent study predicting miRNA target genes in
  humans has listed various histone
  methyltransferases, methyl CpG-binding
  proteins,CD-containing proteins, and histone
  deacetylases

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Unanswered questions and future directions

• One central problem that has yet to be critically
  addressed is how epigenetic marks are templated
  during DNA replication and faithfully inherited
  during cell division.
• Resolving the biology underlying how RNA mediates
  such an array of circumstances within the cell is
  imperative in order to bring us one step closer
  to decoding epigenetic processes.

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RNA interference machinery influences the nuclear
organization of a chromatin insulator
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Main content

• The evidence for a functional relationship
  between RNAi and the gypsy insulator of D.
  melanogaster.

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Introduction to gypsy insulator

• Two functional properties
• 1. the abilities to interfere with
  promoter-enhancer interactions
• 2. shield transgenes from position effects caused
  by surrounding chromatin.

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The protein complex that binding to the gypsy
insulator

• DNA-binding protein Suppressor of Hairy wing
  (Su(Hw))
• DNA-binding Centrosomal protein 190 (CP190)
• Modifier of mdg4 2.2 (Mod(mdg4)2.2)
• They existing as a complex, insulator proteins
  concentrate in nuclear foci termed insulator
  bodies, and insulator activity correlates with
  the ability to form these higher-order
  structures. Proper localization of insulator
  bodies requires an intact nuclear matrix
  scaffold, and in particular, the presence of
  lamin as well as RNA.

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The insulator complex interaction component

• Rm62 proteinpurified by immunoaffinity
  purification with cp190 and identified by
  MALDI-TOF mass spectrometry.
• (It is required for dsRNA-mediated silencing,
  heterochromatin formation and transposon
  silencing. Given that Rm62 is a putative
  RNA-binding protein, they repeated the
  purifications in the presence or absence of RNase
  A.)
• Small RNAproved by RNaseA digestion assay

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The data evidence
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• They proposed these complex to act as attachment
  regions that bridge two or more DNA sequences,
  causing DNA looping and the creation of a
  distinct chromatin domain.

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The model of insulator chromatin domain formation
Su
Mod2.2
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The experimental approvements for this theory
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Model choose

• gypsy insulator of D. melanogaster.
• black pigmentation were visible in the abdomen
  owing to intermediate y2 expression
• ct6 is not fully expressed, resulting in a
  notched wing margin
• Insertion of the gypsy retrotransposon into y2
  and ct6 results in enhancer-specific gene
  expression defects dependent on insulator
  activity. The insulator blocks enhancer promoter
  communication of y2 and ct6, resulting in
  decreased expression.

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Mutation background

• Rm62E/Rm62sh(3)029,resulted in decreased
  pigmentation and larger notches in the wing
  margin compared with mod(mdg4)u1 alone,
  indicating improvement of insulator activity
• Mod(mdg4)u1 null mutant can reduced
  insulator activity .moderate levels of black
  pigmentation were visible in the abdomen owing to
  intermediate y2 expression
• CP1904-1/CP190P11 loss-of-function
  mutants caused by a nonsense mutation in one copy
  of CP190 and deletion of the second copy showed
  reduced insulator activity
• piwi1/piwi2mod(mdg4)u1 and aubQC42/
  mod(mdg4)u1 double mutants showed increased
  pigmentation and restoration to a round wing
  margin, indicating reduced insulator activity
• aubDP-3a/, piwi1/ and piwi2/
  heterozygous mutants reduced insulator activity

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These results suggest that wild-type Rm62
activity negatively affects insulator function in
vivo.
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Triple aubQC42/ Rm62Emod(mdg4)u1/Rm62sh(3)029
mod(mdg4)u1 and piwi2/Rm62Emod(mdg4)u1/Rm62sh(3)
029mod(mdg4)u1 flies showed the same effects on
y2 and ct6 as double aubQC42/mod(mdg4)u1 and
piwi2/ mod(mdg4)u1 mutants, respectively
This epistasis results suggest that these genes
affect insulator activity through a common
RNAi-dependent pathway and further show that piwi
and aub act upstream of Rm62 with respect to
insulator function
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gypsy insulator protects a white transgene from
the repressive effects of omb regulatory elements
Combination of either piwi2/ or aubQC42/ with
mod(mdg4)T6 resulted in a further increase of
white repression, confirming that insulator
function is reduced.
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effects on insulator function are not caused by
altered expression of insulator component genes
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• Finally, they examined the effect of loquacious,
  which encodes the protein partner of the enzyme
  Dicer-1 involved primarily in microRNA
  processing. They did not observe any effect on y2
  or ct6 in loqf00791 mod(mdg4)u1 compared with
  mod(mdg4)u1 flies, suggesting that the microRNA
  pathway does not directly or indirectly influence
  insulator function (data not shown)

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polytene chromosomes.
myc-Piwi localizes to condensed regions of DNA
GFP-Aub (data not shown) and myc-Piwi
localization patterns were consistent with their
roles in centromeric heterochromatin silencing
and possibly throughout the genome.
In contrast, the localization patterns of
Mod(mdg4)2.2 and Su(Hw) were distinct from that
of GFP-Aub and myc-Piwi, with minimal staining at
the chromocenter (Fig. 3 and data not shown).
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Comparison of localization patterns of Rm62 and
CP190, which binds to hundreds of euchromatic
sites, showed a limited degree of overlap,
suggesting that these proteins interact
transiently or outside the context of polytenized
chromosomes (Fig. 3b). Rm62 and CP190 may
interact during earlier stages of development,
such as in embryos, from which we purified
insulator complexes.
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Polytene chromosomes
These results and the finding that Argonautes,
Rm62 and insulator proteins all show distinct
patterns of genomic localization suggest that the
RNAi machinery does not target insulator proteins
directly to their genomic binding sites.
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These results suggest that Argonaute proteins
contribute to higher-order insulator complex
formation and that Rm62 negatively affects
insulator function by hindering its ability
to produce these structures.
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Conclusion

• Taken together, the results suggest the existence
  of an RNA species required for the formation or
  integrity of insulator bodies, perhaps a product
  of processing by Argonautes and the other RNAi
  machinery.
• The putative RNA helicase Rm62 may be recruited
  to insulator complexes through physical
  interaction with CP190 and RNA. Although it is
  unknown at what mechanistic step Rm62 acts in
  RNAi, Rm62 may act downstream of Argonautes to
  unwind or remodel RNA-insulator protein
  complexes, thereby disrupting gypsy insulator
  activity and nuclear organization.

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• Proper insulator body localization requires an
  intact nuclear matrix, and early observations
  identified RNA as an important component of this
  nuclear scaffold.
• Future studies should determine the identity of
  putative gypsy insulator associated RNAs. The
  results suggest a previously unknown function of
  the RNAi machinery in the control of nuclear
  architecture to effect changes in gene
  expression.

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• Thank you !