Bio 525 spring 2010 nuclear architecture and genomic function
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Bio 525/ Spring, 2010 Nuclear Architecture and Genomic Function. Session 8: Dynamics of Genomic Organization & Function in Living Cells II; Background and Figures for Kaiser et al., 2008; Tsukamoto et al., 2000 . Formation of nuclear structures T Misteli, Nature 456, 333-334.

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Bio 525 spring 2010 nuclear architecture and genomic function

Bio 525/ Spring, 2010Nuclear Architecture and Genomic Function

Session 8: Dynamics of Genomic Organization & Function in Living Cells II; Background and Figures for Kaiser et al., 2008; Tsukamoto et al., 2000


Bio 525 spring 2010 nuclear architecture and genomic function

Formation of nuclear structuresT Misteli, Nature 456, 333-334


Bio 525 spring 2010 nuclear architecture and genomic function

  • Functions of Cajal Bodies (Coiled Bodies)

  • Assembly of several snRNP

  • Modification of U snRNA

  • 3′-end processing of histone mRNA

  • Cell cycle regulation

  • Assembly of the transcription factors

  • Apoptotic signaling

DL Spector, Cajal Bodies, Cell 2006 127:1071


Bio 525 spring 2010 nuclear architecture and genomic function

Lac-Operator-Repressor interactions


Bio 525 spring 2010 nuclear architecture and genomic function

Kaiser et al., Science. 2008 322:1713-1717

De Novo Formation of a Subnuclear Body


Bio 525 spring 2010 nuclear architecture and genomic function

  • Major Conclusions of Kaiser et al 2008

  • Immobilization of a single structural component is often sufficient for a

  • nuclear body formation (Fig 1 & 3 & 4)

  • 2) Cajal bodies formed de novo via tethering coilin are functional (Fig 2)

  • 3) Association of proteins with tethered components of Cajal Bodies is suggestive

  • of self-organization model (Fig 4)


Bio 525 spring 2010 nuclear architecture and genomic function

Conclusion 1: Immobilization of a single structural component is often sufficient for a nuclear body formation (Fig 1 & 3 & 4)


Bio 525 spring 2010 nuclear architecture and genomic function

Fig 1 (A-E) Immobilization of a single structural

component leads to

nuclear body formation.

Immunofluorescence

microscopy on HeLa cells

transiently transfected with

various GFP-LacI fusion

proteins (green),

Cherry-LacI (red) and

stained with the

indicated antibody (A to E).

(Insets) High magnification

view of formed CB.


Bio 525 spring 2010 nuclear architecture and genomic function

Fig 1 (F) Quantification of de novo CB formation


Bio 525 spring 2010 nuclear architecture and genomic function

Conclusion 2: Cajal bodies formed de novo via tethering coilin are functional (Fig 2)


Bio 525 spring 2010 nuclear architecture and genomic function

Fig 2 (A-E). Tethering coilin forms functional CBs.


Bio 525 spring 2010 nuclear architecture and genomic function

Fig 2 (F, G). Tethering coilin forms functional CBs.


Bio 525 spring 2010 nuclear architecture and genomic function

Fig. 3. Coilin and SMN are required for Cajal body formation.


Bio 525 spring 2010 nuclear architecture and genomic function

Conclusion 3: Association of proteins with tethered components of Cajal Bodies is suggestive of self-organization model (Suppl Fig 3, Fig 4)


Bio 525 spring 2010 nuclear architecture and genomic function

Suppl Figure 3 The kinetics of de novo CB formation.


Bio 525 spring 2010 nuclear architecture and genomic function

Fig. 4. Other Cajal body components are capable of forming Cajal bodies de novo. Immunofluorescence microscopy on HeLa cells transiently transfected with CB components fused with GFP-LacI, Cherry-lacI, and coilin-specific antibody (A to G). Arrows indicate the location of de novo formed CB.


Bio 525 spring 2010 nuclear architecture and genomic function

Suppl Table 1 Summary of fusion proteins immobilized on chromatin and their abilities to form de novo CBs

.


Bio 525 spring 2010 nuclear architecture and genomic function

Formation of nuclear structuresT Misteli, Nature 456, 333-334


Bio 525 spring 2010 nuclear architecture and genomic function

  • Major Conclusions of Kaiser et al 2008

  • Immobilization of a single structural component is often sufficient for a

  • nuclear body formation (Fig 1 & 3 & 4)

  • 2) Cajal bodies formed de novo via tethering coilin are functional (Fig 2)

  • 3) Association of proteins with tethered components of Cajal Bodies is suggestive

  • of self-organization model (Fig 4)


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al., Nat Cell Biol. 2000 2:871-8.

Visualization of gene activity in living cells


Bio 525 spring 2010 nuclear architecture and genomic function

Visualization of extranucleolar transcription sites in HeLa cell


Bio 525 spring 2010 nuclear architecture and genomic function

Major Conclusions of Tsukamoto et al 2000

1) Based on lac operator/repressor system and two kinds of fluorescent proteins it

was developed an experimental tool which allows for direct visualization of gene and

its protein product in living cells.

2) Activation of gene cluster coincides with “opening” of its structure. Open chromatin

structure persists while gene cluster is expressed.

3) Accumulation of exogenous proteins including tetracycline transactivator complex

and EYFP-lac-repressor at a gene cluster induces co-localization with promyelocytic

leukemia (PML) nuclear bodies


Bio 525 spring 2010 nuclear architecture and genomic function

Conclusion 1 Figures 1-2

Based on lac operator/repressor system and two kinds of fluorescent proteins it

was developed an experimental tool which allows for direct visualization of gene and

its protein product in living cells.


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al Nat Cell Biol 2000, Figure 1.


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al Nat Cell Biol 2000, Figure 2. Characterization of isolated clones.a, Southern blotting of isolated clones. b, Induction of CFP–SKL protein by doxycycline (24 h after transfection). c, Time course of induction of clone-2 cells.


Bio 525 spring 2010 nuclear architecture and genomic function

Conclusion 2; Figures 3-6, Table1

Activation of gene cluster coincides with “opening” of its structure. Open chromatin

structure persists while gene cluster is expressed


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al Nat Cell Biol 2000, Figure 3. Visualization of the genetic locus.


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al Nat Cell Biol 2000, Figure 4

Comparison of different methods of fixation and staining.

.


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al Nat Cell Biol 2000, Figure 5

Changes in chromatin organization during gene activation.

.


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al Nat Cell Biol 2000, Table1. Correlation between chromatin

organization and CFP/SKL gene expression


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al Nat Cell Biol 2000, Figure 6 .

The open chromatin structure is static.


Bio 525 spring 2010 nuclear architecture and genomic function

Conclusion 3 Figure 7, Table 2

Accumulation of exogenous proteins including tetracycline transactivator complex

and EYFP-lac-repressor at a gene cluster induces co-localization with promyelocytic leukemia (PML) nuclear bodies


Bio 525 spring 2010 nuclear architecture and genomic function

PML bodies are shown in red and lac repressor is shown in green.

The p3216PCb integration site was visualized in clone-22 cells by in vivo

expressed EYFP/ lac repressor (a–c), by EGFP/ lac repressor overlay staining (d, e)

or by RNA FISH (f) after the addition of doxycycline.

CFP–SKL expression was shown as a cyan signal.

Tsukamoto et al Nat Cell Biol 2000, Figure 7 Relationship between the integrated locus and PML bodies.


Bio 525 spring 2010 nuclear architecture and genomic function

Tsukamoto et al Nat Cell Biol 2000, Table2. Association of a PML body with the integrated genetic locus


Bio 525 spring 2010 nuclear architecture and genomic function

Major Conclusions of Tsukamoto et al 2000

1) Based on lac operator/repressor system and two kinds of fluorescent proteins it

was developed an experimental tool which allows for direct visualization of gene and

its product in living cells.

2) Activation of gene cluster coincides with “opening” of its structure. Open chromatin

structure persists while gene cluster is expressed

3) Accumulation of exogenous proteins including tetracycline transactivator complex

and EYFP-lac-repressor at a gene cluster induces co-localization with promyelocytic

leukemia (PML) nuclear bodies


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