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

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 signalingDL Spector, Cajal Bodies, Cell 2006 127:1071

Lac-Operator-Repressor interactions

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

De Novo Formation of a Subnuclear Body

Major Conclusions of Kaiser et al 2008

1) 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)

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

Fig 1 (A-E) Immobilization of a single structural component leads to nuclear body formation.Immunofluorescencemicroscopy on HeLa cells transiently transfected with various GFP-LacI fusion proteins (green), Cherry-LacI (red) and stained with theindicated antibody (A to E).(Insets) High magnificationview of formed CB.

Fig 1 (F) Quantification of de novo CB formation

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

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

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

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

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

Suppl Figure 3 The kinetics of de novo CB formation.

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.

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

Formation of nuclear structures T Misteli, Nature 456, 333-334

Major Conclusions of Kaiser et al 2008

1) 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)

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

Visualization of gene activity in living cells

Visualization of extranucleolar transcription sites in HeLa cell

Major Conclusions of Tsukamoto et al 2000

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

Conclusion 1 Figures 1-2

Based on lac operator/repressor system and two kinds of fluorescent proteins itwas developed an experimental tool which allows for direct visualization of gene and

its protein product in living cells.

Tsukamoto et al Nat Cell Biol 2000, Figure 1.

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.

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

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

Tsukamoto et al Nat Cell Biol 2000, Figure 4 Comparison of different methods of fixation and staining.

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Tsukamoto et al Nat Cell Biol 2000, Figure 5 Changes in chromatin organization during gene activation.

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Tsukamoto et al Nat Cell Biol 2000, Table1. Correlation between chromatin organization and CFP/SKL gene expression

Tsukamoto et al Nat Cell Biol 2000, Figure 6 .The open chromatin structure is static.

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

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

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 vivoexpressed 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, Table2. Association of a PML body with the integrated genetic locus

Major Conclusions of Tsukamoto et al 2000

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