1. Session 6 Dynamics of Genomic Organization & Function in Living Cells I Background & Figures for: Zink et al., Hum Genet 102 (1998) 241-51. Structure and dynamics of human interphase chromosome territories in vivo

2. Domains)

3. Visualization of Replication Sites (Fluorescence and Electron Microscopy) Early S Mid S Late S

4. Double Labeling of DNA During Replication. CldU: Early S Replicating DNA chaseIdU: Mid/Late S Replicating DNA CldU: Early S Replicating DNA chase IdU: Mid/Late S Replicating DNA Mid S Late S G2 Mitosis Ma et al J Cell Biol. 1998 143:1415-25.

6. Schematic illustration of the comparative measurement of motion of chromatin domains within individual chromosome territories. Cell is labeled in two channels for early- (red) and mid- or late- (green) S-phase replicating chromatin. Several generations following the labeling, segregation of the label allows to distinguish individual chromosome territories.

7. Zink D, Cremer T, Saffrich R, Fischer R, Trendelenburg MF, Ansorge W, Stelzer EH. Structure and dynamics of human interphase chromosome territories in vivo. Hum Genet. 1998;102:241-51.

8. Conclusions of Zink et al., 1998 1) Replication-labeled chromatin domains maintain structural integrity following replication (Fig 1 & 2). 2) Replication labeled chromatin domains undergo constant motion and configuration changes (Fig 3 & 4). 3) This motion is constrained to the spatial limits within individual chromosome territories (Fig 3 & 4).

9. Conclusion 1 Replication-labeled chromatin domains maintain structural integrity following replication (Fig 1 & 2).

10. Zink et al, Hum Genet, 1998, Figure 1. Replication-labeled and segregated human chromatids Cultures of human diploid fibroblasts were grown after initial replication labeling for several cell cycles. All panels show chromatids of fixed cells labelled with IdU/CldU according to the scheme: 2 h IdU pulse/4 h chase/2 h CldU pulse. Both the IdU and the CldU label are depicted in green in panels A–E. C Light optical section through a pre-S-phase nucleus with replication-labeled chromatid territories (green). D Subsequent light optical section from the same nuclear plane showing the two painted 15q territories (red; arrow and arrowhead). E Overlay of the images shown in C and D. In panels F–H the IdU label is shown in green and the CldU label is depicted in red.

11. Zink et al, Hum Genet, 1998. Figure 2: Replication-labelled and segregated human chromatids of human neuroblastoma cells (SH-EP N14; A, B) and diploid fibroblasts (C, D). Panel D shows BrdU labelled chromatid territories; the other panels depict Cy3-AP3-dUTP-labelled chromatids. Panels A and D show fixed chromatids, while panels B and C display in vivo images. A Anaphase depicting two completely replication-labelled chromatids. The inset shows the DAPI counterstain. B Optical section (Cy3 detection) of an interphase nucleus from the same slide as the anaphase shown in A. The inset shows an enlargement of one territory (arrowhead). C Video image (non-confocal) of several typical replication- labelled chromatid territories observed in a nucleus 1 week after microinjection of Cy3-AP3- dUTP. D Optical section of a nucleus (containing whole BrdU- labelled chromatid territories.

12. Conclusions 2 & 3 (Fig 3 & 4) Replication labeled chromatin domains undergo constant motion and configuration changes. This motion is constrained to the spatial limits within individual chromosome territories.

13. Zink et al, Hum Genet, 1998 Figure 3: In vivo images of HeLa cells grown for several cell cycles after Cy3-AP3- dUTP microinjection. Time-Lapse Microscopy

14. Zink et al, Hum Genet, 1998 Figure 3 II: In vivo images of HeLa cells grown for several cell cycles after Cy3-AP3-dUTP microinjection. Time-Lapse Microscopy

15. Zink et al, Hum Genet, 1998 Figure 4: Time-lapse recording of one nucleus from the neuroblastoma cell line. Time-Lapse Microscopy

16. Conclusions of Zink et al., 1998 1) Replication-labeled chromatin domains maintain structural integrity following replication (Fig 1 & 2). 2) Replication labeled chromatin domains undergo constant motion and configuration changes (Fig 3 & 4). 3) This motion is constrained to the spatial limits within individual chromosome territories (Fig 3 & 4).

17. Background & Figures for: Li G, Sudlow G, Belmont AS. Interphase Cell Cycle Dynamics of a Late-Replicating, Heterochromatic Homogeneously Staining Region: Precise Choreography of Condensation/Decondensation and Nuclear Positioning J Cell Biol (1998) 140, 975-989..

19. Belmont, TRENDS in Cell Biology 2001, 11 250-257; Figure 1

20. Major conclusions of Li et al 1998 1) Lac-operator is amplified within a late replicating homogeneously Staining ~90 Mb region (HSR) and is replicated in the late S-phase. 2) Intranuclear position and conformation of HSR is changed in early G1, mid to late S late G2 and M phases. While in G1, G2 and M phases these changes are related to mitotic condensation and cell division, in S-phase they coincide with replication of HSR.

21. Conclusion 1 Lac-operator is amplified within a late replicating homogeneously staining ~90 Mb region (HSR) and is replicated in the late S-phase.

22. Li et al JCB 1998, Figure 1. A03_1 HSR characterization: (A) HSR (arrow) is on a distinctive, late- replicating chromosome, present in the parental DG44 cell line (top inset) and is itself late replicating. D E

23. Conclusion 2 Intranuclear position and conformation of HSR is changed in early G1, mid to late S late G2 and M phases. While in G1, G2 and M phases these changes are related to mitotic condensation and cell division, in S-phase they coincide with replication of HSR.

24. Li et al JCB 1998, Figure 2. (A) Metaphase HSR, ~1  m in length. Staining is concentrated on periphery. (B) “Uncoiling” conformation”; Initial uncoiling of HSR (40 min); (C) “Linear conformation”; Extension of HSR to ~0.3-  m diam, ~2-  m-long fiber (80 min). (D) “Loose conformation”; Intermediate, possibly irregularly coiled (see text) structure leading towards condensed chromatin mass (180 min). (E) Uniform, condensed mass with no substructure evident atlight microscopy resolution (4 h). (F) HSRs in A–E at higher magnification. Bars, 2  m.

25. Li et al JCB 1998, Table 2. A03_1 HSR Conformation Versus Nuclear Location during G1 Phase

26. (A) 0 h, pattern A: compact, HSR near NE; (B) 6 h, pattern B: decondensed, ball-shaped HSR with internal fibrillar appearance; (C) 6 h, pattern C: linear HSR suggestive of intertwined chromatids; (D) 9 h, pattern D: linear HSR with parallel chromatids; (E) 9 h, showing larger condensed mass in late G2/early prophase nucleus; and (F) 9 h, prophase HSR with extended, parallel chromatids. Bar, 2  m. Li et al JCB 1998, Figure 4. A03_1 HSR changes during S through G2.

27. Li et al JCB 1998, Figure 4. A03_1 HSR changes during S through G2. Merged lac repressor (red) and BrdU pulse label (green) images.

28. Li et al JCB 1998, Table III. Statistics Relating A03_1 HSR Conformation, Intranuclear Position, and HSR DNA Replication

29. Belmont, TRENDS in Cell Biology 2001, 11 250-257; Figure 7. Direct observation of a translocation of HSR. (a–f) Images represent specific time points, measured from release from an early S-phase block. (a–f) correspond to t = 1, 5, 5.5, 6, 9 and 9.5 hrs, respectively.

30. Li et al JCB 1998, Figure 4. Figure 7. In vivo dynamics of A03_1 HSR.

31. Li et al JCB 1998, Figure 9. Summary of changes in A03_1 HSR conformation and intranuclear position as a function of cell cycle progression.

32. Major conclusions of Li et al 1998 1) Lac-operator is amplified within a late replicating homogeneously staining ~90 Mb region (HSR) and is replicated in the late S-phase. 2) Intranuclear position and conformation of HSR is changed in early G1, mid to late S late G2 and M phases. While in G1, G2 and M phases these changes are related to mitotic condensation and cell division, in S-phase they coincide with replication of HSR.

33. Tsukamoto T, Hashiguchi N, Janicki SM, Tumbar T, Belmont AS, Spector DL. Visualization of gene activity in living cells. Nat Cell Biol. 2000 2:871-8.

34. 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 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

35. 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 direct visualization of gene and its product in living cells.

36. Tsukamoto et al Nat Cell Biol 2000, Figure 1.

37. 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.

38. 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

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

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

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

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

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

44. 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

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

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

47. 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 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