Session 6 (4/21/09) Dynamics of Genomic Organization & Function in Living Cells I Papers covered: 1.Zink et al., Hum Genetics 102 (1998)24 – Pliss et al., Chromosoma, 2009

Domains)

Double Labeling of DNA During Replication. Ma et al J Cell Biol : 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

Visualization of Replication Sites (Fluorescence and Electron Microscopy) Early SMid SLate S

Zink et al., Hum Genetics 102 (1998) 24 – 51. Structure and dynamics of human interphase chromosome territories in vivo

Major conclusions of Zink et al., ) 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).

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

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: 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.

Zink et al, Hum Genet, 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.

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.

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

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.

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

Conclusions of Zink et al., ) 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).

Chromatin dynamics is correlated with replication timing Pliss A, Malyavantham K, Bhattacharya S, Zeitz M, Berezney R. Chromosoma Mar 19.

Major Conclusions of Pliss et al., Chromosoma )Chromatin configuration includes both discrete domains and “relaxed” or “diffuse” pool (Fig 1 & 2) 2)Chromatin dynamics includes both linear motion of chromatin domains and transformations of configuration (Fig 1 & 2) 3)Both these parameters of chromatin dynamics are significantly higher in chromatin replicated in early S then in mid and late S-phase (Fig 2 & 3). 4)Motion of chromatin is in complex relation with gene expression (Fig 4)

Conclusion 1 Chromatin configuration includes both discrete domains and “relaxed” or “diffuse” pool (Fig 1 & 2) Conclusion 2 Chromatin dynamics includes both linear motion of chromatin domains and transformations of configuration (Fig 1 & 2)

Fig. 1(a) Early S-phase labeled chromatin in living HeLa cells 3 days following microinjection of Cy3- dUTP. Left: 2D maximal intensity projection of a deconvolved z-stack of optical sections acquired with an inverted fluorescence microscope. Right: a single confocal section.

Fig. 1(b)Comparison of the dynamics of early, mid, and late S-phase replicating chromatin domains

Fig 1(c) Chromatin fluctuates between discrete and diffuse configurations in live cells. HeLa cell labeled in early S-phase was acquired in time-lapse mode and a representative area was cropped.

Conclusion 3 Both chromatin linear motion and reconfigurations are significantly higher in chromatin replicated in early S then in mid and late S-phase (Fig 2 & 3).

Fig 2(a) Schematic illustrating the segregation of replication labeled chromatin. 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.

Fig 2 (b) Chromosome territory exhibiting signal for both early and mid S-replicating chromatin domains (ChrD) (red and green channels, respectively)acquired with 3-min intervals (upper row). Second and fourth row: mid and early S-replicating ChrD, respectively, were segmented, corrected for cell movement, and merged. Earlier time point contours (red), subsequent time point (green). Third and fifth rows: enlarged centers of gravity (centroids) of contoured early and mid S replicating ChrD at consecutive 3-min intervals were merged as described above. Centroids of early (row 5, right panel) and mid S (row 3, right panel) replicating ChrD at 0 and 12 min were merged. Corresponding centroids that shifted 4 pixels (~0.3 μm) or less were colored white. (c) White outlined area on the upper row was enlarged for each channel individually to show pronounced differences in the degree of configuration and position changes of early versus mid S-labeled ChrD.

Fig. 3 Comparison of the average translation motion of early, mid, and late S-phase replicating chromatin domains (ChrD).

Fig 3 (e, f ) Effect of transcription inhibitors on chromatin dynamics. (e) Translational motion of the early S-replicated ChrD is significantly reduced by actinomycin D treatment with no significant reduction by α- amanitin. (f ) Time-lapse analysis for motion following actinomycin D treatment.

Conclusion 4 Motion of chromatin is in complex relation with gene expression (Fig 4)

Fig. 4 Visualization of the transcription sites, cDNA, and early S-labeled chromatin indicates association of transcription with relaxed chromatin surrounding the discrete ChrD.

BrdU Pulse 25 min, then chase 150 min; BrdU green channel, cDNA red channel

Major Conclusions of Pliss et al., Chromosoma )Chromatin configuration includes both discrete domains and “relaxed” or “diffuse” pool (Fig 1 & 2) 2)Chromatin dynamics includes both linear motion of chromatin domains and transformations of configuration (Fig 1 & 2) 3)Both these parameters of chromatin dynamics are significantly higher in chromatin replicated in early S then in mid and late S-phase (Fig 2 & 3). 4)Motion of chromatin is in complex relation with gene expression (Fig 4)

Background & Figures for: Phair & Misteli Nature (2000), 404: High mobility of proteins in the mammalian cell nucleus

Mapping of the DNA and proteins in the living cells: Immunocytochemical tools: Labeling with antibodies at both light and electron microscopy level (proteins, nascent DNA and RNA in fixed cells) FISH approach (specific DNA and RNA sequences in fixed cells) Fluorescent proteins fusions (GFP, EGFP, CFP, YFP etc in both fixed and live cells)

Major Conclusions of Phair and Misteli, ) GFP constructs of SF2/ASF, fibrillarin, and HMG-17, co-localize with endogenous proteins and apparently their function is not altered 2) FRAP and FLIP experiments indicate energy independent high mobility for SF2/ASF, fibrillarin, and HMG-17 proteins. FLIP experiments also demonstrate the absence of long term associations of these molecules with various nuclear compartments

Conclusion 1, Figure 1 GFP constructs of SF2/ASF, fibrillarin, and HMG-17, co-localize with endogenous proteins and apparently their function is not altered

Phair and Misteli 2000, Figure 1, Colocalization of GFP-fusion constructs with endogenous proteins.

Conclusion 2, Figure FRAP and FLIP experiments indicate energy independent high mobility for SF2/ASF, fibrillarin, and HMG-17 proteins. FLIP experiments also demonstrate the absence of long term associations of these molecules with various nuclear compartments

FRAP FLIP Schematic representation of FRAP and FLIP techniques

Phair and Misteli 2000, Figure 2, FRAP of nucleoplasmic regions and kinetics of recovery GFP-HMG-17 GFP-SF2/ASF GFP-Fibrillarin

Phair and Misteli 2000, Figure 2 cont, FRAP of nucleoplasmic regions and kinetics of recovery

Phair and Misteli 2000, Figure 3 FRAP of nuclear compartments and kinetics of recovery GFP-SF2/ASF GFP-Fibrillarin

Phair and Misteli 2000, Figure 4 FLIP after bleaching of nucleoplasmic area

Major Conclusions of Phair and Misteli, ) GFP constructs of SF2/ASF, fibrillarin, and HMG-17, co-localize with endogenous proteins and apparently their function is not altered 2) FRAP and FLIP experiments indicate energy independent high mobility for SF2/ASF, fibrillarin, and HMG-17 proteins. FLIP experiments also demonstrate the absence of long term associations of these molecules with various nuclear compartments