Figure 22
Spatial relation between different histone lysine methylation sites (green) and nascent RNA (red) visualized in DLD-1 cells. The graphs on the right of each image show the quantitative assessment of overlap between nascent-RNA (red) and each histone methylation site (green) as the percentage of the co-localizing volume for
each color channel. All images are deconvolved. Bar indicates 5µm
To check whether distinct histone lysine methylation sites can be assigned to transcriptionally active sites, the spatial relationship between different methylation sites (the same six sites already investigated together with centromeres) and nascent RNA was analyzed. Cells were pulse labeled by application of Br-UTP scratch transcription labeling (Schermelleh et al., 2001) as described in 3.3. Representative mid-sections of merged confocal images for each histone modification and nascent-RNA are shown in figure 22. Attached are their respective co-localization coefficients M1 (Br-UTP labeled nascent-RNA, red) and M2 (histone methylation, green) from 6-8 investigated nuclei. As was expected the highest co-localization coefficient with nascent-RNA was found for H3K4me3 (A) (M1=40%),
confirming its connection to actively transcribed chromatin. Nascent-RNA displayed similar overlap to H4K20me1/H3K9me1 (B, C) as well as H3K27me3 (D) (M1=17%). Very low co- localization coefficients were obtained for H3K9me3/H4K20me3 (E, F). This is as estimated because in constitutive heterochromatic regions only low transcriptional activity occurs.
4.1.4
Pattern formation in cycling and quiescent nuclei
While inspecting the histone methylation patterns in the epifluorescent microscope two different shaped pattern of H3K9me3 and H4K20me3 were eye-catching. The first idea was that the variable pattern formation depends on different cell cycle stages. MCF-7 cells providing the biggest and most distinct H4K20me3 antibody pattern were our choice for evaluation. Cycling MCF-7 cells were discriminated from quiescent cells by their positive pKi67 staining pattern. In both cycling and quiescent cells centromeres are in close contact and at least partially embedded in constitutive heterochromatin visualized by H4K20me3 staining. In quiescent cells the heterochromatic clusters appear bigger and in a more ring-like structure (23 B) compared to cycling cells (23 A). Additionally cycling cells displayed more foci throughout the nucleus and remote from centromeres. For the other antibody patterns no difference between cycling and quiescent cells was observable by visual inspection.
Figure 23
3D reconstructions of chromatin patterns after H4K20me3 immunostaining (green) together with centromeres (red) in a nucleus from a cycling MCF-7 cell (A) and from a quiescent cell (B). In both nuclei all centromeres are spatially associated to H4K20me3 stained chromatin clusters, but show only partial embedding. H4K20me3 clusters appear more dispersed in (A) compared to (B), where clusters frequently form ring-like structures. The
inset magnifications point out in more detail the characteristic structure and spatial relation between H4K20me3 and centromeres. The 3D reconstruction was made from deconvolved images. Bars indicate 5µm
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Figure 24
Comparison of pattern formation between nuclei of cycling (left panel, red) and of quiescent MCF-7 cells (mid panel, green) after immunostaining with H4K20me1 (A), H3K27me3 (B), H4K20me3 (C) and H3K9me3 (D). All
pixels above threshold are shown as red or green respectively.
The graphs of the right panel illustrate the results of the applied radial autocorrelation function (RAC) summarized from ten nuclei each. The x-axis denotes relative distance intervals of the normalized nuclear length, the y-axis the percentage of pairs of pixels that fall into each relative distance interval (red curves = cycling cells, green curves =quiescent cells).
Similar pixel distribution between cycling and quiescent cells was obtained for mono-H4K20 (A3). The curves of
the H3K27me3 signals show a slight increase of larger distances in quiescent cells (B3). Differences in the small
distances are observed for H4K20me3 and more pronounced for H3K9me3 stained chromatin between the red and the green curves (C3 and D3). The sharp peaks and consecutive drops of the green curves (marked with asterisks in C3 and D3) in contrast to the smoother red curves reflect the increase of small relative distances in
together with the lack of relative distances > 0.8 indicates that the nuclear periphery is devoid of these big clusters. All images are deconvolved. Bar indicates 5µm.
As evaluation method to assess the difference between cycling and quiescent cells the radial autocorrelation (RAC) function analysis described by Walter et al., (Walter et al., 2003) (see methods and protocols 3.14.5) was applied. The degree of clustering was evaluated for the following histone methylation sites: H4K20me1, H3K9me3, H3K27 and H4K20 (figure 24). Each of the four modifications in nuclei from ten cycling and ten quiescent MCF-7 cells was investigated. Therefore projections were made from five subsequent optical sections representing about 1µm of the middle part of a given nucleus. After threshold was set manually and normalization of the size of these projections was performed, 2D distance measurements were carried out between all possible pairs of pixels representing a distinct histone modification. Distance values were grouped into 50 intervals of increasing relative distances (corresponding to approximately 200-300nm for each interval). RAC was established as a measure of the frequencies of pairs of pixels belonging to each interval. figure 24 (right panel) illustrates the results of RAC analysis summarized from ten nuclei each.
As expected already from judgement by eye, no difference between cycling (A1, red) and quiescent (A2, green) cells was detectable as reflected by the almost identical curves in the graph A3. For H3K27me3 (B1 and B2) a small shift of the staining signals towards the nuclear periphery in quiescent cells seems to occur (B3). For both H3K9me3 and H4K20me3 an increase in smaller distances was discovered. This result is mirrored in the respective graphs (C3 and D3) by the sharp peaks followed immediate by clear drops (marked by asterisks) and in contrast to the smoother curves shown for cycling cells.
4.1.5 Interrelationship of different lysine methylation sites
To test whether patterns of different histone methylation sites represent distinct nuclear zones, combinations of antibodies were performed by double immunostaining experiments (figure 25). Apart from that the following questions were addressed: share H3K9me3 and H4K20me3 which both represent constitutive heterochromatin the same 3D nuclear topology and can H4K20me1 be verified as a marker for active chromatin by co-localization with H3K4me3? Both pairs of these histone modifications displayed globally similar distribution patterns in single staining experiments (compare with figures 19 and 20). To compare histone modifications pairwise and directly in individual DLD-1 nuclei, a double immunostaining protocol had to be applied. This was necessary because all used primary