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Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope.

Backlund MP, Joyner R, Weis K, Moerner WE - Mol. Biol. Cell (2014)

Bottom Line: As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes.This relative increase has potentially important biological implications, as it might suggest coupling via shared silencing factors or association with decoupled machinery upon activation.We also found that on the time scale studied (∼0.1-30 s), the loci moved with significantly higher subdiffusive mean square displacement exponents than previously reported, which has implications for the application of polymer theory to chromatin motion in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305.

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Distributions of interlocus distances for each condition. (A) PDFs with 100-nm binning. (B) CDFs with 100-nm binning. Dashed lines give ± SEM for each bin as determined from 100 bootstrapped samples.
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Figure 3: Distributions of interlocus distances for each condition. (A) PDFs with 100-nm binning. (B) CDFs with 100-nm binning. Dashed lines give ± SEM for each bin as determined from 100 bootstrapped samples.

Mentions: Cis-acting regulatory elements were recently reported to induce clustering of distinct gene loci in yeast (Brickner et al., 2012). It was therefore of interest to investigate whether transcriptional activation of the GAL loci may induce a similar change in the 3D distance between the two loci in diploid yeast. To test this hypothesis, we computed the time average of the 3D interlocus distance over the first 10 frames (when signal is highest) of each track pair. These values were then binned to produce the distributions shown in Figure 3. Figure 3A shows the probability density functions (PDFs), and Figure 3B shows the associated cumulative distribution functions (CDFs) under each condition. The narrowly peaked PDF of the cross-talk experiment (black) gives the lower limit for our estimates of interlocus distance set by localization and registration errors. The mean of this distribution is <Rcross-talk> = 48 ± 2 nm (mean ± SEM determined from 100 bootstrapped samples), which is on the order of what is expected from the approximate localization errors given in the previous section. The two DLSC cases both peak to the right of this limit, in the expected order (mean and SEM rounded to nearest 10 nm): <RDLSC-30 kbp> = 300 ± 20 nm and <RDLSC-108 kbp> = 430 ± 40 nm. A previous study using fluorescence in situ hybridization (FISH) found results similar to our DLSC-108 kbp case for yeast loci separated by a similar genomic distance along chromosome VI (436 nm for 103-kbp separation; Bystricky et al., 2004). However, a pair of loci separated by 30 kbp in Bystricky et al. (2004) was reported to have an average Euclidean separation of ∼160–190 nm, somewhat smaller than the DLSC-30 kbp case we found here. Possible reasons for this discrepancy are several. First, the FISH portion of their study reported distances from a single 2D confocal section. This procedure could cause deflation of the true Euclidean value, since the z-extent of a confocal section (i.e., depth of focus, ∼500–700 nm) is nonzero due to diffraction. Second, the differences in labels may contribute, since the TetO and LacO inserts themselves have a nonnegligible length and could conceivably affect the compaction of the chromatin differently than the FISH probes used in Bystricky et al. (2004). Note that this finite label size and integration method also makes our estimates of minimal genomic distance approximate, which should discourage overinterpretation of this aspect of our results beyond a validation of the two-color DH-PSF microscope in exploring the flexibility of the chromatin fiber. We also note that residual registration and localization error will falsely inflate the computed distances and that this error will have a greater relative effect on more closely separated loci. Finally, the difference may be explained in part by heterogeneity of chromatin compaction. The relevant portion of Bystricky et al. (2004) only dealt with chromosomes V, VI, and XIV, whereas we looked at chromosome II. In any case, it is interesting that the distribution of RDLSC-108 kbp is noticeably broader than that of RDLSC-30 kbp. As the distance between loci on the same chromatin fiber becomes significantly larger than the persistence length of the polymer (170–220 nm according to Bystricky et al., 2004), the floppier section can access more conformations covering larger separations.


Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope.

Backlund MP, Joyner R, Weis K, Moerner WE - Mol. Biol. Cell (2014)

Distributions of interlocus distances for each condition. (A) PDFs with 100-nm binning. (B) CDFs with 100-nm binning. Dashed lines give ± SEM for each bin as determined from 100 bootstrapped samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230621&req=5

Figure 3: Distributions of interlocus distances for each condition. (A) PDFs with 100-nm binning. (B) CDFs with 100-nm binning. Dashed lines give ± SEM for each bin as determined from 100 bootstrapped samples.
Mentions: Cis-acting regulatory elements were recently reported to induce clustering of distinct gene loci in yeast (Brickner et al., 2012). It was therefore of interest to investigate whether transcriptional activation of the GAL loci may induce a similar change in the 3D distance between the two loci in diploid yeast. To test this hypothesis, we computed the time average of the 3D interlocus distance over the first 10 frames (when signal is highest) of each track pair. These values were then binned to produce the distributions shown in Figure 3. Figure 3A shows the probability density functions (PDFs), and Figure 3B shows the associated cumulative distribution functions (CDFs) under each condition. The narrowly peaked PDF of the cross-talk experiment (black) gives the lower limit for our estimates of interlocus distance set by localization and registration errors. The mean of this distribution is <Rcross-talk> = 48 ± 2 nm (mean ± SEM determined from 100 bootstrapped samples), which is on the order of what is expected from the approximate localization errors given in the previous section. The two DLSC cases both peak to the right of this limit, in the expected order (mean and SEM rounded to nearest 10 nm): <RDLSC-30 kbp> = 300 ± 20 nm and <RDLSC-108 kbp> = 430 ± 40 nm. A previous study using fluorescence in situ hybridization (FISH) found results similar to our DLSC-108 kbp case for yeast loci separated by a similar genomic distance along chromosome VI (436 nm for 103-kbp separation; Bystricky et al., 2004). However, a pair of loci separated by 30 kbp in Bystricky et al. (2004) was reported to have an average Euclidean separation of ∼160–190 nm, somewhat smaller than the DLSC-30 kbp case we found here. Possible reasons for this discrepancy are several. First, the FISH portion of their study reported distances from a single 2D confocal section. This procedure could cause deflation of the true Euclidean value, since the z-extent of a confocal section (i.e., depth of focus, ∼500–700 nm) is nonzero due to diffraction. Second, the differences in labels may contribute, since the TetO and LacO inserts themselves have a nonnegligible length and could conceivably affect the compaction of the chromatin differently than the FISH probes used in Bystricky et al. (2004). Note that this finite label size and integration method also makes our estimates of minimal genomic distance approximate, which should discourage overinterpretation of this aspect of our results beyond a validation of the two-color DH-PSF microscope in exploring the flexibility of the chromatin fiber. We also note that residual registration and localization error will falsely inflate the computed distances and that this error will have a greater relative effect on more closely separated loci. Finally, the difference may be explained in part by heterogeneity of chromatin compaction. The relevant portion of Bystricky et al. (2004) only dealt with chromosomes V, VI, and XIV, whereas we looked at chromosome II. In any case, it is interesting that the distribution of RDLSC-108 kbp is noticeably broader than that of RDLSC-30 kbp. As the distance between loci on the same chromatin fiber becomes significantly larger than the persistence length of the polymer (170–220 nm according to Bystricky et al., 2004), the floppier section can access more conformations covering larger separations.

Bottom Line: As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes.This relative increase has potentially important biological implications, as it might suggest coupling via shared silencing factors or association with decoupled machinery upon activation.We also found that on the time scale studied (∼0.1-30 s), the loci moved with significantly higher subdiffusive mean square displacement exponents than previously reported, which has implications for the application of polymer theory to chromatin motion in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus