Relationship between nuclear volume and pairing.

<p>a, Rank-order plot of the percentage ± SD of large nuclei. A nuclei was considered large if its volume was at or greater than the 95^th percentile volume of control cells. X-axis denotes the RNAi target. <i>P</i> values were determined by an unpaired <i>t</i> test. Inset, the frequency of single-signal nuclei was plotted against the frequency of large nuclei. The coefficient of determination <i>R²</i> is a measure of how well the data fit a linear regression, with values close to or exactly one representing a perfect fit. As <i>R²</i> = 0.004, there is no significant correlation between the percentages of paired nuclei and large nuclei. A minimum number of 250 nuclei were scored for each dsRNA. b, The number of FISH signals was plotted against the volume of each nucleus following <i>borr</i> and <i>scra</i> RNAi. No correlation was found between the degree of unpairing (number of FISH signals) and the size of the nuclei.</p

RNAi of a subset of pairing promoters causes Cap-H2–dependent pairing disruption.

<p>a, Representative FISH images are shown for RNAi knockdown of candidate pairing promoters <i>slmb</i> and <i>pav</i>, where the number of single-signal dodeca FISH signals per nucleus (noted) is decreased as compared to <i>lacZ</i> RNAi control (<i>P</i><0.05). Co-depletion of Cap-H2 increases the number of single-signal dodeca FISH signals per nucleus (<i>P</i><0.05 compared to <i>slmb</i> and <i>pav</i> RNAi alone). <i>pav</i> RNAi also produces multi-nucleated cells and large nuclei (hashed circles), characteristic of cytokinesis defects that lead to aneuploidy, which are also observed following <i>pav cap-H2</i> double RNAi treatment. n denotes number of nuclei scored. Scale bars equal 5 µm. Also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002667#pgen-1002667-t001" target="_blank">Table 1</a>. b, Relative frequencies of interhomolog distances (unpaired = two signals >1.0 µm apart) based on dodeca FISH ± SD for three tests. Cap-H2 co-depletion also reduces the distances between signals following <i>slmb</i> and <i>pav</i> RNAi (<i>P</i><0.05), another indication that pairing is restored. c, The percentage of large nuclei ± SD following <i>pav</i> and <i>polo</i> RNAi in the presence and absence of <i>cap-H2</i> RNAi. Although the frequency of large nuclei in <i>pav cap-H2</i> is significantly reduced as compared to that of <i>pav</i> (<i>P</i> = 0.0072), both were significantly increased compared to controls (<i>P</i><0.0001). The frequency of large nuclei in <i>polo cap-H2</i> was not significantly different as compared to that of <i>polo</i> (<i>P</i> = 0.3791). A minimum number of 500 nuclei were scored for each experiment.</p

Pairing models involving both candidate pairing promoting and anti-pairing factors.

<p>a, Summary of candidate pairing factors identified in the screen. Green boxes denote candidate pairing promoters (hatched green were those identified only in the primary screen) and red boxes denote candidate anti-pairers. A representative sampling of pairing promoters were tested (italicized) and found (asterisk) to be important for euchromatic pairing. Proteins are grouped based on either a known function or localization pattern. Candidate pairing promoters found to elicit RNAi phenotypes dependent on Cap-H2 are presented as potential condensin II regulators (question marks). Note one dsRNA targets both CG42550 and CG14463 (separated by comma). b, Model for how compaction and intrachromosomal interactions compete with homolog pairing. Although all chromosomal regions may transiently unpair prior to or during S-phase, homolog pairing (red circles) of heterochromatic centromeric regions (grey lines) may be in competition with intrachromosomal interactions (black circles), causing pairing to occur less often, more slowly, or with less stability than homolog pairing of less compacted euchromatic regions (blue lines), where the paucity of repeated sequences reduces the likelihood of intrachromosomal interactions. This figure is not meant to imply a causal or dependent relationship between heterochromatic and euchromatic pairing, although such a relationship may exist. c, Model for pairing through the cell cycle. Proper spindle formation and chromosome segregation during anaphase/telophase of mitosis may bundle centromeric heterochromatic regions to spindle poles and directly facilitate or accelerate homolog recognition. Such interactions would then be maintained through G1. During S-phase, however, the pairing of regions is perhaps more dynamic, becoming antagonized and then re-paired subsequently. In this case, not all pairing interactions would be reestablished until the following mitosis. Euchromatic pairing is not depicted.</p

RNAi of candidate pairing promoters disrupts pairing.

<p>a, Representative FISH images are shown for RNAi knockdown of candidate pairing promoters (<i>slmb</i>, <i>lin19</i>, <i>shtd</i>, <i>klp61f</i>, <i>dhc64C</i>, <i>mcph1</i>, <i>borr</i>), where the number of FISH signals per nucleus is increased compared to control. The percentage of single-signal nuclei is noted for both 359 and dodeca. n denotes number of nuclei scored. Scale bars equal 5 µm. b, Relative frequencies of interhomolog distances (unpaired = two signals >1.0 µm apart) based on dodeca FISH ± SD for three tests. dsRNA targets are either grouped based on known interactions (SCF, APC, CPC) or localization patterns of the proteins they encode (MTOC). All significantly reduced the percentage of paired nuclei compared to control (<i>P</i><0.05). c, Chromosomal targets of euchromatic FISH probes 16E and 28B and graph displaying the percentage of single-signal nuclei ± SD following RNAi. Asterisks denote a significant reduction from control (<i>P</i><0.05). A minimum number of 100 nuclei were scored for each dsRNA.</p

RNAi of candidate anti-pairers enhances heterochromatic pairing frequencies.

<p>a, Representative FISH images are shown for RNAi depletion of anti-pairers (<i>cdk8</i>, <i>cap-H2</i>, and <i>orc1</i>), where the number of FISH signals per nucleus is decreased as compared to that of control. Each resulted in a significant increase in the percentage of single-signal nuclei (noted) for both 359 and dodeca (<i>P</i><0.05). n denotes number of nuclei scored. Scale bars equal 5 µm. b, FACS plot (upper) of Kc₁₆₇ cells sorted into G1, early S (S1), late S (S2), and G2/M subpopulations and the percentage of nuclei producing a single FISH signal ± SD when targeting 359, AACAC, and dodeca in each. <i>P</i> values were determined by an unpaired <i>t</i> test. A minimum number of 100 nuclei were scored for each subpopulation. c, Example of a nucleus in which inter-signal distances were measured. Dot-plot displays the average inter-signal distances per nucleus ± the standard error of the mean (SEM). <i>Cap-H2</i>, <i>ORC1</i> and <i>lacZ</i> RNAi results are noted for reference and red box denotes hits that exhibited a significant shift in the distances per nucleus within the population (<i>P</i><0.01) based on an unpaired <i>t</i> test with unequal variance. Insets, relative frequencies of inter-signal distances following <i>Cap-H2</i> and <i>ORC1</i> RNAi compared to a <i>lacZ</i> RNAi control. d, Representative FISH images of a nucleus that produced a single signal for each probe (paired) and a nucleus with partially or fully overlapped 359 and dodeca signals (clustered). No significant difference in clustering levels was observed by this assay following depletion of any anti-pairer as compared to control. Graph displays results for the 16 candidate anti-pairers found to produce a significant reduction in inter-signal distances following RNAi in c (red box). A minimum number of 300 nuclei were scored for each dsRNA.</p

Solvent-Dependent Pyranopterin Cyclization in Molybdenum Cofactor Model Complexes

The conserved pterin dithiolene ligand that coordinates molybdenum (Mo) in the cofactor (Moco) of mononuclear Mo enzymes can exist in both a tricyclic pyranopterin dithiolene form and as a bicyclic pterin–dithiolene form as observed in protein crystal structures of several bacterial molybdoenzymes. Interconversion between the tricyclic and bicyclic forms via pyran scission and cyclization has been hypothesized to play a role in the catalytic mechanism of Moco. Therefore, understanding the interconversion between the tricyclic and bicyclic forms, a type of ring–chain tautomerism, is an important aspect of study to understand its role in catalysis. In this study, equilibrium constants (<i>K</i>_eq) as well as enthalpy, entropy, and free energy values are obtained for pyran ring tautomerism exhibited by two Moco model complexes, namely, (Et₄N)­[Tp*Mo­(O)­(S₂BMOPP)] (<b>1</b>) and (Et₄N)­[Tp*Mo­(O)­(S₂PEOPP)] (<b>2</b>), as a solvent-dependent equilibrium process. <i>K</i>_eq values obtained from ¹H NMR data in seven deuterated solvents show a correlation between solvent polarity and tautomer form, where solvents with higher polarity parameters favor the pyran form