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Comparing the Assembly and Handedness Dynamics of (H3.3-H4)2 Tetrasomes to Canonical Tetrasomes.

Vlijm R, Lee M, Ordu O, Boltengagen A, Lusser A, Dekker NH, Dekker C - PLoS ONE (2015)

Bottom Line: Recent findings that H3.3-containing nucleosomes result in less stable and less condensed chromatin further underline the need to study the microscopic underpinnings of H3.3-containing tetrasomes and nucleosomes.We also examine the effect of free NAP1, H3.3, and H4 in solution on flipping behavior and conclude that the probability for a tetrasome to occupy the left-handed state is only slightly enhanced by the presence of free protein.These data demonstrate that the incorporation of H3.3 does not alter the structural dynamics of tetrasomes, and hence that the preferred incorporation of this histone variant in transcriptionally active regions does not result from its enhanced ability to accommodate torsional stress, but rather may be linked to specific chaperone or remodeler requirements or communication with the nuclear environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.

ABSTRACT
Eukaryotic nucleosomes consists of an (H3-H4)2 tetramer and two H2A-H2B dimers, around which 147 bp of DNA are wrapped in 1.7 left-handed helical turns. During chromatin assembly, the (H3-H4)2 tetramer binds first, forming a tetrasome that likely constitutes an important intermediate during ongoing transcription. We recently showed that (H3-H4)2 tetrasomes spontaneously switch between a left- and right-handed wrapped state of the DNA, a phenomenon that may serve to buffer changes in DNA torque induced by RNA polymerase in transcription. Within nucleosomes of actively transcribed genes, however, canonical H3 is progressively replaced by its variant H3.3. Consequently, one may ask if and how the DNA chirality dynamics of tetrasomes is altered by H3.3. Recent findings that H3.3-containing nucleosomes result in less stable and less condensed chromatin further underline the need to study the microscopic underpinnings of H3.3-containing tetrasomes and nucleosomes. Here we report real-time single-molecule studies of (H3.3-H4)2 tetrasome dynamics using Freely Orbiting Magnetic Tweezers and Electromagnetic Torque Tweezers. We find that the assembly of H3.3-containing tetrasomes and nucleosomes by the histone chaperone Nucleosome Assembly Protein 1 (NAP1) occurs in an identical manner to that of H3-containing tetrasomes and nucleosomes. Likewise, the flipping behavior of DNA handedness in tetrasomes is not impacted by the presence of H3.3. We also examine the effect of free NAP1, H3.3, and H4 in solution on flipping behavior and conclude that the probability for a tetrasome to occupy the left-handed state is only slightly enhanced by the presence of free protein. These data demonstrate that the incorporation of H3.3 does not alter the structural dynamics of tetrasomes, and hence that the preferred incorporation of this histone variant in transcriptionally active regions does not result from its enhanced ability to accommodate torsional stress, but rather may be linked to specific chaperone or remodeler requirements or communication with the nuclear environment.

No MeSH data available.


Related in: MedlinePlus

NAP1-assisted (H3.3-H4)2 tetrasome assembly.(A) Schematic of the in vitro assay showing a single DNA molecule (blue) tethered between a glass surface and a paramagnetic bead. The circular magnet above the bead applies a stretching force to the bead (and hence to the DNA), but leaves it free to rotate about the DNA-tether axis. A nonmagnetic reference bead is fixed to the surface to allow for drift correction. After flushing in NAP1 preincubated with histones H3.3-H4, tetrasomes are loaded onto the DNA. (B) Time-dependence of the end-to-end length z (μm) (left) of a single DNA tether during the assembly of two (H3.3-H4)2 tetrasomes. The step sizes are -25 and -27 ± 5 nm. The green arrow at t = 420 s indicates the flushing in of the proteins. Data was acquired at 100 Hz, and red lines indicate the mean values of each assembly step. The histogram on the right derives from 19 independent assembly experiments (69 steps). A Gaussian fit shows that the average step in z during tetramer assembly is -25 ± 6 nm. (C) Time-dependence of bead rotations θ (turns) (left) of the same DNA tether as in B). Compaction of the DNA (shown in B)) occurs concurrently with a change in linking number (changes in θ). The step sizes in θ are -1.17 ± 0.24 and -0.97 ± 0.24 turns. The green arrow at t = 420 s indicates the flushing in of the proteins. Data was acquired at 100 Hz, and red lines indicate the mean values of each assembly step. The histogram on the right derives from 15 independent assembly experiments (23 steps). It can be fitted to Gaussian peaks. The most likely step in θ during tetramer assembly is -0.8 ± 0.1 turns. A small number of steps appears to result from the simultaneous assembly of two tetramers, with a mean step size in θ of -1.9 ± 0.1 turns. (D) The total degree of compaction (Δz) plotted versus the total change in linking number (Δθassembly) on 25 individual DNA molecules following the assembly of tetrasomes (black squares). Fits to a linear relationship yield Δz/Δθassembly = 32 ± 2 nm (solid red line).
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pone.0141267.g002: NAP1-assisted (H3.3-H4)2 tetrasome assembly.(A) Schematic of the in vitro assay showing a single DNA molecule (blue) tethered between a glass surface and a paramagnetic bead. The circular magnet above the bead applies a stretching force to the bead (and hence to the DNA), but leaves it free to rotate about the DNA-tether axis. A nonmagnetic reference bead is fixed to the surface to allow for drift correction. After flushing in NAP1 preincubated with histones H3.3-H4, tetrasomes are loaded onto the DNA. (B) Time-dependence of the end-to-end length z (μm) (left) of a single DNA tether during the assembly of two (H3.3-H4)2 tetrasomes. The step sizes are -25 and -27 ± 5 nm. The green arrow at t = 420 s indicates the flushing in of the proteins. Data was acquired at 100 Hz, and red lines indicate the mean values of each assembly step. The histogram on the right derives from 19 independent assembly experiments (69 steps). A Gaussian fit shows that the average step in z during tetramer assembly is -25 ± 6 nm. (C) Time-dependence of bead rotations θ (turns) (left) of the same DNA tether as in B). Compaction of the DNA (shown in B)) occurs concurrently with a change in linking number (changes in θ). The step sizes in θ are -1.17 ± 0.24 and -0.97 ± 0.24 turns. The green arrow at t = 420 s indicates the flushing in of the proteins. Data was acquired at 100 Hz, and red lines indicate the mean values of each assembly step. The histogram on the right derives from 15 independent assembly experiments (23 steps). It can be fitted to Gaussian peaks. The most likely step in θ during tetramer assembly is -0.8 ± 0.1 turns. A small number of steps appears to result from the simultaneous assembly of two tetramers, with a mean step size in θ of -1.9 ± 0.1 turns. (D) The total degree of compaction (Δz) plotted versus the total change in linking number (Δθassembly) on 25 individual DNA molecules following the assembly of tetrasomes (black squares). Fits to a linear relationship yield Δz/Δθassembly = 32 ± 2 nm (solid red line).

Mentions: We directly monitored tetrasome formation upon flushing in H3.3 and H4 pre-incubated with the histone chaperone NAP1 into the flow cell using FOMT, a technique that allows one to simultaneously measure dynamical changes in the end-to-end length and linking number of single tethered DNA molecules. In this approach, a vertically oriented magnetic field is used to apply a stretching force, without constraining the free rotation of the DNA molecule (Fig 2A). The DNA molecules employed (1.9 kbp in length) did not contain specific nucleosome-positioning sequences. We limited the applied stretching force to 0.8 pN, well below the 3 pN above which DNA begins to peel off from the nucleosome [36]. Upon flushing in NAP1/histone complexes, this experimental configuration allowed us to observe a distinct, stepwise decrease in the end-to-end length z of the DNA, indicating compaction, accompanied by a clockwise rotation θ of the bead, reflecting a decrease in the linking number of the DNA tether (left panels in Fig 2B and 2C). From several independent (H3.3-H4)2 assembly experiments, we obtained an average extension change <Δz> = -25 ± 6 nm (Fig 2B, right) and linking number change <Δθassembly> = -0.8 ± 0.2 turns (Fig 2C, right). By changing the histone concentration, we assembled varying numbers of tetrasomes per DNA molecule. The total degree of compaction Δz and the overall change in linking number Δθassembly following assembly were found to be linearly correlated with a slope Δz/Δθassembly of 32 ± 2 nm/turn (Fig 2D).


Comparing the Assembly and Handedness Dynamics of (H3.3-H4)2 Tetrasomes to Canonical Tetrasomes.

Vlijm R, Lee M, Ordu O, Boltengagen A, Lusser A, Dekker NH, Dekker C - PLoS ONE (2015)

NAP1-assisted (H3.3-H4)2 tetrasome assembly.(A) Schematic of the in vitro assay showing a single DNA molecule (blue) tethered between a glass surface and a paramagnetic bead. The circular magnet above the bead applies a stretching force to the bead (and hence to the DNA), but leaves it free to rotate about the DNA-tether axis. A nonmagnetic reference bead is fixed to the surface to allow for drift correction. After flushing in NAP1 preincubated with histones H3.3-H4, tetrasomes are loaded onto the DNA. (B) Time-dependence of the end-to-end length z (μm) (left) of a single DNA tether during the assembly of two (H3.3-H4)2 tetrasomes. The step sizes are -25 and -27 ± 5 nm. The green arrow at t = 420 s indicates the flushing in of the proteins. Data was acquired at 100 Hz, and red lines indicate the mean values of each assembly step. The histogram on the right derives from 19 independent assembly experiments (69 steps). A Gaussian fit shows that the average step in z during tetramer assembly is -25 ± 6 nm. (C) Time-dependence of bead rotations θ (turns) (left) of the same DNA tether as in B). Compaction of the DNA (shown in B)) occurs concurrently with a change in linking number (changes in θ). The step sizes in θ are -1.17 ± 0.24 and -0.97 ± 0.24 turns. The green arrow at t = 420 s indicates the flushing in of the proteins. Data was acquired at 100 Hz, and red lines indicate the mean values of each assembly step. The histogram on the right derives from 15 independent assembly experiments (23 steps). It can be fitted to Gaussian peaks. The most likely step in θ during tetramer assembly is -0.8 ± 0.1 turns. A small number of steps appears to result from the simultaneous assembly of two tetramers, with a mean step size in θ of -1.9 ± 0.1 turns. (D) The total degree of compaction (Δz) plotted versus the total change in linking number (Δθassembly) on 25 individual DNA molecules following the assembly of tetrasomes (black squares). Fits to a linear relationship yield Δz/Δθassembly = 32 ± 2 nm (solid red line).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4623960&req=5

pone.0141267.g002: NAP1-assisted (H3.3-H4)2 tetrasome assembly.(A) Schematic of the in vitro assay showing a single DNA molecule (blue) tethered between a glass surface and a paramagnetic bead. The circular magnet above the bead applies a stretching force to the bead (and hence to the DNA), but leaves it free to rotate about the DNA-tether axis. A nonmagnetic reference bead is fixed to the surface to allow for drift correction. After flushing in NAP1 preincubated with histones H3.3-H4, tetrasomes are loaded onto the DNA. (B) Time-dependence of the end-to-end length z (μm) (left) of a single DNA tether during the assembly of two (H3.3-H4)2 tetrasomes. The step sizes are -25 and -27 ± 5 nm. The green arrow at t = 420 s indicates the flushing in of the proteins. Data was acquired at 100 Hz, and red lines indicate the mean values of each assembly step. The histogram on the right derives from 19 independent assembly experiments (69 steps). A Gaussian fit shows that the average step in z during tetramer assembly is -25 ± 6 nm. (C) Time-dependence of bead rotations θ (turns) (left) of the same DNA tether as in B). Compaction of the DNA (shown in B)) occurs concurrently with a change in linking number (changes in θ). The step sizes in θ are -1.17 ± 0.24 and -0.97 ± 0.24 turns. The green arrow at t = 420 s indicates the flushing in of the proteins. Data was acquired at 100 Hz, and red lines indicate the mean values of each assembly step. The histogram on the right derives from 15 independent assembly experiments (23 steps). It can be fitted to Gaussian peaks. The most likely step in θ during tetramer assembly is -0.8 ± 0.1 turns. A small number of steps appears to result from the simultaneous assembly of two tetramers, with a mean step size in θ of -1.9 ± 0.1 turns. (D) The total degree of compaction (Δz) plotted versus the total change in linking number (Δθassembly) on 25 individual DNA molecules following the assembly of tetrasomes (black squares). Fits to a linear relationship yield Δz/Δθassembly = 32 ± 2 nm (solid red line).
Mentions: We directly monitored tetrasome formation upon flushing in H3.3 and H4 pre-incubated with the histone chaperone NAP1 into the flow cell using FOMT, a technique that allows one to simultaneously measure dynamical changes in the end-to-end length and linking number of single tethered DNA molecules. In this approach, a vertically oriented magnetic field is used to apply a stretching force, without constraining the free rotation of the DNA molecule (Fig 2A). The DNA molecules employed (1.9 kbp in length) did not contain specific nucleosome-positioning sequences. We limited the applied stretching force to 0.8 pN, well below the 3 pN above which DNA begins to peel off from the nucleosome [36]. Upon flushing in NAP1/histone complexes, this experimental configuration allowed us to observe a distinct, stepwise decrease in the end-to-end length z of the DNA, indicating compaction, accompanied by a clockwise rotation θ of the bead, reflecting a decrease in the linking number of the DNA tether (left panels in Fig 2B and 2C). From several independent (H3.3-H4)2 assembly experiments, we obtained an average extension change <Δz> = -25 ± 6 nm (Fig 2B, right) and linking number change <Δθassembly> = -0.8 ± 0.2 turns (Fig 2C, right). By changing the histone concentration, we assembled varying numbers of tetrasomes per DNA molecule. The total degree of compaction Δz and the overall change in linking number Δθassembly following assembly were found to be linearly correlated with a slope Δz/Δθassembly of 32 ± 2 nm/turn (Fig 2D).

Bottom Line: Recent findings that H3.3-containing nucleosomes result in less stable and less condensed chromatin further underline the need to study the microscopic underpinnings of H3.3-containing tetrasomes and nucleosomes.We also examine the effect of free NAP1, H3.3, and H4 in solution on flipping behavior and conclude that the probability for a tetrasome to occupy the left-handed state is only slightly enhanced by the presence of free protein.These data demonstrate that the incorporation of H3.3 does not alter the structural dynamics of tetrasomes, and hence that the preferred incorporation of this histone variant in transcriptionally active regions does not result from its enhanced ability to accommodate torsional stress, but rather may be linked to specific chaperone or remodeler requirements or communication with the nuclear environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.

ABSTRACT
Eukaryotic nucleosomes consists of an (H3-H4)2 tetramer and two H2A-H2B dimers, around which 147 bp of DNA are wrapped in 1.7 left-handed helical turns. During chromatin assembly, the (H3-H4)2 tetramer binds first, forming a tetrasome that likely constitutes an important intermediate during ongoing transcription. We recently showed that (H3-H4)2 tetrasomes spontaneously switch between a left- and right-handed wrapped state of the DNA, a phenomenon that may serve to buffer changes in DNA torque induced by RNA polymerase in transcription. Within nucleosomes of actively transcribed genes, however, canonical H3 is progressively replaced by its variant H3.3. Consequently, one may ask if and how the DNA chirality dynamics of tetrasomes is altered by H3.3. Recent findings that H3.3-containing nucleosomes result in less stable and less condensed chromatin further underline the need to study the microscopic underpinnings of H3.3-containing tetrasomes and nucleosomes. Here we report real-time single-molecule studies of (H3.3-H4)2 tetrasome dynamics using Freely Orbiting Magnetic Tweezers and Electromagnetic Torque Tweezers. We find that the assembly of H3.3-containing tetrasomes and nucleosomes by the histone chaperone Nucleosome Assembly Protein 1 (NAP1) occurs in an identical manner to that of H3-containing tetrasomes and nucleosomes. Likewise, the flipping behavior of DNA handedness in tetrasomes is not impacted by the presence of H3.3. We also examine the effect of free NAP1, H3.3, and H4 in solution on flipping behavior and conclude that the probability for a tetrasome to occupy the left-handed state is only slightly enhanced by the presence of free protein. These data demonstrate that the incorporation of H3.3 does not alter the structural dynamics of tetrasomes, and hence that the preferred incorporation of this histone variant in transcriptionally active regions does not result from its enhanced ability to accommodate torsional stress, but rather may be linked to specific chaperone or remodeler requirements or communication with the nuclear environment.

No MeSH data available.


Related in: MedlinePlus