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

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.

真核核小体（eukaryotic nucleosomes）由一个(H3-H4)2四聚体与两个H2A-H2B二聚体构成，其外围以1.7个左手螺旋圈缠绕147 bp的DNA。在染色质组装过程中，(H3-H4)2四聚体率先结合DNA，形成四聚体（tetrasome），该结构可能是活跃转录进程中的关键中间体。我们此前的研究显示，(H3-H4)2四聚体可在DNA左手缠绕与右手缠绕状态间自发切换，这一现象或可缓冲转录过程中RNA聚合酶诱导的DNA扭矩变化。然而，在活跃转录基因的核小体中，经典组蛋白H3会逐步被其变体H3.3所替代。据此我们不禁发问：H3.3是否会改变四聚体的DNA手性动力学？若会，具体机制又是什么？近期研究发现，包含H3.3的核小体所形成的染色质稳定性更弱、压缩程度更低，这进一步凸显了研究含H3.3四聚体与核小体微观机制的必要性。本研究借助自由悬浮磁镊（Freely Orbiting Magnetic Tweezers）与电磁扭矩镊（Electromagnetic Torque Tweezers），开展了(H3.3-H4)2四聚体动力学的实时单分子研究。我们发现，组蛋白伴侣核小体组装蛋白1（Nucleosome Assembly Protein 1, NAP1）介导含H3.3的四聚体与核小体的组装过程，其机制与含经典H3的四聚体和核小体完全一致。同样，四聚体中DNA手性的翻转行为亦不受H3.3存在的影响。我们还探究了溶液中游离的NAP1、H3.3与H4对该翻转行为的影响，并得出结论：游离蛋白的存在仅会轻微提升四聚体处于左手缠绕状态的概率。上述数据表明，H3.3的掺入并不会改变四聚体的结构动力学。因此，该组蛋白变体在转录活跃区域的偏好性掺入，并非源于其更强的扭转应力容纳能力，而是可能与特定的组蛋白伴侣或染色质重塑因子需求，或是与核内环境的信号通讯相关。