Nucleosomes represent a challenge in regard to transcription. Histone eviction enables RNA polymerase II (RNAPII) progression through DNA, but compromises chromatin integrity. Here, we used the SNAP-tag system to distinguish new and old histones and monitor chromatin reassembly coupled to transcription in human cells. We uncovered a transcription-dependent loss of old histone variants H3.1 and H3.3. At transcriptionally active domains, H3.3 enrichment reflected both old H3.3 retention and new deposition. Mechanistically, we found that the histone regulator A (HIRA) chaperone is critical to processing both new and old H3.3 via different pathways. De novo H3.3 deposition is totally dependent on HIRA trimerization as well as on its partner ubinuclein 1 (UBN1), while antisilencing function 1 (ASF1) interaction with HIRA can be bypassed. By contrast, recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows absolute dependency on ASF1–HIRA interaction. We propose a model whereby HIRA coordinates these distinct pathways during transcription to fine-tune chromatin states.

核小体代表了转录方面的挑战。组蛋白逐出使 RNA 聚合酶 II (RNAPII) 通过 DNA 进展，但会损害染色质的完整性。在这里，我们使用 SNAP-tag 系统来区分新的和旧的组蛋白，并监测与人类细胞转录相关的染色质重组。我们发现了旧组蛋白变体 H3.1 和 H3.3 的转录依赖性丢失。在转录活性域，H3.3 富集反映了旧的 H3.3 保留和新的沉积。从机制上讲，我们发现组蛋白调节剂 A (HIRA) 伴侣对于通过不同途径处理新旧 H3.3 至关重要。从头 H3.3 沉积完全依赖于 HIRA 三聚化及其伙伴泛核蛋白 1 (UBN1)，而可以绕过抗沉默功能 1 (ASF1) 与 HIRA 的相互作用。相比之下，H3.3 的回收需要 HIRA，但独立于 UBN1 或 HIRA 三聚化进行，并且绝对依赖于 ASF1-HIRA 相互作用。我们提出了一个模型，其中 HIRA 在转录过程中协调这些不同的途径以微调染色质状态。