Heterochromatin diversity modulates genome compartmentalization and loop extrusion barriers [Protect-seq]

Two dominant processes organizing chromosomes are loop extrusion and the compartmental segregation of active and inactive chromatin. The molecular players involved in loop extrusion during interphase, cohesin and CTCF, have been extensively studied and experimentally validated. However, neither the molecular determinants nor the functional roles of compartmentalization are well understood. Here, we distinguish three inactive chromatin states using contact frequency profiling, comprising two types of heterochromatin and a previously uncharacterized inactive state exhibiting a neutral interaction preference. We find that heterochromatin marked by long continuous stretches of H3K9me3, HP1α and HP1β correlates with a conserved signature of strong compartmentalization and is abundant in HCT116 colon cancer cells. We demonstrate that disruption of DNA methyltransferase activity dramatically remodels genome compartmentalization as a consequence of the loss of H3K9me3 and HP1 binding. Interestingly, H3K9me3-HP1α/β is replaced by the neutral inactive state and retains late replication timing. Furthermore, we show that H3K9me3-HP1α/β heterochromatin is permissive to loop extrusion by cohesin but refractory to CTCF, explaining a paucity of visible loop extrusion-associated patterns in Hi-C. Accordingly, CTCF loop extrusion barriers are reactivated upon loss of H3K9me3-HP1α/β, not as a result of canonical demethylation of the CTCF binding motif but due to an intrinsic resistance of H3K9me3-HP1α/β heterochromatin to CTCF binding. Together, our work reveals a dynamic structural and organizational diversity of the inactive portion of the genome and establishes new connections between the regulation of chromatin state and chromosome organization, including an interplay between DNA methylation, compartmentalization and loop extrusion. We compare genome structure and chromatin state genome-wide using Hi-C, ChIP-seq, Repli-seq, and Protect-seq in HCT116 colon cancer cells with its genetic derivative DKO cells which lack DNMT3B and DNMT1 activity and HCT116 cells treated with the DNA methylation inhibitor 5-Azacytidine (5Aza).

介导染色体组装的两大核心过程为环挤出(loop extrusion)与活性、非活性染色质的区室化分离。间期内环挤出过程的核心分子因子黏连蛋白(cohesin)与CCCTC结合因子(CTCF)已被广泛研究并得到实验验证。然而，染色质区室化的分子决定因素与功能机制均尚未被充分阐明。本研究通过接触频率谱分析，区分出三种非活性染色质状态：两类异染色质，以及一种此前未被表征、呈现中性相互作用偏好的非活性状态。研究发现，以长连续H3K9me3、异染色质蛋白1α(HP1α)与异染色质蛋白1β(HP1β)标记的异染色质，与强区室化的保守特征显著相关，且在HCT116结肠癌细胞中富集度较高。本研究证实，DNA甲基转移酶活性的丧失会导致H3K9me3与HP1结合丢失，进而显著重塑基因组区室化结构。有趣的是，H3K9me3-HP1α/β异染色质会被该中性非活性状态替代，并保留复制时序延迟的特征。进一步研究表明，H3K9me3-HP1α/β异染色质可容许黏连蛋白介导的环挤出，但对CTCF结合具有抗性，这解释了Hi-C实验中难以观测到与环挤出相关的典型模式的原因。相应地，当H3K9me3-HP1α/β异染色质丧失后，CTCF环挤出屏障会被重新激活——这一过程并非源于CTCF结合基序的经典去甲基化，而是由于H3K9me3-HP1α/β异染色质本身对CTCF结合具有抗性。综上，本研究揭示了基因组非活性区域的动态结构与组织多样性，并建立了染色质状态调控与染色体组装之间的新关联，涵盖DNA甲基化、区室化与环挤出之间的相互调控关系。本研究在HCT116结肠癌细胞、其遗传衍生的DNMT3B与DNMT1活性缺失的DKO细胞，以及经DNA甲基化抑制剂5-氮杂胞苷(5-Azacytidine, 5Aza)处理的HCT116细胞中，通过Hi-C、染色质免疫共沉淀测序(ChIP-seq)、复制时序测序(Repli-seq)与Protect-seq技术，开展了全基因组范围的基因组结构与染色质状态比较分析。