Many biomolecular condensates, including transcriptional condensates, are formed in elastic mediums. In this work, we study the nonequilibrium condensate dynamics in a chromatin-like environment modeled as a heterogeneous elastic medium. We demonstrate that the ripening process in such an elastic medium exhibits a temporal power-law scaling of the average condensate radius, depending on the local stiffness distribution and different from Ostwald ripening. Moreover, we incorporate an active process to model the dissolution of transcriptional condensates upon RNA accumulation. Intriguingly, three types of kinetics of condensate growth emerge, corresponding to constitutively expressed, transcriptional-bursting, and silenced genes. Furthermore, the simulated burst frequency decreases exponentially with the local stiffness, through which we infer a lognormal distribution of local stiffness in living cells using the transcriptome-wide distribution of burst frequency. Under the inferred stiffness distribution, the simulated distributions of bursting kinetic parameters agree reasonably well with the experimental data. Our findings reveal the interplay between biomolecular condensates and elastic mediums, yielding far-reaching implications for gene expression.

Promyelocytic leukemia (PML) protein constitutes an indispensable element within PML-nuclear bodies (PML-NBs), playing a pivotal role in the regulation of multiple cellular functions while coordinating the innate immune response against viral invasions. Simultaneously, numerous viruses elude immune detection by targeting PML-NBs. Japanese encephalitis virus (JEV) is a flavivirus that causes Japanese encephalitis, a severe neurological disease that affects humans and animals. However, the mechanism through which JEV evades immunity via PML-NBs has been scarcely investigated. In the present study, PK15 cells were infected with JEV, and the quantity of intracellular PML-NBs was enumerated. The immunofluorescence results indicated that the number of PML-NBs was significantly reduced in JEV antigen-positive cells compared to viral antigen-negative cells. Subsequently, ten JEV proteins were cloned and transfected into PK15 cells. The results revealed that JEV non-structural proteins, NS2B, NS3, NS4A, NS4B, and NS5, significantly diminished the quantity of PML-NBs. Co-transfection was performed with the five JEV proteins and various porcine PML isoforms. The results demonstrated that NS2B colocalized with PML4 and PML5, NS4A colocalized with PML1 and PML4, NS4B colocalized with PML1, PML3, PML4, and PML5, while NS3 and NS5 interacted with all five PML isoforms. Furthermore, ectopic expression of PML isoforms confirmed that PML1, PML3, PML4, and PML5 inhibited JEV replication. These findings suggest that JEV disrupts the structure of PML-NBs through interaction with PML isoforms, potentially leading to the attenuation of the host\'s antiviral immune response.

Alternative lengthening of telomeres (ALTs) mechanism is activated in some somatic, germ cells, and human cancer cells. However, the key regulators and mechanisms of the ALT pathway remain elusive. Here we demonstrated that ZBTB40 is a novel telomere-associated protein and binds to telomeric dsDNA through its N-terminal BTB (BR-C, ttk and bab) or POZ (Pox virus and Zinc finger) domain in ALT cells. Notably, the knockout or knockdown of ZBTB40 resulted in the telomere dysfunction-induced foci and telomere lengthening in the ALT cells. The results also show that ZBTB40 is associated with ALT-associated promyelocytic leukemia nuclear bodies, and the loss of ZBTB40 induces the accumulation of the ALT-associated promyelocytic leukemia nuclear bodies in U2OS cells. Taken together, our results implicate that ZBTB40 is a key player of telomere protection and telomere lengthening regulation in human ALT cells.

Growing evidence suggests prevalence of transcriptional condensates on chromatin, yet their mechanisms of formation and functional significance remain largely unclear. In human cancer, a series of mutations in the histone acetylation reader ENL create gain-of-function mutants with increased transcriptional activation ability. Here, we show that these mutations, clustered in ENL\'s structured acetyl-reading YEATS domain, trigger aberrant condensates at native genomic targets through multivalent homotypic and heterotypic interactions. Mechanistically, mutation-induced structural changes in the YEATS domain, ENL\'s two disordered regions of opposing charges, and the incorporation of extrinsic elongation factors are all required for ENL condensate formation. Extensive mutagenesis establishes condensate formation as a driver of oncogenic gene activation. Furthermore, expression of ENL mutants beyond the endogenous level leads to non-functional condensates. Our findings provide new mechanistic and functional insights into cancer-associated condensates and support condensate dysregulation as an oncogenic mechanism.

The retrovirus HIV-1 integrates into the host genome and establishes a latent viral reservoir that escapes immune surveillance. Molecular mechanisms of HIV-1 latency have been studied extensively to achieve a cure for the acquired immunodeficiency syndrome (AIDS). Latency-reversing agents (LRAs) have been developed to reactivate and eliminate the latent reservoir by the immune system. To develop more promising LRAs, it is essential to evaluate new therapeutic targets. Here, we find that CBX4, a component of the Polycomb Repressive Complex 1 (PRC1), contributes to HIV-1 latency in seven latency models and primary CD4+ T cells. CBX4 forms nuclear bodies with liquid-liquid phase separation (LLPS) properties on the HIV-1 long terminal repeat (LTR) and recruits EZH2, the catalytic subunit of PRC2. CBX4 SUMOylates EZH2 utilizing its SUMO E3 ligase activity, thereby enhancing the H3K27 methyltransferase activity of EZH2. Our results indicate that CBX4 acts as a bridge between the repressor complexes PRC1 and PRC2 that act synergistically to maintain HIV-1 latency. Dissolution of phase-separated CBX4 bodies could be a potential intervention to reactivate latent HIV-1.

Human p53 is a transcription factor regulating the transcription of a variety of target genes. Under various stresses, its tumor suppressor function was activated by the phosphorylation of p53. In this study, we found that full-length wild-type p53 could form phase-separated condensates with the aggregation tendency in vitro and in vivo. The LLPS of p53 was regulated by multiple functional domains. Specific DNA could promote the formation of p53 condensates. Fluorescence recovery data after photobleaching revealed that the Ser392 phosphorylation enhanced the fluidity of p53 condensates. Fluorescence analysis suggested that Ser392 phosphorylation increased the p53 concentration in condensates involved in transcription initiation and the stability of p53-mediated transcriptional condensates. The experiments in cells showed that p53 was evenly dispersed in the nucleus, it formed the dynamic condensates under the UV radiation-induced DNA damage, and the Ser392 nonphosphorylatable mutant S392A p53 formed condensates with significantly reduced number and size. These findings revealed that p53 phosphorylation modified its LLPS behavior, and suggested a mechanism that phosphorylation regulated condensate preference.

Paralogs that arise from gene duplications during genome evolution enable genetic redundancy and phenotypic robustness. Variation in the coding or regulatory sequence of paralogous transcriptional regulators diversifies their functions and relationships, which provides developmental robustness against genetic or environmental perturbation. The fate transition of plant shoot stem cells for flowering and reproductive success requires a robust transcriptional control. However, how paralogs function and interact to achieve such robustness is unknown.
Here, we explore the genetic relationship and protein behavior of ALOG family transcriptional factors with diverse transcriptional abundance in shoot meristems. A mutant spectrum covers single and higher-order mutant combinations of five ALOG paralogs and creates a continuum of flowering transition defects, showing gradually enhanced precocious flowering, along with inflorescence simplification from wild-type-like to progressively fewer flowers until solitary flower with sterile floral organs. Therefore, these paralogs play unequal roles and act together to achieve a robust genetic canalization. All five proteins contain prion-like intrinsically disordered regions (IDRs) and undergo phase separation. Accumulated mutations following gene duplications lead to IDR variations among ALOG paralogs, resulting in divergent phase separation and transcriptional regulation capabilities. Remarkably, they retain the ancestral abilities to assemble into a heterotypic condensate that prevents precocious activation of the floral identity gene ANANTHA.
Our study reveals a novel genetic canalization mechanism enabled by heterotypic transcriptional condensates formed by paralogous protein interactions and phase separation, uncovering the molecular link between gene duplication caused IDR variation and robust transcriptional control of stem cell fate transition.

Acute promyelocytic leukemia (APL) is driven by the oncoprotein PML/RARα, which destroys the architecture of PML nuclear bodies (NBs). PML NBs are critical to tumor suppression, and their disruption mediated by PML/RARα accelerates APL pathogenesis. However, the mechanisms of PML NB disruption remain elusive. Here, we reveal that the failure of NB assembly in APL results from neddylation-induced aberrant phase separation of PML/RARα. Mechanistically, PML/RARα is neddylated in the RARα moiety, and this neddylation enhances its DNA-binding ability and further impedes the phase separation of the PML moiety, consequently disrupting PML NB construction. Accordingly, deneddylation of PML/RARα restores its phase separation process to reconstruct functional NBs and activates RARα signaling, thereby suppressing PML/RARα-driven leukemogenesis. Pharmacological inhibition of neddylation by MLN4924 eradicates APL cells both in vitro and in vivo. Our work elucidates the neddylation-destroyed phase separation mechanism for PML/RARα-driven NB disruption and highlights targeting neddylation for APL eradication.

Nuclear bodies are membrane-free nuclear substructures that are localized in the mammalian nuclear matrix region. They are multiprotein complexes that recruit other proteins to participate in various cellular activities, such as transcription, RNA splicing, epigenetic regulation, tumorigenesis and antiviral defense. It is of great significance to clarify the functions and regulatory mechanisms of nuclear bodies to probe related diseases and virus-host interactions. This review takes several nuclear bodies associated proteins as examples, summarizes the formation process, structure and functions of nuclear bodies, and focuses on their important roles in antiviral infection. It is expected to provide new insight into host antiviral mechanisms.

Biomolecules specifically aggregate in the cytoplasm and nucleus, driving liquid-liquid phase separation (LLPS) formation and diverse biological processes. Extensive studies have focused on revealing multiple functional membraneless organelles in both the nucleus and cytoplasm. Condensation compositions of LLPS, such as proteins and RNAs affecting the formation of phase separation, have been gradually unveiled. LncRNAs possessing abundant second structures usually promote phase separation formation by providing architectural scaffolds for diverse RNAs and proteins interaction in both the nucleus and cytoplasm. Beyond scaffolds, lncRNAs may possess more diverse functions, such as functioning as enhancer RNAs or buffers. In this review, we summarized current studies on the function of phase separation and its related lncRNAs, mainly in the nucleus. This review will facilitate our understanding of the formation and function of phase separation and the role of lncRNAs in these processes and related biological activities. A deeper understanding of the formation and maintaining of phase separation will be beneficial for disease diagnosis and treatment.