Brown adipose tissue (BAT) dissipates energy through non-shivering thermogenesis and holds promise as a therapeutic target for obesity. The transcription factor Early B Cell Factor 2 (EBF2) is a key regulator of the thermogenic gene program, yet its underlying transcriptional regulatory mechanisms and potential for pharmacological targeting remain incompletely defined. Here, we identify a low-complexity C-terminal domain (CTD) within EBF2 that drives biomolecular condensation critical for thermogenic activation. Deletion of the CTD (ΔCTD) or mutation of conserved proline residues disrupts EBF2 phase separation without affecting genomic occupancy, thereby impairing brown adipocyte differentiation and thermogenic capacity both in vitro and in male mice. Remarkably, fusion of the intrinsically disordered region (IDR) of FUS to the EBF2 ΔCTD mutant fully rescues its function, whereas fusion with the MED1-IDR produces distinct transcriptional and phenotypic outcomes. Mechanistically, EBF2 condensates sequester the transcriptional repressor ZFP423 while excluding HDAC1, creating a permissive chromatin environment that promotes thermogenic gene expression. Finally, a phenotypic small-molecule screen targeting phase separation identifies compounds that modulate EBF2 condensate dynamics and thermogenic programming. Together, our findings reveal phase separation as an emerging regulatory mechanism underlying brown fat determination and suggest that targeting biomolecular condensation offers a potential therapeutic strategy for obesity.

Origin Recognition Complex subunit 1 (ORC1) is essential for ORC assembly at sequence-independent mammalian origins and regulates diverse epigenetic functions. Thus, mapping its genomic distribution is critical to elucidate the mechanisms underlying mammalian replication origin selection and its broader epigenetic roles. However, existing genomic profiles exhibit considerable variation due to inherent technical challenges. To address this, we generated a mouse embryonic stem (mES) cell line with an HA-MNase-mAID tag knocked into the endogenous Orc1 locus and optimized a ChEC-seq approach to map ORC1 chromatin binding with high specificity and reproducibility. Our profiling identified approximately 70,000 ORC1 binding sites broadly distributed across euchromatin and heterochromatin, spanning both early- and late-replicating regions. Intriguingly, cell-cycle-resolved profiling revealed that the ORC1 binding landscape remains remarkably stable, including in S phase. Through repli-ChEC-seq, we found that ORC1 rapidly recovers chromatin binding following DNA replication. Despite this persistent chromatin occupancy, ORC1 is dispensable for S phase progression in mES cells. Instead, we uncovered that ORC1 is involved in the epigenetic inheritance of H3K9me3 and the transcriptional repression of endogenous retroviruses (ERVs). Together, these findings provide a comprehensive spatiotemporal binding map of ORC1 in mES cells, and support the expansion of ORC1’s functional repertoire ranging from canonical DNA replication to heterochromatin maintenance and ERV silencing.

SUN1/2, core components of the linker of nucleoskeleton and cytoskeleton complex, transmit extracellular mechanical forces to nuclear lamina and chromatin. However, their role in regulating peripheral chromatin in mechanosensing and mechanoadaptation remains unclear. Using CRISPR/Cas9-mediated knockout of Sun1 or Sun2 in myoblasts, we identified a SUN1/2-dependent mechano-feedback loop. SUN1/2 depletion down-regulates genes for cell adhesion (e.g., integrin alpha-4) and for mechanotransduction (e.g., cell division cycle 42 and Ras homolog family member A). The primary mechanism involves redistribution of heterochromatin from nuclear periphery to the nucleoplasm and remodeling of lamina-associated domains (LADs), as an adaptive response to the loss of SUN proteins. Furthermore, lamin A/C acts as a key downstream effector, consistently modulating adhesion-related gene expression through the remodeling of LADs. Functionally, knockout of either Sun1/2 or Lmna aggravates differentiation defects in C2C12 myoblasts and abolishes adaptive responses to mechanical cues. This study provides proof of concept that nuclear mechanotransduction proteins can modulate cellular mechanoadaptation via a mechano-feedback loop, which coordinates LAD reorganization with the expression of upstream mechanotransduction genes.

Microglial phagocytosis exerts essential roles in neurodegeneration, but how phagocytic processes may reciprocally regulate microglia remains incompletely understood. Here, we report that microglial response in the mouse model of pathological axonal degeneration depends on the phagocytic receptor MerTK. The MerTK-triggered downstream phospholipase C signal is sufficient to induce the up-regulation of PU.1 and IRF8, the two central transcription factors governing microglial functions. Chromatin immunoprecipitation-sequencing analyses identify that PU.1 and IRF8 directly target the gene locus of TGFβ1, and disruption of this PU.1-IRF8 targeting site abolishes the induction of microglial TGFβ1 during neurodegeneration. Of importance, neurodegeneration-induced TGFβ1 acts in an autocrine manner, and the microglia-specific deletion of TGFβ1 or its receptors TGFβR1 or TGFβR2 blocks microglial response. Moreover, microglial TGFβ1 autocrine signaling similarly occurs in the 5×FAD mouse model of Alzheimer's disease and in human patients. These results have delineated an important mechanism underlying microglial response to neurodegeneration.

The 3D architecture of the eukaryotic genome is largely shaped by cohesin complexes containing either STAG1 or STAG2 subunits. Yet, their roles in post-mitotic genome refolding remain unclear. Here, we establish STAG2 as the predominant paralog and primary orchestrator of this process. We find that upon mitotic exit, STAG1 depletion imposes negligible effects on genome refolding or transcription reactivation, whereas STAG2 regulates genome remodeling in a stage- and chromatin-context-dependent manner. In early-G1, STAG2 promotes small euchromatic structural loops, enhancer-promoter contacts and transcriptional refiring; in late-G1, it suppresses large loops by limiting the more processive STAG1-cohesin. STAG2 processivity is constrained by CTCF roadblocks rather than genomic traveling distance. Co-depletion causes synergistic loss of structural loops and stronger transcriptional dysregulation, yet residual chromatin-bound cohesin retains measurable extrusion capacity. Together, these results establish STAG2 as the principal regulator of post-mitotic spatiotemporal chromatin reorganization, while STAG1 provides compensatory support for robustness.

NIPBL promotes chromatin loop extrusion by the cohesin complex until it stalls at convergently oriented CTCF sites, forming structural loops. While a large fraction of loops connecting cis-regulatory elements (CREs) can be maintained in cohesin-depleted cells, whether the loop extrusion process contributes to the de novo establishment of CRE loops remains unclear. To address this question, we characterized the formation of structural and CRE loops in NIPBL-depleted cells during the mitosis-to-G1-phase transition. Structural loop formation was impaired proportionally to loop length. Computational modeling supports these observations, suggesting that NIPBL promotes both cohesin loading and extrusion. Notably, most CRE loops, regardless of length, were established normally upon NIPBL degradation. While a subset of contacts among weak CREs were formed with delayed kinetics in NIPBL-depleted cells, generally gene activation was only mildly impaired. Collectively, our findings suggest that postmitotic establishment of regulatory contacts and gene transcription can occur independently of chromatin loop extrusion.

Condensin complexes are indispensable for mitotic chromosome condensation. In this review, we summarize their structural and functional features, focusing on ATP-dependent loop extrusion and mitotic chromatin folding. We discuss current models of mitotic chromosome formation with an emphasis on the emerging role of condensin in suppressing interphase chromatin contact patterns during mitosis. Additionally, we outline regulatory mechanisms governing condensin activity, including phosphorylation-dependent and -independent regulation of chromatin loading and unloading. Finally, we connect condensin dysfunction to chromosomal instability, cancer, and neurodevelopmental disorders. Our discussions underscore condensin’s significance as a genome architect and a key player in disease pathogenesis.

Replication stress (RS), if not effectively and timely addressed, could result in DNA damage in mitosis. However, the relationship between RS and other mitotic events, such as nuclear envelope (NE) breakdown and reassembly, remains poorly understood. Here we report that RS can lead to NE defect. Importantly, rather than de novo NE rupture, the defect per se is a result of nuclear envelope reassembly defect (NERD) during mitosis. Interestingly, NERD is associated with mitotic DNA damage, and repair of the damage by DNA polymerase theta (Polθ)-mediated end joining (TMEJ) ameliorates NERD. Genomic mapping of lamina associated domains (LADs) by cleavage under targets and tagmentation (CUT&Tag) identifies a population of replication stress-sensitive LADs (RESSLADs). Strikingly, a substantial portion of RESSLADs reside in the common fragile sites (CFSs). The loss of RESSLADs-NE interaction under RS might be attributed to the sustained phosphorylation of Lamin A/C at the sites of NERD. In addition, prominent NE defect is observed under multiple conditions of synthetic lethality. Altogether, these findings establish a link between genome instability and nuclear vulnerability under replication stress.

Mammalian genomes are folded through the distinct actions of structural maintenance of chromosome (SMC) complexes, which include the chromatin loop-extruding cohesin (extrusive cohesin), the sister chromatid cohesive cohesin and the mitotic chromosome-associated condensins1-3. Although these complexes function at different stages of the cell cycle, they exist together on chromatin during the G2-to-M phase transition, when the genome structure undergoes substantial reorganization1,2. Yet, how the different SMC complexes affect each other and how their interactions orchestrate the dynamic folding of the three-dimensional genome remain unclear. Here we engineered all possible cohesin and condensin configurations on mitotic chromosomes to delineate the concerted, mutually influential action of SMC complexes. We show that condensin disrupts the binding of extrusive cohesin at CCCTC-binding factor (CTCF) sites, thereby promoting the disassembly of interphase topologically associating domains (TADs) and loops during mitotic progression. Conversely, extrusive cohesin impedes condensin-mediated mitotic chromosome spiralization. Condensin reduces peaks of cohesive cohesin, whereas cohesive cohesin antagonizes condensin-mediated longitudinal shortening of mitotic chromosomes. The presence of both extrusive and cohesive cohesin synergizes these effects and inhibits mitotic chromosome condensation. Extrusive cohesin positions cohesive cohesin at CTCF-binding sites. However, cohesive cohesin by itself cannot be arrested by CTCF molecules and is insufficient to establish TADs or loops. Moreover, it lacks loop-extrusion capacity, which indicates that cohesive cohesin has nonoverlapping functions with extrusive cohesin. Finally, cohesive cohesin restricts chromatin loop expansion mediated by extrusive cohesin. Collectively, our data describe a three-way interaction among major SMC complexes that dynamically modulates chromatin architecture during cell cycle progression.

The clustering of multiple transcription factor binding sites (TFBSs) for the same TF has proved to be a pervasive feature of cis-regulatory elements in the eukaryotic genome. However, the contribution of binding sites within the homotypic clusters of TFBSs (HCTs) to TF binding and target gene expression remains to be understood. Here, we characterize the CHD4 enhancers that harbor unique functional ZNF410 HCTs genome wide. We uncover that ZNF410 controls chromatin accessibility and activity of the CHD4 enhancer regions. We demonstrate that ZNF410 binds to the HCTs in a collaborative fashion, further conferring transcriptional activation. In particular, three ZNF410 motifs (sub-HCTs) located at 3' end of the distal enhancer act as "switch motifs" to control chromatin accessibility and enhancer activity. Mechanistically, the SWI/SNF complex is selectively required to mediate cooperative ZNF410 binding for CHD4 expression. Together, our findings expose a complex functional hierarchy of homotypic clustered motifs, which cooperate to fine-tune target gene expression.

NIPBL promotes chromatin loop extrusion by the cohesin complex until it stalls at convergently oriented CTCF sites, leading to the formation of structural loops. However, to what extent loop extrusion contributes to the establishment vs maintenance of cis-regulatory element (CRE) connectivity is poorly understood. Here, we explored the de novo establishment of chromatin folding patterns at the mitosis-to-G1-phase transition upon acute NIPBL loss. NIPBL depletion primarily impaired the formation of cohesion-mediated structural loops with NIPBL dependence being proportional to loop length. In contrast, the majority of CRE loops were established independently of loop extrusion regardless of length. However, NIPBL depletion slowed the re-formation of CRE loops with weak enhancers. Transcription of genes at NIPBL-independent loop anchors was activated normally in the absence of NIPBL. In sum, establishment of most regulatory contacts and gene transcription following mitotic exit is independent of loop extrusion.

The prevalence and severity of liver fibrosis appear higher in men than in premenopausal women, while postmenopausal women exhibit the worsened disease. However, the pathophysiological mechanism underlying such clinical observations remains incompletely understood. Here, we show that sex hormone depletion in adult female mice exaggerates the model of liver fibrosis, while estradiol replacement in castrated male mice is sufficient to mitigate the disease severity. Transcriptomic analyses and immunohistochemistry then demonstrate that both human and mouse hepatic stellate cells (HSCs), the primary cell type responsible for extracellular fibrous depositions, predominantly express the estrogen receptor 1 (ESR1). Of importance, genetic deletion of ESR1 in mouse HSCs markedly promotes liver fibrosis. Moreover, chromatin immunoprecipitation followed by sequencing (ChIP-seq) and in vitro manipulations reveal that ESR1 can directly target the expression of fibrosis-related genes in HSCs. Together, this study has elucidated a critical aspect of ESR1 signaling in the sexual dimorphism of liver fibrosis. ### Competing Interest Statement The authors have declared no competing interest.

How specific enhancer-promoter pairing is established remains mostly unclear. Besides the CTCF/cohesin machinery, few nuclear factors have been studied for a direct role in physically connecting regulatory elements. Using a murine erythroid cell model, we show via acute degradation experiments that LDB1 directly and broadly promotes connectivity among regulatory elements. Most LDB1-mediated contacts, even those spanning hundreds of kb, can form in the absence of CTCF, cohesin, or YY1 as determined using multiple degron systems. Moreover, an engineered LDB1-driven chromatin loop is cohesin independent. Cohesin-driven loop extrusion does not stall at LDB1-occupied sites but aids the formation of a subset of LDB1-anchored loops. Leveraging the dynamic reorganization of nuclear architecture during the transition from mitosis to G1 phase, we observe that loop formation and de novo LDB1 occupancy correlate and can occur independently of structural loops. Tri-C and Region Capture Micro-C reveal that LDB1 organizes multi-enhancer networks to activate transcription. These findings establish LDB1 as a driver of spatial connectivity.

As cells exit mitosis and enter G1, chromosomes decompact and transcription is reestablished. Hi-C studies have indicated that all interphase three-dimensional genome features, including A/B compartments, topologically associating domains and CCCTC-binding factor loops, are lost during mitosis. However, Hi-C is insensitive to features such as microcompartments, nested focal interactions between cis-regulatory elements. Here we apply region capture Micro-C to mouse erythroblasts from mitosis to G1. We unexpectedly observe microcompartments in prometaphase, which strengthen in anaphase and telophase before weakening throughout G1. Microcompartment anchors coincide with transcriptionally spiking promoters during mitosis. Loss of condensin loop extrusion differentially impacts microcompartments and A/B compartments, suggesting that they are partially distinct. Polymer modeling shows that microcompartment formation is favored by chromatin compaction and disfavored by loop extrusion, providing a basis for strong microcompartmentalization in anaphase and telophase. Our results suggest that compaction and homotypic affinity drive microcompartment formation, which may explain transient transcriptional spiking at mitotic exit.

Few transcription factors have been examined for their direct roles in physically connecting enhancers and promoters. Here acute degradation of Yin Yang 1 (YY1) in erythroid cells revealed its requirement for the maintenance of numerous enhancer-promoter loops, but not compartments or domains. Despite its reported ability to interact with cohesin, the formation of YY1-dependent enhancer-promoter loops does not involve stalling of cohesin-mediated loop extrusion. Integrating mitosis-to-G1-phase dynamics, we observed partial retention of YY1 on mitotic chromatin, predominantly at gene promoters, followed by rapid rebinding during mitotic exit, coinciding with enhancer-promoter loop establishment. YY1 degradation during the mitosis-to-G1-phase interval revealed a set of enhancer-promoter loops that require YY1 for establishment during G1-phase entry but not for maintenance in interphase, suggesting that cell cycle stage influences YY1's architectural function. Thus, as revealed here for YY1, chromatin architectural functions of transcription factors can vary in their interplay with CTCF and cohesin as well as by cell cycle stage. Yin Yang 1 (YY1) aids in the formation of enhancer-promoter (E-P) loops independently of cohesin. YY1 maintains a subset of E-P interactions in interphase and establishes an overlapping yet distinct set after mitotic exit.

ABSTRACTDuring mitosis, condensin activity interferes with interphase chromatin structures. Here, we generated condensin-free mitotic chromosomes to investigate genome folding principles. Co- depletion of condensin I and II, but neither alone, triggered mitotic chromosome compartmentalization in ways that differ from interphase. Two distinct euchromatic compartments, indistinguishable in interphase, rapidly emerged upon condensin loss with different interaction preferences and dependence on H3K27ac. Constitutive heterochromatin gradually self-aggregated and co-compartmentalized with the facultative heterochromatin, contrasting with their separation during interphase. While topologically associating domains (TADs) and CTCF/cohesin mediated structural loops remained undetectable, cis-regulatory element contacts became apparent, providing an explanation for their quick re-establishment during mitotic exit. HP1 proteins, which are thought to partition constitutive heterochromatin, were absent from mitotic chromosomes, suggesting, surprisingly, that constitutive heterochromatin can self-aggregate without HP1. Indeed, in cells traversing from M- to G1-phase in the combined absence of HP1α, HP1Π and HP1γ, re-established constitutive heterochromatin compartments normally. In sum, “clean-slate” condensin-deficient mitotic chromosomes illuminate mechanisms of genome compartmentalization not revealed in interphase cells.

All measurable features of higher-order chromosomal architecture undergo drastic reorganization as cells enter and exit mitosis. During mitosis, gene transcription is temporarily halted, the nuclear envelope is dismantled, and chromosomes undergo condensation. At this time, chromatin compartments, topologically associating domains (TADs), and loops that connect enhancers with promoters as well as CTCF/cohesin loops are dissolved. Upon G1 entry, genome organization is rebuilt in the daughter nuclei to resemble that of the mother nucleus. We survey recent studies that traced these features in relation to gene expression during the mitosis-to-G1-phase transition at high temporal resolution. Dissection of fluctuating architectural features informed the hierarchical relationships of chromosomal organization, the mechanisms by which they are formed, and their mutual (in-) dependence. These studies highlight the importance of considering the cell cycle dynamics for studies of chromosomal organization.

Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is not well understood. Here, by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters. Primary transcript RNA-FISH shows that CTCF lowers burst fraction but not burst intensity of uasTrx and that co-bursting of sense and antisense transcripts is disfavored. Genome editing, chromatin conformation studies and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its architectural function. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription. Luan et al. find that CTCF shapes the transcriptional landscape in part by suppressing the initiation of upstream antisense transcription at hundreds of divergent gene promoters.

Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4–NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation. Depletion of BRD4 reduces the chromatin occupancy of NIPBL, resulting in aberrant genome folding. Loss of BRD4 impedes neural crest differentiation, which can be rescued by depletion of WAPL.

During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. We exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to genome re-sculpting in newborn nuclei. Depletion of CTCF during the M- to G1-phase transition alters short-range compartmentalization after mitosis. Chromatin domain boundary re-formation is impaired upon CTCF loss, but a subset of boundaries, characterized by transitions in chromatin states, is established normally. Without CTCF, structural loops fail to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persist deeply into G1-phase in CTCF-depleted cells. CTCF loss-associated gains in transcription are often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declines upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitate formation of CRE loops nested within them, especially those involving weak CREs. Transcription inhibition does not significantly affect global architecture or transcription start site-associated boundaries. However, ongoing transcription contributes considerably to the formation of gene domains, regions of enriched contacts along gene bodies. Notably, gene domains emerge in ana/telophase prior to completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yields insights into the contributions of CTCF and transcription to chromatin architecture dynamics during the mitosis to G1-phase progression.