Nuclear morphology is shaped by loop-extrusion programs.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

Transcriptional network governing extraembryonic endoderm cell fate choice.

The mechanism by which transcription factor (TF) network instructs cell-type-specific transcriptional programs to drive primitive endoderm (PrE) progenitors to commit to parietal endoderm (PE) versus visceral endoderm (VE) cell fates remains poorly understood. To address the question, we analyzed the single-cell transcriptional signatures defining PrE, PE, and VE cell states during the onset of the PE-VE lineage bifurcation. By coupling with the epigenomic comparison of active enhancers unique to PE and VE cells, we identified GATA6, SOX17, and FOXA2 as central regulators for the lineage divergence. Transcriptomic analysis of cXEN cells, an in vitro model for PE cells, after the acute depletion of GATA6 or SOX17 demonstrated that these factors induce Mycn, imparting the self-renewal properties of PE cells. Concurrently, they suppress the VE gene program, including key genes like Hnf4a and Ttr, among others. We proceeded with RNA-seq analysis on cXEN cells with FOXA2 knockout, in conjunction with GATA6 or SOX17 depletion. We found FOXA2 acts as a potent suppressor of Mycn while simultaneously activating the VE gene program. The antagonistic gene regulatory activities of GATA6/SOX17 and FOXA2 in promoting alternative cell fates, and their physical co-bindings at the enhancers provide molecular insights to the plasticity of the PrE lineage. Finally, we show that the external cue, BMP signaling, promotes the VE cell fate by activation of VE TFs and repression of PE TFs including GATA6 and SOX17. These data reveal a putative core gene regulatory module that underpins PE and VE cell fate choice.

Enhancer-instructed epigenetic landscape and chromatin compartmentalization dictate a primary antibody repertoire protective against specific bacterial pathogens.

Antigen receptor loci are organized into variable (V), diversity (D) and joining (J) gene segments that rearrange to generate antigen receptor repertoires. Here, we identified an enhancer (E34) in the murine immunoglobulin kappa (Igk) locus that instructed rearrangement of Vκ genes located in a sub-topologically associating domain, including a Vκ gene encoding for antibodies targeting bacterial phosphorylcholine. We show that E34 instructs the nuclear repositioning of the E34 sub-topologically associating domain from a recombination-repressive compartment to a recombination-permissive compartment that is marked by equivalent activating histone modifications. Finally, we found that E34-instructed Vκ-Jκ rearrangement was essential to combat Streptococcus pneumoniae but not methicillin-resistant Staphylococcus aureus or influenza infections. We propose that the merging of Vκ genes with Jκ elements is instructed by one-dimensional epigenetic information imposed by enhancers across Vκ and Jκ genomic regions. The data also reveal how enhancers generate distinct antibody repertoires that provide protection against lethal bacterial infection.

Fe-Catalyzed Three-Component Coupling Reaction of α,ß,γ,δ-Unsaturated Carbonyl Compounds and Conjugate Dienes with Alkylsilyl Peroxides and Nucleophiles.

An Fe(OTf)2-catalyzed three-component coupling reaction of α,ß,γ,δ-unsaturated carbonyl compounds with alkylsilyl peroxides in the presence of certain heteronucleophiles (ROH and indole) is realized under mild reaction conditions. A variety of α,ß,γ,δ-diene carbonyl substrates with different substituents were successfully employable via combination with several different alkylsilyl peroxides. This new approach is also applicable to the double functionalization of diene substrates.

Engagement of the costimulatory molecule ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells.

Elevated gene expression of the costimulatory receptor Icos is a hallmark of CD8+ tissue-resident memory (Trm) T cells. Here, we examined the contribution of ICOS in Trm cell differentiation. Upon transfer into WT mice, Icos-/- CD8+ T cells exhibited defective Trm generation but produced recirculating memory populations normally. ICOS deficiency or ICOS-L blockade compromised establishment of CD8+ Trm cells but not their maintenance. ICOS ligation during CD8+ T cell priming did not determine Trm induction; rather, effector CD8+ T cells showed reduced Trm differentiation after seeding into Icosl-/- mice. IcosYF/YF CD8+ T cells were compromised in Trm generation, indicating a critical role for PI3K signaling. Modest transcriptional changes in the few Icos-/- Trm cells suggest that ICOS-PI3K signaling primarily enhances the efficiency of CD8+ T cell tissue residency. Thus, local ICOS signaling promotes production of Trm cells, providing insight into the contribution of costimulatory signals in the generation of tissue-resident populations.

The copper-catalyzed selective monoalkylation of active methylene compounds with alkylsilyl peroxides.

A novel method for a mild copper-catalyzed selective monoalkylation of active methylene compounds with various alkylsilyl peroxides has been developed. The reaction has a broad substrate scope and our mechanistic studies suggest the participation of radical species in this alkylation reaction.

Calcium signaling instructs NIPBL recruitment at active enhancers and promoters via distinct mechanisms to reconstruct genome compartmentalization.

During developmental progression the genomes of immune cells undergo large-scale changes in chromatin folding. However, insights into signaling pathways and epigenetic control of nuclear architecture remain rudimentary. Here, we found that in activated neutrophils calcium influx rapidly recruited the cohesin-loading factor NIPBL to thousands of active enhancers and promoters to dictate widespread changes in compartment segregation. NIPBL recruitment to enhancers and promoters occurred with distinct kinetics. The induction of NIPBL-binding was coordinate with increased P300, BRG1 and RNA polymerase II occupancy. NIPBL-bound enhancers were associated with NFAT, PU.1, and CEBP cis elements, whereas NIPBL-bound promoters were enriched for GC-rich DNA sequences. Using an acute degradation system, we found that the histone acetyltransferases P300 and CBP maintained H3K27ac abundance and facilitated NIPBL occupancy at enhancers and that active transcriptional elongation is essential to maintain H3K27ac abundance. Chromatin remodelers, containing either of the mutually exclusive BRG1 and BRM ATPases, promoted NIPBL recruitment at active enhancers. Conversely, at active promoters, depletion of BRG1 and BRM showed minimal effect on NIPBL occupancy. Finally, we found that calcium signaling in both primary innate and adaptive immune cells swiftly induced NIPBL occupancy. Collectively, these data reveal how transcriptional regulators, histone acetyltransferases, chromatin remodelers, and transcription elongation promote NIPBL occupancy at active enhancers while the induction of NIPLB occupancy at promoters is primarily associated with GC-rich DNA sequences.

Upon microbial challenge, human neutrophils undergo rapid changes in nuclear architecture and chromatin folding to orchestrate an immediate inflammatory gene program.

Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.

A B-Cell-Specific Enhancer Orchestrates Nuclear Architecture to Generate a Diverse Antigen Receptor Repertoire.

The genome is organized into topologically associated domains (TADs) that enclose smaller subTADs. Here, we identify and characterize an enhancer that is located in the middle of the V gene region of the immunoglobulin kappa light chain (Igκ) locus that becomes active preceding the stage at which this locus undergoes V(D)J recombination. This enhancer is a hub of long-range chromatin interactions connecting subTADs in the V gene region with the recombination center at the J genes. Deletion of this element results in a highly altered long-range chromatin interaction pattern across the locus and, importantly, affects individual V gene utilization locus-wide. These results indicate the existence of an enhancer-dependent framework in the Igκ locus and further suggest that the composition of the diverse antibody repertoire is regulated in a subTAD-specific manner. This enhancer thus plays a structural role in orchestrating the proper folding of the Igκ locus in preparation for V(D)J recombination.

RNAs interact with BRD4 to promote enhanced chromatin engagement and transcription activation.

The bromodomain and extra-terminal motif (BET) protein BRD4 binds to acetylated histones at enhancers and promoters via its bromodomains (BDs) to regulate transcriptional elongation. In human colorectal cancer cells, we found that BRD4 was recruited to enhancers that were co-occupied by mutant p53 and supported the synthesis of enhancer-directed transcripts (eRNAs) in response to chronic immune signaling. BRD4 selectively associated with eRNAs that were produced from BRD4-bound enhancers. Using biochemical and biophysical methods, we found that BRD4 BDs function cooperatively as docking sites for eRNAs and that the BDs of BRD2, BRD3, BRDT, BRG1, and BRD7 directly interact with eRNAs. BRD4-eRNA interactions increased BRD4 binding to acetylated histones in vitro and augmented BRD4 enhancer recruitment and transcriptional cofactor activities. Our results suggest a mechanism by which eRNAs are directly involved in gene regulation by modulating enhancer interactions and transcriptional functions of BRD4.

Transcriptome-Wide Analysis Reveals the Origin of Peloria in Chinese Cymbidium (Cymbidium sinense).

An orchid flower exhibits a zygomorphic corolla with a well-differentiated labellum. In Cymbidium sinense, many varieties with peloric or pseudopeloric flowers have been bred during centuries of domestication. However, little is known about the molecular basis controlling orchid floral zygomorphy and the origin of these varieties. Here, we studied the floral morphogenesis of C. sinense and transcriptome-wide enriched differentially expressed genes among different varieties. The floral zygomorphy of C. sinense is established in the early developmental process. Out of 27 MIKCC-MADS factors, we found two homeotic MADS genes whose expression was down-regulated in peloric varieties but up-regulated in pseudopeloric varieties. CsAP3-2 expressed in the inner floral organs co-operates with a labellum-specific factor CsAGL6-2, asymmetrically promoting the differentiation of inner tepals. Interestingly, we detected exon deletions on CsAP3-2 in peloric varieties, indicating that loss of B-function results in the origin of peloria. Additional petaloid structures developed when we ectopically expressed these genes in Arabidopsis, suggesting their roles in floral morphogenesis. These findings indicate that the interplay among MADS factors would be crucial for orchid floral zygomorphy, and mutations in these factors may have maintained during artificial selection.

Mutant p53 regulates enhancer-associated H3K4 monomethylation through interactions with the methyltransferase MLL4.

Monomethylation of histone H3 lysine 4 (H3K4me1) is enriched at enhancers that are primed for activation and the levels of this histone mark are frequently altered in various human cancers. Yet, how alterations in H3K4me1 are established and the consequences of these epigenetic changes in tumorigenesis are not well understood. Using ChIP-Seq in human colon cancer cells, we demonstrate that mutant p53 depletion results in decreased H3K4me1 levels at active enhancers that reveal a striking colocalization of mutant p53 and the H3K4 monomethyltransferase MLL4 following chronic tumor necrosis factor alpha (TNFα) signaling. We further reveal that mutant p53 forms physiological associations and direct interactions with MLL4 and promotes the enhancer binding of MLL4, which is required for TNFα-inducible H3K4me1 and histone H3 lysine 27 acetylation (H3K27ac) levels, enhancer-derived transcript (eRNA) synthesis, and mutant p53-dependent target gene activation. Complementary in vitro studies with recombinant chromatin and purified proteins demonstrate that binding of the MLL3/4 complex and H3K4me1 deposition is enhanced by mutant p53 and p300-mediated acetylation, which in turn reflects a MLL3/4-dependent enhancement of mutant p53 and p300-dependent transcriptional activation. Collectively, our findings establish a mechanism in which mutant p53 cooperates with MLL4 to regulate aberrant enhancer activity and tumor-promoting gene expression in response to chronic immune signaling.

Mutant p53 shapes the enhancer landscape of cancer cells in response to chronic immune signaling.

Inflammation influences cancer development, progression, and the efficacy of cancer treatments, yet the mechanisms by which immune signaling drives alterations in the cancer cell transcriptome remain unclear. Using ChIP-seq, RNA-seq, and GRO-seq, here we demonstrate a global overlap in the binding of tumor-promoting p53 mutants and the master proinflammatory regulator NFκB that drives alterations in enhancer and gene activation in response to chronic TNF-α signaling. We show that p53 mutants interact directly with NFκB and that both factors impact the other's binding at diverse sets of active enhancers. In turn, the simultaneous and cooperative binding of these factors is required to regulate RNAPII recruitment, the synthesis of enhancer RNAs, and the activation of tumor-promoting genes. Collectively, these findings establish a mechanism by which chronic TNF-α signaling orchestrates a functional interplay between mutant p53 and NFκB that underlies altered patterns of cancer-promoting gene expression.Inflammation is known to affect cancer development, yet the mechanisms by which immune signaling drives transformation remain unclear. Here, the authors provide evidence that chronic TNF-α signaling promotes the enhancer binding and transcriptional interplay between mutant p53 and NFκB.