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.

Active maintenance of CD8+ T cell naivety through regulation of global genome architecture.

The differentiation of naive CD8+ T lymphocytes into cytotoxic effector and memory CTL results in large-scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organization underpin these transcriptional programs. We use Hi-C to map changes in the spatial organization of long-range genome contacts within naive, effector, and memory virus-specific CD8+ T cells. We observe that the architecture of the naive CD8+ T cell genome is distinct from effector and memory genome configurations, with extensive changes within discrete functional chromatin domains associated with effector/memory differentiation. Deletion of BACH2, or to a lesser extent, reducing SATB1 DNA binding, within naive CD8+ T cells results in a chromatin architecture more reminiscent of effector/memory states. This suggests that key transcription factors within naive CD8+ T cells act to restrain T cell differentiation by actively enforcing a unique naive chromatin state.

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.

Active maintenance of CD8+ T cell naïvety through regulation of global genome architecture.

The differentiation of naïve CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naïve, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naïve chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naïve CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naïve chromatin state.

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.

A proposed unified interphase nucleus chromosome structure: Preliminary preponderance of evidence.

Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-µm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [https://en.wikipedia.org/wiki/Slinky; motif used in Bowerman et al., eLife 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.

A proposed unified mitotic chromosome architecture.

A molecular architecture is proposed for a representative mitotic chromosome, human chromosome 10. This architecture is built on an interphase chromosome structure based on cryo-electron microscopy (cryo-EM) cellular tomography [J. Sedat et al., Proc. Natl. Acad. Sci. U.S.A., in press], thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle for mitotic chromosomes is specific coiling of the 11-nm nucleosome fiber into large scale, ∼200-nm interphase structures, a Slinky [https://en.wikipedia.org/wiki/Slinky; motif cited in S. Bowerman et al., eLife 10, e65587 (2021)], then further modified with subsequent additional coiling for the final mitotic chromosome structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well-known cytological configurations. In addition, proof is experimentally provided by digital PCR technology that G1 T cell nuclei are diploid with one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.

Helix-Loop-Helix Proteins in Adaptive Immune Development.

The E/ID protein axis is instrumental for defining the developmental progression and functions of hematopoietic cells. The E proteins are dimeric transcription factors that activate gene expression programs and coordinate changes in chromatin organization. Id proteins are antagonists of E protein activity. Relative levels of E/Id proteins are modulated throughout hematopoietic development to enable the progression of hematopoietic stem cells into multiple adaptive and innate immune lineages including natural killer cells, B cells and T cells. In early progenitors, the E proteins promote commitment to the T and B cell lineages by orchestrating lineage specific programs of gene expression and regulating VDJ recombination of antigen receptor loci. In mature B cells, the E/Id protein axis functions to promote class switch recombination and somatic hypermutation. E protein activity further regulates differentiation into distinct CD4+ and CD8+ T cells subsets and instructs mature T cell immune responses. In this review, we discuss how the E/Id proteins define the adaptive immune system lineages, focusing on their role in directing developmental gene programs.

Bursty gene expression and mRNA decay pathways orchestrate B cell activation.

It is well established that the helix-loop-helix proteins, E2A and E2-2, promote B cell activation. Here, we examined how during the course of B cell activation E2A and E2-2 gene expression is regulated. We found that E2A and E2-2 mRNA abundance concomitantly increased in activated B cells. The increase in E2A and E2-2 mRNA abundance correlated with increased cell growth. Elevated E2A and E2-2 mRNA abundance was instructed by increased transcriptional bursting frequencies and elevated E2A and E2-2 mRNA half-lives. The increase in E2A and E2-2 bursting frequencies often occurred at shared interchromosomal transcriptional hubs. We suggest that in naïve B cells low E2A and E2-2 bursting frequencies and high E2A and E2-2 mRNA decay rates instruct noisy gene expression that allows a clonal and swift response to invading pathogens whereas in activated B cells increased transcriptional bursting and low mRNA decay rates dictate an activated B lineage gene program.

A regulatory network of microRNAs confers lineage commitment during early developmental trajectories of B and T lymphocytes.

The commitment of hematopoietic multipotent progenitors (MPPs) toward a particular lineage involves activation of cell type-specific genes and silencing of genes that promote alternate cell fates. Although the gene expression programs of early-B and early-T lymphocyte development are mutually exclusive, we show that these cell types exhibit significantly correlated microRNA (miRNA) profiles. However, their corresponding miRNA targetomes are distinct and predominated by transcripts associated with natural killer, dendritic cell, and myeloid lineages, suggesting that miRNAs function in a cell-autonomous manner. The combinatorial expression of miRNAs miR-186-5p, miR-128-3p, and miR-330-5p in MPPs significantly attenuates their myeloid differentiation potential due to repression of myeloid-associated transcripts. Depletion of these miRNAs caused a pronounced de-repression of myeloid lineage targets in differentiating early-B and early-T cells, resulting in a mixed-lineage gene expression pattern. De novo motif analysis combined with an assay of promoter activities indicates that B as well as T lineage determinants drive the expression of these miRNAs in lymphoid lineages. Collectively, we present a paradigm that miRNAs are conserved between developing B and T lymphocytes, yet they target distinct sets of promiscuously expressed lineage-inappropriate genes to suppress the alternate cell-fate options. Thus, our studies provide a comprehensive compendium of miRNAs with functional implications for B and T lymphocyte development.

Spatial Organization of Chromatin: Transcriptional Control of Adaptive Immune Cell Development.

Higher-order spatial organization of the genome into chromatin compartments (permissive and repressive), self-associating domains (TADs), and regulatory loops provides structural integrity and offers diverse gene regulatory controls. In particular, chromatin regulatory loops, which bring enhancer and associated transcription factors in close spatial proximity to target gene promoters, play essential roles in regulating gene expression. The establishment and maintenance of such chromatin loops are predominantly mediated involving CTCF and the cohesin machinery. In recent years, significant progress has been made in revealing how loops are assembled and how they modulate patterns of gene expression. Here we will discuss the mechanistic principles that underpin the establishment of three-dimensional (3D) chromatin structure and how changes in chromatin structure relate to alterations in gene programs that establish immune cell fate.

The E protein-TCF1 axis controls γδ T cell development and effector fate.

TCF1 plays a critical role in T lineage commitment and the development of αß lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis.

FOXO1 constrains activation and regulates senescence in CD8 T cells.

Naive and memory T cells are maintained in a quiescent state, yet capable of rapid response and differentiation to antigen challenge via molecular mechanisms that are not fully understood. In naive cells, the deletion of Foxo1 following thymic development results in the increased expression of multiple AP-1 family members, rendering T cells less able to respond to antigenic challenge. Similarly, in the absence of FOXO1, post-infection memory T cells exhibit the characteristics of extended activation and senescence. Age-based analysis of human peripheral T cells reveals that levels of FOXO1 and its downstream target, TCF7, are inversely related to host age, whereas the opposite is found for AP-1 factors. These characteristics of aging also correlate with the formation of T cells manifesting features of cellular senescence. Our work illustrates a role for FOXO1 in the active maintenance of stem-like properties in T cells at the timescales of acute infection and organismal life span.

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.

Heterogeneity and clonal relationships of adaptive immune cells in ulcerative colitis revealed by single-cell analyses.

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

Early precursors and molecular determinants of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing.

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.

Plasma Cell Fate Is Orchestrated by Elaborate Changes in Genome Compartmentalization and Inter-chromosomal Hubs.

The transition from the follicular B to the plasma cell stage is associated with large-scale changes in cell morphology. Here, we examine whether plasma cell development is also associated with changes in nuclear architecture. We find that the onset of plasma cell development is concomitant with a decline in remote genomic interactions; a gain in euchromatic character at loci encoding for factors that specify plasma cell fate, including Prdm1 and Atf4; and establishment of de novo inter-chromosomal hubs. We find that, in developing plasma cells and concurrent with transcriptional silencing, the Ebf1 locus repositions from an euchromatic to peri-centromeric heterochromatic environment. Finally, we find that inter-chromosomal hubs are enriched for the deposition of either H3K27Ac or H3K27me3. These data indicate that plasma cell fate is orchestrated by elaborate changes in genome topology and that epigenetic marks, linked with super-enhancers or transcriptionally repressed regions, are enriched at inter-chromosomal hubs.

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.

Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy.

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure's lateral elements (LEs). While the components of the mammalian chromosome axis/LE-including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2-are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.