Aire in Autoimmunity.

The role of the autoimmune regulator (Aire) in central immune tolerance and thymic self-representation was first described more than 20 years ago, but fascinating new insights into its biology continue to emerge, particularly in the era of advanced single-cell genomics. We briefly describe the role of human genetics in the discovery of Aire, as well as insights into its function gained from genotype-phenotype correlations and the spectrum of Aire-associated autoimmunity-including insights from patients with Aire mutations with broad and diverse implications for human health. We then highlight emerging trends in Aire biology, focusing on three topic areas. First, we discuss medullary thymic epithelial diversity and the role of Aire in thymic epithelial development. Second, we highlight recent developments regarding the molecular mechanisms of Aire and its binding partners. Finally, we describe the rapidly evolving biology of the identity and function of extrathymic Aire-expressing cells (eTACs), and a novel eTAC subset called Janus cells, as well as their potential roles in immune homeostasis.

The CD4 Versus CD8 T Cell Fate Decision: A Multiomics-Informed Perspective.

The choice of developing thymocytes to become CD8+ cytotoxic or CD4+ helper T cells has been intensely studied, but many of the underlying mechanisms remain to be elucidated. Recent multiomics approaches have provided much higher resolution analysis of gene expression in developing thymocytes than was previously achievable, thereby offering a fresh perspective on this question. Focusing on our recent studies using CITE-seq (cellular indexing of transcriptomes and epitopes) analyses of mouse thymocytes, we present a detailed timeline of RNA and protein expression changes during CD8 versus CD4 T cell differentiation. We also revisit our current understanding of the links between T cell receptor signaling and expression of the lineage-defining transcription factors ThPOK and RUNX3. Finally, we propose a sequential selection model to explain the tight linkage between MHC-I versus MHC-II recognition and T cell lineage choice. This model incorporates key aspects of previously proposed kinetic signaling, instructive, and stochastic/selection models.

Gene Regulatory Circuits in Innate and Adaptive Immune Cells.

Cell identity and function largely rely on the programming of transcriptomes during development and differentiation. Signature gene expression programs are orchestrated by regulatory circuits consisting of cis-acting promoters and enhancers, which respond to a plethora of cues via the action of transcription factors. In turn, transcription factors direct epigenetic modifications to revise chromatin landscapes, and drive contacts between distal promoter-enhancer combinations. In immune cells, regulatory circuits for effector genes are especially complex and flexible, utilizing distinct sets of transcription factors and enhancers, depending on the cues each cell type receives during an infection, after sensing cellular damage, or upon encountering a tumor. Here, we review major players in the coordination of gene regulatory programs within innate and adaptive immune cells, as well as integrative omics approaches that can be leveraged to decipher their underlying circuitry.

CD4 Helper and CD8 Cytotoxic T Cell Differentiation.

A fundamental question in developmental immunology is how bipotential thymocyte precursors generate both CD4+ helper and CD8+ cytotoxic T cell lineages. The MHC specificity of αß T cell receptors (TCRs) on precursors is closely correlated with cell fate-determining processes, prompting studies to characterize how variations in TCR signaling are linked with genetic programs establishing lineage-specific gene expression signatures, such as exclusive CD4 or CD8 expression. The key transcription factors ThPOK and Runx3 have been identified as mediating development of helper and cytotoxic T cell lineages, respectively. Together with increasing knowledge of epigenetic regulators, these findings have advanced our understanding of the transcription factor network regulating the CD4/CD8 dichotomy. It has also become apparent that CD4+ T cells retain developmental plasticity, allowing them to acquire cytotoxic activity in the periphery. Despite such advances, further studies are necessary to identify the molecular links between TCR signaling and the nuclear machinery regulating expression of ThPOK and Runx3.

Thymic Epithelial Cells.

Intrathymic T cell development is a complex process that depends upon continuous guidance from thymus stromal cell microenvironments. The thymic epithelium within the thymic stroma comprises highly specialized cells with a high degree of anatomic, phenotypic, and functional heterogeneity. These properties are collectively required to bias thymocyte development toward production of self-tolerant and functionally competent T cells. The importance of thymic epithelial cells (TECs) is evidenced by clear links between their dysfunction and multiple diseases where autoimmunity and immunodeficiency are major components. Consequently, TECs are an attractive target for cell therapies to restore effective immune system function. The pathways and molecular regulators that control TEC development are becoming clearer, as are their influences on particular stages of T cell development. Here, we review both historical and the most recent advances in our understanding of the cellular and molecular mechanisms controlling TEC development, function, dysfunction, and regeneration.

Dual-role transcription factors stabilize intermediate expression levels.

Precise control of gene expression levels is essential for normal cell functions, yet how they are defined and tightly maintained, particularly at intermediate levels, remains elusive. Here, using a series of newly developed sequencing, imaging, and functional assays, we uncover a class of transcription factors with dual roles as activators and repressors, referred to as condensate-forming level-regulating dual-action transcription factors (TFs). They reduce high expression but increase low expression to achieve stable intermediate levels. Dual-action TFs directly exert activating and repressing functions via condensate-forming domains that compartmentalize core transcriptional unit selectively. Clinically relevant mutations in these domains, which are linked to a range of developmental disorders, impair condensate selectivity and dual-action TF activity. These results collectively address a fundamental question in expression regulation and demonstrate the potential of level-regulating dual-action TFs as powerful effectors for engineering controlled expression levels.

Inherited blood cancer predisposition through altered transcription elongation.

Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a ∼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.

DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.

Chromatin context-dependent regulation and epigenetic manipulation of prime editing.

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.

Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor.

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ T cells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ T cells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ T cell subset by coupled T cell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ T cells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ T cell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ T cell exhaustion and could be targeted to potentiate cancer immunotherapy.

ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of anti-tumor immunity.

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.

Cooperative assembly confers regulatory specificity and long-term genetic circuit stability.

A ubiquitous feature of eukaryotic transcriptional regulation is cooperative self-assembly between transcription factors (TFs) and DNA cis-regulatory motifs. It is thought that this strategy enables specific regulatory connections to be formed in gene networks between otherwise weakly interacting, low-specificity molecular components. Here, using synthetic gene circuits constructed in yeast, we find that high regulatory specificity can emerge from cooperative, multivalent interactions among artificial zinc-finger-based TFs. We show that circuits "wired" using the strategy of cooperative TF assembly are effectively insulated from aberrant misregulation of the host cell genome. As we demonstrate in experiments and mathematical models, this mechanism is sufficient to rescue circuit-driven fitness defects, resulting in genetic and functional stability of circuits in long-term continuous culture. Our naturally inspired approach offers a simple, generalizable means for building high-fidelity, evolutionarily robust gene circuits that can be scaled to a wide range of host organisms and applications.

A transcription factor atlas of directed differentiation.

Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.

Global identification of SWI/SNF targets reveals compensation by EP400.

Mammalian SWI/SNF chromatin remodeling complexes move and evict nucleosomes at gene promoters and enhancers to modulate DNA access. Although SWI/SNF subunits are commonly mutated in disease, therapeutic options are limited by our inability to predict SWI/SNF gene targets and conflicting studies on functional significance. Here, we leverage a fast-acting inhibitor of SWI/SNF remodeling to elucidate direct targets and effects of SWI/SNF. Blocking SWI/SNF activity causes a rapid and global loss of chromatin accessibility and transcription. Whereas repression persists at most enhancers, we uncover a compensatory role for the EP400/TIP60 remodeler, which reestablishes accessibility at most promoters during prolonged loss of SWI/SNF. Indeed, we observe synthetic lethality between EP400 and SWI/SNF in cancer cell lines and human cancer patient data. Our data define a set of molecular genomic features that accurately predict gene sensitivity to SWI/SNF inhibition in diverse cancer cell lines, thereby improving the therapeutic potential of SWI/SNF inhibitors.

Orphan quality control shapes network dynamics and gene expression.

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.

CAND1 orchestrates CRLs through rock and roll.

Proper regulation of protein degradation is essential for cell physiology. In the current issue of Cell, Baek et al. elucidated how a large class of ubiquitin ligase, known as CRL, is assembled and disassembled through a key regulator, CAND1.

Chromosome-level organization of the regulatory genome in the Drosophila nervous system.

Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations.

Super-enhancers include classical enhancers and facilitators to fully activate gene expression.

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.

Systemwide disassembly and assembly of SCF ubiquitin ligase complexes.

Cells respond to environmental cues by remodeling their inventories of multiprotein complexes. Cellular repertoires of SCF (SKP1-CUL1-F box protein) ubiquitin ligase complexes, which mediate much protein degradation, require CAND1 to distribute the limiting CUL1 subunit across the family of ∼70 different F box proteins. Yet, how a single factor coordinately assembles numerous distinct multiprotein complexes remains unknown. We obtained cryo-EM structures of CAND1-bound SCF complexes in multiple states and correlated mutational effects on structures, biochemistry, and cellular assays. The data suggest that CAND1 clasps idling catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF. New SCF production proceeds in reverse, through SKP1-F box allosterically destabilizing CAND1. The CAND1-SCF conformational ensemble recycles CUL1 from inactive complexes, fueling mixing and matching of SCF parts for E3 activation in response to substrate availability. Our data reveal biogenesis of a predominant family of E3 ligases, and the molecular basis for systemwide multiprotein complex assembly.

Human microglial state dynamics in Alzheimer's disease progression.

Altered microglial states affect neuroinflammation, neurodegeneration, and disease but remain poorly understood. Here, we report 194,000 single-nucleus microglial transcriptomes and epigenomes across 443 human subjects and diverse Alzheimer's disease (AD) pathological phenotypes. We annotate 12 microglial transcriptional states, including AD-dysregulated homeostatic, inflammatory, and lipid-processing states. We identify 1,542 AD-differentially-expressed genes, including both microglia-state-specific and disease-stage-specific alterations. By integrating epigenomic, transcriptomic, and motif information, we infer upstream regulators of microglial cell states, gene-regulatory networks, enhancer-gene links, and transcription-factor-driven microglial state transitions. We demonstrate that ectopic expression of our predicted homeostatic-state activators induces homeostatic features in human iPSC-derived microglia-like cells, while inhibiting activators of inflammation can block inflammatory progression. Lastly, we pinpoint the expression of AD-risk genes in microglial states and differential expression of AD-risk genes and their regulators during AD progression. Overall, we provide insights underlying microglial states, including state-specific and AD-stage-specific microglial alterations at unprecedented resolution.