Transcriptional and Metabolic Control of Memory B Cells and Plasma Cells.

For many infections and almost all vaccines, neutralizing-antibody-mediated immunity is the primary basis and best functional correlate of immunological protection. Durable long-term humoral immunity is mediated by antibodies secreted by plasma cells that preexist subsequent exposures and by memory B cells that rapidly respond to infections once they have occurred. In the midst of the current pandemic of coronavirus disease 2019, it is important to define our current understanding of the unique roles of memory B cells and plasma cells in immunity and the factors that control the formation and persistence of these cell types. This fundamental knowledge is the basis to interpret findings from natural infections and vaccines. Here, we review transcriptional and metabolic programs that promote and support B cell fates and functions, suggesting points at which these pathways do and do not intersect.

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.

An RNA polymerase that became a Swiss army knife.

RNA polymerases (RNAPs) control the first step of gene expression in all forms of life by transferring genetic information from DNA to RNA, a process known as transcription. In this issue of Cell, Webster et al. and Wu et al. report three-dimensional structures of RNAP complexes from chloroplasts.

Structure of the plant plastid-encoded RNA polymerase.

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined. Here, we present cryoelectron microscopy (cryo-EM) structures of native 21-subunit PEP and a PEP transcription elongation complex from white mustard (Sinapis alba). We identify that PAPs encase the core polymerase, forming extensive interactions that likely promote complex assembly and stability. During elongation, PAPs interact with DNA downstream of the transcription bubble and with the nascent mRNA. The models reveal details of the superoxide dismutase, lysine methyltransferase, thioredoxin, and amino acid ligase enzymes that are subunits of PEP. Collectively, these data provide a foundation for the mechanistic understanding of chloroplast transcription and its role in plant growth and adaptation.

Direct observation of a condensate effect on super-enhancer controlled gene bursting.

Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.

Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.

Leveraging AAVs' versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screening with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 vectors across AAV serotypes combined with a transposon system, we substantially amplified labeling efficacy and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function.

Molecular Mechanisms of Transcription-Coupled Repair.

Transcription-coupled repair (TCR), discovered as preferential nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers located in transcribed mammalian genes compared to those in nontranscribed regions of the genome, is defined as faster repair of the transcribed strand versus the nontranscribed strand in transcribed genes. The phenomenon, universal in model organisms including Escherichia coli, yeast, Arabidopsis, mice, and humans, involves a translocase that interacts with both RNA polymerase stalled at damage in the transcribed strand and nucleotide excision repair proteins to accelerate repair. Drosophila, a notable exception, exhibits TCR but lacks an obvious TCR translocase. Mutations inactivating TCR genes cause increased damage-induced mutagenesis in E. coli and severe neurological and UV sensitivity syndromes in humans. To date, only E. coli TCR has been reconstituted in vitro with purified proteins. Detailed investigations of TCR using genome-wide next-generation sequencing methods, cryo-electron microscopy, single-molecule analysis, and other approaches have revealed fascinating mechanisms.

Transcription-Coupled Nucleotide Excision Repair and the Transcriptional Response to UV-Induced DNA Damage.

Ultraviolet (UV) irradiation and other genotoxic stresses induce bulky DNA lesions, which threaten genome stability and cell viability. Cells have evolved two main repair pathways to remove such lesions: global genome nucleotide excision repair (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER). The modes by which these subpathways recognize DNA lesions are distinct, but they converge onto the same downstream steps for DNA repair. Here, we first summarize the current understanding of these repair mechanisms, specifically focusing on the roles of stalled RNA polymerase II, Cockayne syndrome protein B (CSB), CSA and UV-stimulated scaffold protein A (UVSSA) in TC-NER. We also discuss the intriguing role of protein ubiquitylation in this process. Additionally, we highlight key aspects of the effect of UV irradiation on transcription and describe the role of signaling cascades in orchestrating this response. Finally, we describe the pathogenic mechanisms underlying xeroderma pigmentosum and Cockayne syndrome, the two main diseases linked to mutations in NER factors.

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.

Functional partitioning of transcriptional regulators by patterned charge blocks.

Components of transcriptional machinery are selectively partitioned into specific condensates, often mediated by protein disorder, yet we know little about how this specificity is achieved. Here, we show that condensates composed of the intrinsically disordered region (IDR) of MED1 selectively partition RNA polymerase II together with its positive allosteric regulators while excluding negative regulators. This selective compartmentalization is sufficient to activate transcription and is required for gene activation during a cell-state transition. The IDRs of partitioned proteins are necessary and sufficient for selective compartmentalization and require alternating blocks of charged amino acids. Disrupting this charge pattern prevents partitioning, whereas adding the pattern to proteins promotes partitioning with functional consequences for gene activation. IDRs with similar patterned charge blocks show similar partitioning and function. These findings demonstrate that disorder-mediated interactions can selectively compartmentalize specific functionally related proteins from a complex mixture of biomolecules, leading to regulation of a biochemical pathway.

RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size.

A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.

A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery.

In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome.

Transcriptional regulation of innate and adaptive lymphocyte lineages.

The lymphocyte family has expanded significantly in recent years to include not only the adaptive lymphocytes (T cells, B cells) and NK cells, but also several additional innate lymphoid cell (ILC) types. ILCs lack clonally distributed antigen receptors characteristic of adaptive lymphocytes and instead respond exclusively to signaling via germline-encoded receptors. ILCs resemble T cells more closely than any other leukocyte lineage at the transcriptome level and express many elements of the core T cell transcriptional program, including Notch, Gata3, Tcf7, and Bcl11b. We present our current understanding of the shared and distinct transcriptional regulatory mechanisms involved in the development of adaptive T lymphocytes and closely related ILCs. We discuss the possibility that a core set of transcriptional regulators common to ILCs and T cells establish enhancers that enable implementation of closely aligned effector pathways. Studies of the transcriptional regulation of lymphopoiesis will support the development of novel therapeutic approaches to correct early lymphoid developmental defects and aberrant lymphocyte function.

Non-coding 7S RNA inhibits transcription via mitochondrial RNA polymerase dimerization.

The mitochondrial genome encodes 13 components of the oxidative phosphorylation system, and altered mitochondrial transcription drives various human pathologies. A polyadenylated, non-coding RNA molecule known as 7S RNA is transcribed from a region immediately downstream of the light strand promoter in mammalian cells, and its levels change rapidly in response to physiological conditions. Here, we report that 7S RNA has a regulatory function, as it controls levels of mitochondrial transcription both in vitro and in cultured human cells. Using cryo-EM, we show that POLRMT dimerization is induced by interactions with 7S RNA. The resulting POLRMT dimer interface sequesters domains necessary for promoter recognition and unwinding, thereby preventing transcription initiation. We propose that the non-coding 7S RNA molecule is a component of a negative feedback loop that regulates mitochondrial transcription in mammalian cells.

Parallel analysis of transcription, integration, and sequence of single HIV-1 proviruses.

HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.

Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite.

The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.

Designing Biological Circuits: Synthetic Biology Within the Operon Model and Beyond.

In 1961, Jacob and Monod proposed the operon model of gene regulation. At the model's core was the modular assembly of regulators, operators, and structural genes. To illustrate the composability of these elements, Jacob and Monod linked phenotypic diversity to the architectures of regulatory circuits. In this review, we examine how the circuit blueprints imagined by Jacob and Monod laid the foundation for the first synthetic gene networks that launched the field of synthetic biology in 2000. We discuss the influences of the operon model and its broader theoretical framework on the first generation of synthetic biological circuits, which were predominantly transcriptional and posttranscriptional circuits. We also describe how recent advances in molecular biology beyond the operon model-namely, programmable DNA- and RNA-binding molecules as well as models of epigenetic and posttranslational regulation-are expanding the synthetic biology toolkit and enabling the design of more complex biological circuits.

Influenza Virus RNA-Dependent RNA Polymerase and the Host Transcriptional Apparatus.

Influenza virus RNA-dependent RNA polymerase (FluPol) transcribes the viral RNA genome in the infected cell nucleus. In the 1970s, researchers showed that viral transcription depends on host RNA polymerase II (RNAP II) activity and subsequently that FluPol snatches capped oligomers from nascent RNAP II transcripts to prime its own transcription. Exactly how this occurs remains elusive. Here, we review recent advances in the mechanistic understanding of FluPol transcription and early events in RNAP II transcription that are relevant to cap-snatching. We describe the known direct interactions between FluPol and the RNAP II C-terminal domain and summarize the transcription-related host factors that have been found to interact with FluPol. We also discuss open questions regarding how FluPol may be targeted to actively transcribing RNAP II and the exact context and timing of cap-snatching, which is presumed to occur after cap completion but before the cap is sequestered by the nuclear cap-binding complex.

Chromatin contributions to the regulation of innate immunity.

A fundamental property of cells of the innate immune system is their ability to elicit a transcriptional response to a microbial stimulus or danger signal with a high degree of cell type and stimulus specificity. The selective response activates effector pathways to control the insult and plays a central role in regulating adaptive immunity through the differential regulation of cytokine genes. Selectivity is dictated by signaling pathways and their transcription factor targets. However, a growing body of evidence supports models in which different subsets of genes exhibit distinct chromatin features that play active roles in shaping the response. Chromatin also participates in innate memory mechanisms that can promote tolerance to a stimulus or prime cells for a more robust response. These findings have generated interest in the capacity to modulate chromatin regulators with small-molecule compounds for the treatment of diseases associated with innate or adaptive immunity.