5-Formylcytosine is an activating epigenetic mark for RNA Pol III during zygotic reprogramming.

5-Methylcytosine (5mC) is an established epigenetic mark in vertebrate genomic DNA, but whether its oxidation intermediates formed during TET-mediated DNA demethylation possess an instructive role of their own that is also physiologically relevant remains unresolved. Here, we reveal a 5-formylcytosine (5fC) nuclear chromocenter, which transiently forms during zygotic genome activation (ZGA) in Xenopus and mouse embryos. We identify this chromocenter as the perinucleolar compartment, a structure associated with RNA Pol III transcription. In Xenopus embryos, 5fC is highly enriched on Pol III target genes activated at ZGA, notably at oocyte-type tandem arrayed tRNA genes. By manipulating Tet and Tdg enzymes, we show that 5fC is required as a regulatory mark to promote Pol III recruitment as well as tRNA expression. Concordantly, 5fC modification of a tRNA transgene enhances its expression in vivo. The results establish 5fC as an activating epigenetic mark during zygotic reprogramming of Pol III gene expression.

Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

Programmable protein circuit design.

A fundamental challenge in synthetic biology is to create molecular circuits that can program complex cellular functions. Because proteins can bind, cleave, and chemically modify one another and interface directly and rapidly with endogenous pathways, they could extend the capabilities of synthetic circuits beyond what is possible with gene regulation alone. However, the very diversity that makes proteins so powerful also complicates efforts to harness them as well-controlled synthetic circuit components. Recent work has begun to address this challenge, focusing on principles such as orthogonality and composability that permit construction of diverse circuit-level functions from a limited set of engineered protein components. These approaches are now enabling the engineering of circuits that can sense, transmit, and process information; dynamically control cellular behaviors; and enable new therapeutic strategies, establishing a powerful paradigm for programming biology.

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

Cytomegalovirus subverts macrophage identity.

Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.

Revisiting astrocyte to neuron conversion with lineage tracing in vivo.

In vivo cell fate conversions have emerged as potential regeneration-based therapeutics for injury and disease. Recent studies reported that ectopic expression or knockdown of certain factors can convert resident astrocytes into functional neurons with high efficiency, region specificity, and precise connectivity. However, using stringent lineage tracing in the mouse brain, we show that the presumed astrocyte-converted neurons are actually endogenous neurons. AAV-mediated co-expression of NEUROD1 and a reporter specifically and efficiently induces reporter-labeled neurons. However, these neurons cannot be traced retrospectively to quiescent or reactive astrocytes using lineage-mapping strategies. Instead, through a retrograde labeling approach, our results reveal that endogenous neurons are the source for these viral-reporter-labeled neurons. Similarly, despite efficient knockdown of PTBP1 in vivo, genetically traced resident astrocytes were not converted into neurons. Together, our results highlight the requirement of lineage-tracing strategies, which should be broadly applied to studies of cell fate conversions in vivo.

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

Caloric Restriction Reprograms the Single-Cell Transcriptional Landscape of Rattus Norvegicus Aging.

Aging causes a functional decline in tissues throughout the body that may be delayed by caloric restriction (CR). However, the cellular profiles and signatures of aging, as well as those ameliorated by CR, remain unclear. Here, we built comprehensive single-cell and single-nucleus transcriptomic atlases across various rat tissues undergoing aging and CR. CR attenuated aging-related changes in cell type composition, gene expression, and core transcriptional regulatory networks. Immune cells were increased during aging, and CR favorably reversed the aging-disturbed immune ecosystem. Computational prediction revealed that the abnormal cell-cell communication patterns observed during aging, including the excessive proinflammatory ligand-receptor interplay, were reversed by CR. Our work provides multi-tissue single-cell transcriptional landscapes associated with aging and CR in a mammal, enhances our understanding of the robustness of CR as a geroprotective intervention, and uncovers how metabolic intervention can act upon the immune system to modify the process of aging.

Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

Optimal-Transport Analysis of Single-Cell Gene Expression Identifies Developmental Trajectories in Reprogramming.

Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them. We apply the method to reconstruct the landscape of reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18 days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.

Wnt-ß-catenin activation epigenetically reprograms Treg cells in inflammatory bowel disease and dysplastic progression.

The diversity of regulatory T (Treg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt+ Treg cells with elevated expression of ß-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt+ Treg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-ß-catenin signaling in human and murine Treg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt+ Treg cells coexpressing multiple pro-inflammatory cytokines. Binding of the ß-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-ß-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Treg cells. Activation of ß-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

Interleukin-34-orchestrated tumor-associated macrophage reprogramming is required for tumor immune escape driven by p53 inactivation.

As the most frequent genetic alteration in cancer, more than half of human cancers have p53 mutations that cause transcriptional inactivation. However, how p53 modulates the immune landscape to create a niche for immune escape remains elusive. We found that cancer stem cells (CSCs) established an interleukin-34 (IL-34)-orchestrated niche to promote tumorigenesis in p53-inactivated liver cancer. Mechanistically, we discovered that Il34 is a gene transcriptionally repressed by p53, and p53 loss resulted in IL-34 secretion by CSCs. IL-34 induced CD36-mediated elevations in fatty acid oxidative metabolism to drive M2-like polarization of foam-like tumor-associated macrophages (TAMs). These IL-34-orchestrated TAMs suppressed CD8+ T cell-mediated antitumor immunity to promote immune escape. Blockade of the IL-34-CD36 axis elicited antitumor immunity and synergized with anti-PD-1 immunotherapy, leading to a complete response. Our findings reveal the underlying mechanism of p53 modulation of the tumor immune microenvironment and provide a potential target for immunotherapy of cancer with p53 inactivation.

Direct cell reprogramming: approaches, mechanisms and progress.

The reprogramming of somatic cells with defined factors, which converts cells from one lineage into cells of another, has greatly reshaped our traditional views on cell identity and cell fate determination. Direct reprogramming (also known as transdifferentiation) refers to cell fate conversion without transitioning through an intermediary pluripotent state. Given that the number of cell types that can be generated by direct reprogramming is rapidly increasing, it has become a promising strategy to produce functional cells for therapeutic purposes. This Review discusses the evolution of direct reprogramming from a transcription factor-based method to a small-molecule-driven approach, the recent progress in enhancing reprogrammed cell maturation, and the challenges associated with in vivo direct reprogramming for translational applications. It also describes our current understanding of the molecular mechanisms underlying direct reprogramming, including the role of transcription factors, epigenetic modifications, non-coding RNAs, and the function of metabolic reprogramming, and highlights novel insights gained from single-cell omics studies.

Placeholder Nucleosomes Underlie Germline-to-Embryo DNA Methylation Reprogramming.

The fate and function of epigenetic marks during the germline-to-embryo transition is a key issue in developmental biology, with relevance to stem cell programming and transgenerational inheritance. In zebrafish, DNA methylation patterns are programmed in transcriptionally quiescent cleavage embryos; paternally inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal. Here, we provide the mechanism by demonstrating that "Placeholder" nucleosomes, containing histone H2A variant H2A.Z(FV) and H3K4me1, virtually occupy all regions lacking DNA methylation in both sperm and cleavage embryos and reside at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with Placeholder become either active (H3K4me3) or silent (H3K4me3/K27me3). Notably, perturbations causing Placeholder loss confer DNA methylation accumulation, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent gametic and embryonic stages, an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNA methylation, poising parental genes for either gene-specific activation or facultative repression.

Western Diet Triggers NLRP3-Dependent Innate Immune Reprogramming.

Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr-/- mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3-/-/Ldlr-/- mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.

Nudt21 Controls Cell Fate by Connecting Alternative Polyadenylation to Chromatin Signaling.

Cell fate transitions involve rapid gene expression changes and global chromatin remodeling, yet the underlying regulatory pathways remain incompletely understood. Here, we identified the RNA-processing factor Nudt21 as a novel regulator of cell fate change using transcription-factor-induced reprogramming as a screening assay. Suppression of Nudt21 enhanced the generation of induced pluripotent stem cells, facilitated transdifferentiation into trophoblast stem cells, and impaired differentiation of myeloid precursors and embryonic stem cells, suggesting a broader role for Nudt21 in cell fate change. We show that Nudt21 directs differential polyadenylation of over 1,500 transcripts in cells acquiring pluripotency, although only a fraction changed protein levels. Remarkably, these proteins were strongly enriched for chromatin regulators, and their suppression neutralized the effect of Nudt21 during reprogramming. Collectively, our data uncover Nudt21 as a novel post-transcriptional regulator of cell fate and establish a direct, previously unappreciated link between alternative polyadenylation and chromatin signaling.

Cell Reprogramming: The Many Roads to Success.

Cellular reprogramming experiments from somatic cell types have demonstrated the plasticity of terminally differentiated cell states. Recent efforts in understanding the mechanisms of cellular reprogramming have begun to elucidate the differentiation trajectories along the reprogramming processes. In this review, we focus mainly on direct reprogramming strategies by transcription factors and highlight the variables that contribute to cell fate conversion outcomes. We review key studies that shed light on the cellular and molecular mechanisms by investigating differentiation trajectories and alternative cell states as well as transcription factor regulatory activities during cell fate reprogramming. Finally, we highlight a few concepts that we believe require attention, particularly when measuring the success of cell reprogramming experiments.

The angiocrine Rspondin3 instructs interstitial macrophage transition via metabolic-epigenetic reprogramming and resolves inflammatory injury.

Macrophages demonstrate remarkable plasticity that is essential for host defense and tissue repair. The tissue niche imprints macrophage identity, phenotype and function. The role of vascular endothelial signals in tailoring the phenotype and function of tissue macrophages remains unknown. The lung is a highly vascularized organ and replete with a large population of resident macrophages. We found that, in response to inflammatory injury, lung endothelial cells release the Wnt signaling modulator Rspondin3, which activates ß-catenin signaling in lung interstitial macrophages and increases mitochondrial respiration by glutaminolysis. The generated tricarboxylic acid cycle intermediate α-ketoglutarate, in turn, serves as the cofactor for the epigenetic regulator TET2 to catalyze DNA hydroxymethylation. Notably, endothelial-specific deletion of Rspondin3 prevented the formation of anti-inflammatory interstitial macrophages in endotoxemic mice and induced unchecked severe inflammatory injury. Thus, the angiocrine-metabolic-epigenetic signaling axis specified by the endothelium is essential for reprogramming interstitial macrophages and dampening inflammatory injury.

Environmental cues regulate epigenetic reprogramming of airway-resident memory CD8+ T cells.

Tissue-resident memory T cells (TRM cells) are critical for cellular immunity to respiratory pathogens and reside in both the airways and the interstitium. In the present study, we found that the airway environment drove transcriptional and epigenetic changes that specifically regulated the cytolytic functions of airway TRM cells and promoted apoptosis due to amino acid starvation and activation of the integrated stress response. Comparison of airway TRM cells and splenic effector-memory T cells transferred into the airways indicated that the environment was necessary to activate these pathways, but did not induce TRM cell lineage reprogramming. Importantly, activation of the integrated stress response was reversed in airway TRM cells placed in a nutrient-rich environment. Our data defined the genetic programs of distinct lung TRM cell populations and show that local environmental cues altered airway TRM cells to limit cytolytic function and promote cell death, which ultimately leads to fewer TRM cells in the lung.

BATF3 programs CD8+ T cell memory.

Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.