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Mammalian aging is characterized by the progressive loss of tissue function and increased risk for disease. Accumulation of senescent cells in aging tissues partly contributes to this decline, and targeted depletion of senescent cells in vivo ameliorates many age-related phenotypes. The fundamental molecular mechanisms responsible for the decline of cellular health and fitness during senescence and aging are largely unknown. In this study, we investigated whether chromatin-mediated loss of transcriptional fidelity, known to contribute to fitness and survival in yeast and worms, also occurs during human cellular senescence and mouse aging. Our findings reveal aberrant transcription initiation inside genes during senescence and aging that co-occurs with changes in the chromatin landscape. Interventions that alter these spurious transcripts have profound consequences on cellular health, primarily affecting intracellular signal transduction pathways. We propose that age-related spurious transcription promotes a noisy transcriptome and degradation of coherent transcriptional networks.

DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR-Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs to date utilize a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered base editors (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.

Protein aggregation is the hallmark of neurodegeneration, but the molecular mechanisms underlying late-onset Alzheimer's disease (AD) are unclear. Here we integrated transcriptomic, proteomic and epigenomic analyses of postmortem human brains to identify molecular pathways involved in AD. RNA sequencing analysis revealed upregulation of transcription- and chromatin-related genes, including the histone acetyltransferases for H3K27ac and H3K9ac. An unbiased proteomic screening singled out H3K27ac and H3K9ac as the main enrichments specific to AD. In turn, epigenomic profiling revealed gains in the histone H3 modifications H3K27ac and H3K9ac linked to transcription, chromatin and disease pathways in AD. Increasing genome-wide H3K27ac and H3K9ac in a fly model of AD exacerbated amyloid-β42-driven neurodegeneration. Together, these findings suggest that AD involves a reconfiguration of the epigenome, wherein H3K27ac and H3K9ac affect disease pathways by dysregulating transcription- and chromatin-gene feedback loops. The identification of this process highlights potential epigenetic strategies for early-stage disease treatment.

CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells., Reliable, multiplexed gene editing to uncover synergies between targets remains challenging. Here, the authors engineer AsCas12a and the crRNA to improve double knockout for synthetic sick/lethal interaction genetic screening.

The human genome can be segmented into topologically associating domains (TADs), which have been proposed to spatially sequester genes and regulatory elements through chromatin looping. Interactions between TADs have also been suggested, presumably because of variable boundary positions across individual cells. However, the nature, extent and consequence of these dynamic boundaries remain unclear. Here, we combine high-resolution imaging with Oligopaint technology to quantify the interaction frequencies across both weak and strong boundaries. We find that chromatin intermingling across population-defined boundaries is widespread but that the extent of permissibility is locus-specific. Cohesin depletion, which abolishes domain formation at the population level, does not induce ectopic interactions but instead reduces interactions across all boundaries tested. In contrast, WAPL or CTCF depletion increases inter-domain contacts in a cohesin-dependent manner. Reduced chromatin intermingling due to cohesin loss affects the topology and transcriptional bursting frequencies of genes near boundaries. We propose that cohesin occasionally bypasses boundaries to promote incorporation of boundary-proximal genes into neighboring domains.

Features of higher-order chromatin organization-such as A/B compartments, topologically associating domains and chromatin loops-are temporarily disrupted during mitosis1,2. Because these structures are thought to influence gene regulation, it is important to understand how they are re-established after mitosis. Here we examine the dynamics of chromosome reorganization by Hi-C after mitosis in highly purified, synchronous mouse erythroid cell populations. We observed rapid establishment of A/B compartments, followed by their gradual intensification and expansion. Contact domains form from the 'bottom up'-smaller subTADs are formed initially, followed by convergence into multi-domain TAD structures. CTCF is partially retained on mitotic chromosomes and immediately resumes full binding in ana/telophase. By contrast, cohesin is completely evicted from mitotic chromosomes and regains focal binding at a slower rate. The formation of CTCF/cohesin co-anchored structural loops follows the kinetics of cohesin positioning. Stripe-shaped contact patterns-anchored by CTCF-grow in length, which is consistent with a loop-extrusion process after mitosis. Interactions between cis-regulatory elements can form rapidly, with rates exceeding those of CTCF/cohesin-anchored contacts. Notably, we identified a group of rapidly emerging transient contacts between cis-regulatory elements in ana/telophase that are dissolved upon G1 entry, co-incident with the establishment of inner boundaries or nearby interfering chromatin loops. We also describe the relationship between transcription reactivation and architectural features. Our findings indicate that distinct but mutually influential forces drive post-mitotic chromatin reconfiguration.

During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. We exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to genome re-sculpting in newborn nuclei. Depletion of CTCF during the M- to G1-phase transition alters short-range compartmentalization after mitosis. Chromatin domain boundary re-formation is impaired upon CTCF loss, but a subset of boundaries, characterized by transitions in chromatin states, is established normally. Without CTCF, structural loops fail to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persist deeply into G1-phase in CTCF-depleted cells. CTCF loss-associated gains in transcription are often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declines upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitate formation of CRE loops nested within them, especially those involving weak CREs. Transcription inhibition does not significantly affect global architecture or transcription start site-associated boundaries. However, ongoing transcription contributes considerably to the formation of gene domains, regions of enriched contacts along gene bodies. Notably, gene domains emerge in ana/telophase prior to completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yields insights into the contributions of CTCF and transcription to chromatin architecture dynamics during the mitosis to G1-phase progression., Higher-order chromatin structure is temporarily disrupted during mitosis. Here the authors show that loss of the architectural factor CTCF results in failure to form structural loops and leads to inappropriate cis-regulatory contacts and alterations of compartmental interactions after mitosis. Furthermore, they show global 3D architecture is set up without transcription, but that transcription contributes to proper gene domain formation.

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, there is increasing concern about the long-term and sex-specific health implications. Augmenting our IVF mouse model to longitudinally investigate metabolic outcomes in offspring from optimal neonatal litter sizes, we found sex-specific metabolic outcomes in IVF offspring. IVF-conceived females had higher body weight and cholesterol levels compared to naturally conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through adult liver transcriptomics and proteomics, we identified sexually dimorphic dysregulation of the sterol regulatory element-binding protein (SREBP) pathways that are associated with the sex-specific phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.

SUMMARY CCCTC-binding factor (CTCF) is a conserved zinc finger transcription factor implicated in a wide range of functions, including genome organization, transcription activation, and elongation. To explore the basis for CTCF functional diversity, we coupled an auxin-induced degron system with precision nuclear run-on. Unexpectedly, oriented CTCF motifs in gene bodies are associated with transcriptional stalling in a manner independent of bound CTCF. Moreover, CTCF at different binding sites (CBSs) displays highly variable resistance to degradation. Motif sequence does not significantly predict degradation behavior, but location at chromatin boundaries and chromatin loop anchors, as well as co-occupancy with cohesin, are associated with delayed degradation. Single-molecule tracking experiments link chromatin residence time to CTCF degradation kinetics, which has ramifications regarding architectural CTCF functions. Our study highlights the heterogeneity of CBSs, uncovers properties specific to architecturally important CBSs, and provides insights into the basic processes of genome organization and transcription regulation., Graphical Abstract, In brief Using an inducible CTCF degradation approach, Luan et al. show that sensitivity to degradation of CTCF on chromatin is highly variable, reflecting its diverse roles in chromatin architecture and transcription.

Adult stem cells play a critical role in tissue homeostasis and repair. Aging leads to a decline in stem cells’ regenerative capacity that contributes significantly to the maintenance of organ and tissue functions. Age-dependent genomic and epigenetic modifications together play a role in the disruption of critical cellular pathways. However, the epigenetic mechanisms responsible for the decline of adult stem cell functions remain to be well established. Here, we investigated age-dependent, genome-wide alterations in the chromatin accessibility of primary human adipose-derived stem cells (ASCs) in comparison to age-matched fibroblasts via ATAC-seq technology. Our results demonstrate that aging ASCs possess globally more stable chromatin accessibility profiles as compared to aging fibroblasts, suggesting that robust regulatory mechanisms maintain adult stem cell chromatin structure against aging. Furthermore, we observed age-dependent subtle changes in promoter nucleosome positioning in selective pathways during aging, concurrent with altered small ubiquitin-related modifier (SUMO) protein expression under stress conditions. Together, our data suggest a significant role for nucleosome positioning in sumoylation pathway regulation in stress response during adult stem cell aging. The differences described here between the chromatin structure of human ASCs and fibroblasts will further elucidate the mechanisms regulating gene expression during aging in both stem cells and differentiated cells.

Aging is the strongest risk factor for Alzheimer’s disease (AD), although the underlying mechanisms remain unclear. The chromatin state, in particular through the mark H4K16ac, has been implicated in aging and thus may play a pivotal role in age-associated neurodegeneration. Here we compare the genome-wide enrichment of H4K16ac in the lateral temporal lobe of AD individuals against both younger and elderly cognitively normal controls. We found that while normal aging leads to H4K16ac enrichment, AD entails dramatic losses of H4K16ac in the proximity of genes linked to aging and AD. Our analysis highlights the presence of three classes of AD-related changes with distinctive functional roles. Furthermore, we discovered an association between the genomic locations of significant H4K16ac changes with genetic variants identified in prior AD genome-wide association studies and with expression quantitative trait loci. Our results establish the basis for an epigenetic link between aging and AD.

SUMMARY Dosage compensation in Drosophila melanogaster involves a 2-fold transcriptional upregulation of the male X chromosome, which relies on the X-chromosome-binding males-specific lethal (MSL) complex. However, how such 2-fold precision is accomplished remains unclear. Here, we show that a nuclear pore component, Mtor, is involved in setting the correct levels of transcription from the male X chromosome. Using larval tissues, we demonstrate that the depletion of Mtor results in selective upregulation at MSL targets of the male X, beyond the required 2-fold. Mtor and MSL components interact genetically, and depletion of Mtor can rescue the male lethality phenotype of MSL components. Using RNA fluorescence in situ hybridization (FISH) analysis and nascent transcript sequencing, we find that the effect of Mtor is not due to defects in mRNA export but occurs at the level of nascent transcription. These findings demonstrate a physiological role for Mtor in the process of dosage compensation, as a transcriptional attenuator of X chromosome gene expression., Graphical Abstract, In brief During dosage compensation, the expression from the single X chromosome of male flies is upregulated to match the two X’s of females. Aleman et al. show that a nuclear pore protein functions in achieving this transcriptional precision and restricts the upregulation of the male X to the required 2-fold.

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. Here, we report high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. Our investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, we identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. Our findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93.

Active DNA demethylation via Ten-eleven Translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically-engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.

Transcription factors typically regulate a large number of genes. Here we found that transcription factor ZNF410 binds and activates the expression of a single direct target gene, CHD4, to enforce the silencing of the fetal hemoglobin genes (HBG1 and HBG2) in adult erythroid cells. ZNF410 is a pentadactyl DNA binding protein that emerged from a DNA binding domain-focused CRISPR-Cas9 screen aimed at the identification of new regulators of fetal hemoglobin silencing. Depletion of ZNF410 specifically diminished CHD4 expression, leading to reactivation of the normally silent fetal globin genes in both human erythroid culture systems and a human-to-mouse xenotransplant model. Combining RNA-seq and ChIP-seq analyses revealed that CHD4 is the sole direct ZNF410 target gene in erythroid cells, which was further validated by rescue of fetal hemoglobin silencing and other transcriptional changes upon CHD4 restoration in ZNF410-deficient cells. ZNF410 ChIP-seq detected only eight high-confidence peaks with seven associated genes including CHD4. Most strikingly, the two most predominant peaks are located at the CHD4 locus, which contains two highly conserved, dense clusters of ZNF410 binding motifs. The two motif clusters appear to be unique in the human and mouse genomes. Moreover, among the seven ZNF410-bound genes, CHD4 was the only one whose expression was down-regulated upon ZNF410 depletion, indicating that CHD4 is the sole target of ZNF410. Electrophoretic mobility shift assays (EMSAs) showed that the zinc finger (ZF) domain of ZNF410 is necessary and sufficient for DNA binding. When overexpressed, the DNA binding profile of ZF domain alone is very similar to full length ZNF410. Indeed, forced expression of the ZF domain displaced endogenous ZNF410 at all binding sites, including the CHD4 locus. This reduced CHD4 expression to levels comparable to those in ZNF410 deficient cells (and activated the fetal globin genes) but had no effect on the other ZNF410 bound genes, again confirming target specificity. ZNF410 depletion or expression of the dominant negative acting ZF domain lowered CHD4 only by ~65%-70%, which is very well tolerated by erythroid cells, as determined by morphology, cell surface phenotyping, and gene expression profiling. This exposes the fetal globin genes as highly sensitive to CHD4 levels. Lastly, we solved the crystal structure of the ZF domain-DNA complex at 2.75Å resolution pinpointing the protein-DNA contacts and showing that each of the five ZFs make specific DNA contacts. In sum, to our knowledge, ZNF410 is the only transcription factor with just one direct functional target gene in erythroid cells. Given the strong impetus to reactivate fetal globin gene expression in patients with sickle cell disease and some forms of b-thalassemia, it might be possible to exploit the exceptionally high transcriptional selectivity of ZNF410 to raise fetal hemoglobin expression for the treatment of these hemoglobinopathies. Disclosures Weiss: Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company. Blobel:Fulcrum Therapeutics: Consultancy; Pfizer: Research Funding.

Fetal exposure to endocrine disrupting chemicals (EDCs) has been associated with adverse neurobehavioral outcomes across the lifespan and can persist across multiple generations of offspring. However, the underlying mechanisms driving these changes are not well understood. We investigated the molecular perturbations associated with EDC-induced behavioral changes in first (F1) and second (F2) filial generations, using the model EDC bisphenol A (BPA). C57BL/6J dams were exposed to BPA from preconception until lactation through the diet at doses (10 μg/kg bw/d-lower dose or 10 mg/kg bw/d-upper dose) representative of human exposure levels. As adults, F1 male offspring exhibited increased depressive-like behavior, measured by the forced swim test, while females were unaffected. These behavioral changes were limited to the F1 generation and were not associated with altered maternal care. Transcriptome analysis by RNA-sequencing in F1 control and upper dose BPA-exposed adult male hippocampus revealed neurotransmitter systems as major pathways disrupted by developmental BPA exposure. High performance liquid chromatography demonstrated a male-specific reduction in hippocampal serotonin. Administration of the selective serotonin reuptake inhibitor fluoxetine (20 mg/kg bw) rescued the depressive-like phenotype in males exposed to lower, but not upper, dose BPA, suggesting distinct mechanisms of action for each exposure dose. Finally, high resolution mass spectrometry revealed reduced circulating levels of the neuroactive steroid dehydroepiandrosterone in BPA-exposed males, suggesting another potential mechanism underlying the depressive-like phenotype. Thus, behavioral changes associated with early life BPA exposure may be mediated by sex-specific disruptions in the serotonergic system and/or sex steroid biogenesis in male offspring.

Accumulation of senescent cells during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic proteins and performed a high-throughput screen to identify key candidates whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence, and suggests p300 as a potential therapeutic target for senescence and age-related diseases.

Nitrogen acquisition is a major challenge for herbivorous animals, and the repeated origins of herbivory across the ants have raised expectations that nutritional symbionts have shaped their diversification. Direct evidence for N provisioning by internally housed symbionts is rare in animals; among the ants, it has been documented for just one lineage. In this study we dissect functional contributions by bacteria from a conserved, multi-partite gut symbiosis in herbivorous Cephalotes ants through in vivo experiments, metagenomics, and in vitro assays. Gut bacteria recycle urea, and likely uric acid, using recycled N to synthesize essential amino acids that are acquired by hosts in substantial quantities. Specialized core symbionts of 17 studied Cephalotes species encode the pathways directing these activities, and several recycle N in vitro. These findings point to a highly efficient N economy, and a nutritional mutualism preserved for millions of years through the derived behaviors and gut anatomy of Cephalotes ants., Gut bacteria are prevalent across insects including ants, but their precise roles are often unclear. Here, Hu et al. show that microbes aid ants by recycling nitrogen into bio-available amino acids. This function is conserved across the turtle ants, suggesting an ancient nutritional mutualism.

SUMMARY Self-renewing somatic tissues depend upon the proper balance of chromatin-modifying enzymes to coordinate progenitor cell maintenance and differentiation, disruption of which can promote carcinogenesis. As a result, drugs targeting the epigenome hold significant therapeutic potential. The histone demethylase, LSD1 (KDM1A), is overexpressed in numerous cancers, including epithelial cancers; however, its role in the skin is virtually unknown. Here we show that LSD1 directly represses master epithelial transcription factors that promote differentiation. LSD1 inhibitors block both LSD1 binding to chromatin and its catalytic activity, driving significant increases in H3K4 methylation and gene transcription of these fate-determining transcription factors. This leads to both premature epidermal differentiation and the repression of squamous cell carcinoma. Together these data highlight both LSD1’s role in maintaining the epidermal progenitor state and the potential of LSD1 inhibitors for the treatment of keratinocyte cancers, which collectively outnumber all other cancers combined., Graphical Abstract, In Brief Egolf et al. demonstrate that inhibition of the epigenetic regulator and histone demethylase, LSD1, promotes activation of the epidermal differentiation transcriptional program and, in turn, represses the invasion of cutaneous squamous cell carcinoma, one of the most common of all human cancers.