Epigenetic and 3D genome reprogramming during the aging of human hippocampus.

Age-related cognitive decline is associated with altered physiology of the hippocampus. While changes in gene expression have been observed in aging brain, the regulatory mechanisms underlying these changes remain underexplored. We generated single-nucleus gene expression, chromatin accessibility, DNA methylation, and 3D genome data from 40 human hippocampal tissues spanning adult lifespan. We observed a striking loss of astrocytes, OPC, and endothelial cells during aging, including astrocytes that play a role in regulating synapses. Microglia undergo a dramatic switch from a homeostatic state to a primed inflammatory state through DNA methylome and 3D genome reprogramming. Aged cells experience erosion of their 3D genome architecture. Our study identifies age-associated changes in cell types/states and gene regulatory features that provide insight into cognitive decline during human aging.

Evaluating Methods for the Prediction of Cell Type-Specific Enhancers in the Mammalian Cortex.

Identifying cell type-specific enhancers in the brain is critical to building genetic tools for investigating the mammalian brain. Computational methods for functional enhancer prediction have been proposed and validated in the fruit fly and not yet the mammalian brain. We organized the 'Brain Initiative Cell Census Network (BICCN) Challenge: Predicting Functional Cell Type-Specific Enhancers from Cross-Species Multi-Omics' to assess machine learning and feature-based methods designed to nominate enhancer DNA sequences to target cell types in the mouse cortex. Methods were evaluated based on in vivo validation data from hundreds of cortical cell type-specific enhancers that were previously packaged into individual AAV vectors and retro-orbitally injected into mice. We find that open chromatin was a key predictor of functional enhancers, and sequence models improved prediction of non-functional enhancers that can be deprioritized as opposed to pursued for in vivo testing. Sequence models also identified cell type-specific transcription factor codes that can guide designs of in silico enhancers. This community challenge establishes a benchmark for enhancer prioritization algorithms and reveals computational approaches and molecular information that are crucial for the identification of functional enhancers for mammalian cortical cell types. The results of this challenge bring us closer to understanding the complex gene regulatory landscape of the mammalian brain and help us design more efficient genetic tools and potential gene therapies for human neurological diseases.

Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler.

Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses.

Reduction of ZFX levels decreases histone H4 acetylation and increases Pol2 pausing at target promoters.

The ZFX transcriptional activator binds to CpG island promoters, with a major peak at ∼200-250 bp downstream from transcription start sites. Because ZFX binds within the transcribed region, we investigated whether it regulates transcriptional elongation. We used GRO-seq to show that loss or reduction of ZFX increased Pol2 pausing at ZFX-regulated promoters. To further investigate the mechanisms by which ZFX regulates transcription, we determined regions of the protein needed for transactivation and for recruitment to the chromatin. Interestingly, although ZFX has 13 grouped zinc fingers, deletion of the first 11 fingers produces a protein that can still bind to chromatin and activate transcription. We next used TurboID-MS to detect ZFX-interacting proteins, identifying ZNF593, as well as proteins that interact with the N-terminal transactivation domain (which included histone modifying proteins), and proteins that interact with ZFX when it is bound to the chromatin (which included TAFs and other histone modifying proteins). Our studies support a model in which ZFX enhances elongation at target promoters by recruiting H4 acetylation complexes and reducing pausing.

A fast, scalable and versatile tool for analysis of single-cell omics data.

Single-cell omics technologies have revolutionized the study of gene regulation in complex tissues. A major computational challenge in analyzing these datasets is to project the large-scale and high-dimensional data into low-dimensional space while retaining the relative relationships between cells. This low dimension embedding is necessary to decompose cellular heterogeneity and reconstruct cell-type-specific gene regulatory programs. Traditional dimensionality reduction techniques, however, face challenges in computational efficiency and in comprehensively addressing cellular diversity across varied molecular modalities. Here we introduce a nonlinear dimensionality reduction algorithm, embodied in the Python package SnapATAC2, which not only achieves a more precise capture of single-cell omics data heterogeneities but also ensures efficient runtime and memory usage, scaling linearly with the number of cells. Our algorithm demonstrates exceptional performance, scalability and versatility across diverse single-cell omics datasets, including single-cell assay for transposase-accessible chromatin using sequencing, single-cell RNA sequencing, single-cell Hi-C and single-cell multi-omics datasets, underscoring its utility in advancing single-cell analysis.

Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Cell-type-specific 3D-genome organization and transcription regulation in the brain.

3D organization of the genome plays a critical role in regulating gene expression. However, it remains unclear how chromatin organization differs among different cell types in the brain. Here we used genome-scale DNA and RNA imaging to investigate 3D-genome organization in transcriptionally distinct cell types in the primary motor cortex of the mouse brain. We uncovered a wide spectrum of differences in the nuclear architecture and 3D-genome organization among different cell types, ranging from the physical size of the cell nucleus to the active-inactive chromatin compartmentalization and radial positioning of chromatin loci within the nucleus. These cell-type-dependent variations in nuclear architecture and chromatin organization exhibited strong correlation with both total transcriptional activity of the cell and transcriptional regulation of cell-type-specific marker genes. Moreover, we found that the methylated-DNA-binding protein MeCP2 regulates transcription in a divergent manner, depending on the nuclear radial positions of chromatin loci, through modulating active-inactive chromatin compartmentalization.

Adenovirus E1A binding to DCAF10 targets proteasomal degradation of RUVBL1/2 AAA+ ATPases required for quaternary assembly of multiprotein machines, innate immunity, and responses to metabolic stress.

IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

A comparative atlas of single-cell chromatin accessibility in the human brain.

Recent advances in single-cell transcriptomics have illuminated the diverse neuronal and glial cell types within the human brain. However, the regulatory programs governing cell identity and function remain unclear. Using a single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq), we explored open chromatin landscapes across 1.1 million cells in 42 brain regions from three adults. Integrating this data unveiled 107 distinct cell types and their specific utilization of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly a third of the cCREs demonstrated conservation and chromatin accessibility in the mouse brain cells. We reveal strong links between specific brain cell types and neuropsychiatric disorders including schizophrenia, bipolar disorder, Alzheimer's disease (AD), and major depression, and have developed deep learning models to predict the regulatory roles of noncoding risk variants in these disorders.

SnapATAC2: a fast, scalable and versatile tool for analysis of single-cell omics data.

Single-cell omics technologies have ushered in a new era for the study of dynamic gene regulation in complex tissues during development and disease pathogenesis. A major computational challenge in analyzing these datasets is to project the large-scale and high dimensional data into low-dimensional space while retaining the relative relationships between cells in order to decompose the cellular heterogeneity and reconstruct cell-type-specific gene regulatory programs. Conventional dimensionality reduction methods suffer from computational inefficiency, difficulty to capture the full spectrum of cellular heterogeneity, or inability to apply across diverse molecular modalities. Here, we report a fast and nonlinear dimensionality reduction algorithm that not only more accurately captures the heterogeneities of single-cell omics data, but also features runtime and memory usage that is computational efficient and linearly proportional to cell numbers. We implement this algorithm in a Python package named SnapATAC2, and demonstrate its superior performance, remarkable scalability and general adaptability using an array of single-cell omics data types, including single-cell ATAC-seq, single-cell RNA-seq, single-cell Hi-C, and single-cell multiomics datasets.

Comparative single cell epigenomic analysis of gene regulatory programs in the rodent and primate neocortex.

Sequence divergence of cis- regulatory elements drives species-specific traits, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains to be elucidated. We investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset, and mouse with single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome, and chromosomal conformation profiles from a total of over 180,000 cells. For each modality, we determined species-specific, divergent, and conserved gene expression and epigenetic features at multiple levels. We find that cell type-specific gene expression evolves more rapidly than broadly expressed genes and that epigenetic status at distal candidate cis -regulatory elements (cCREs) evolves faster than promoters. Strikingly, transposable elements (TEs) contribute to nearly 80% of the human-specific cCREs in cortical cells. Through machine learning, we develop sequence-based predictors of cCREs in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Lastly, we show that epigenetic conservation combined with sequence similarity helps uncover functional cis -regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Promoter-specific changes in initiation, elongation, and homeostasis of histone H3 acetylation during CBP/p300 inhibition.

Regulation of RNA polymerase II (Pol2) elongation in the promoter-proximal region is an important and ubiquitous control point for gene expression in metazoans. We report that transcription of the adenovirus 5 E4 region is regulated during the release of paused Pol2 into productive elongation by recruitment of the super-elongation complex, dependent on promoter H3K18/27 acetylation by CBP/p300. We also establish that this is a general transcriptional regulatory mechanism that applies to ~7% of expressed protein-coding genes in primary human airway epithelial cells. We observed that a homeostatic mechanism maintains promoter, but not enhancer, H3K18/27ac in response to extensive inhibition of CBP/p300 acetyl transferase activity by the highly specific small molecule inhibitor A-485. Further, our results suggest a function for BRD4 association at enhancers in regulating paused Pol2 release at nearby promoters. Taken together, our results uncover the processes regulating transcriptional elongation by promoter region histone H3 acetylation and homeostatic maintenance of promoter, but not enhancer, H3K18/27ac in response to inhibition of CBP/p300 acetyl transferase activity.

The dopamine receptor antagonist trifluoperazine prevents phenotype conversion and improves survival in mouse models of glioblastoma.

Glioblastoma (GBM) is the deadliest adult brain cancer, and all patients ultimately succumb to the disease. Radiation therapy (RT) provides survival benefit of 6 mo over surgery alone, but these results have not improved in decades. We report that radiation induces a glioma-initiating cell phenotype, and we have identified trifluoperazine (TFP) as a compound that interferes with this phenotype conversion. TFP causes loss of radiation-induced Nanog mRNA expression, and activation of GSK3 with consecutive posttranslational reduction in p-Akt, Sox2, and ß-catenin protein levels. TFP did not alter the intrinsic radiation sensitivity of glioma-initiating cells (GICs). Continuous treatment with TFP and a single dose of radiation reduced the number of GICs in vivo and prolonged survival in syngeneic and patient-derived orthotopic xenograft (PDOX) mouse models of GBM. Our findings suggest that the combination of a dopamine receptor antagonist with radiation enhances the efficacy of RT in GBM by preventing radiation-induced phenotype conversion of radiosensitive non-GICs into treatment-resistant, induced GICs (iGICs).

Dedifferentiation by adenovirus E1A due to inactivation of Hippo pathway effectors YAP and TAZ.

Adenovirus transformed cells have a dedifferentiated phenotype. Eliminating E1A in transformed human embryonic kidney cells derepressed ∼2600 genes, generating a gene expression profile closely resembling mesenchymal stem cells (MSCs). This was associated with a dramatic change in cell morphology from one with scant cytoplasm and a globular nucleus to one with increased cytoplasm, extensive actin stress fibers, and actomyosin-dependent flattening against the substratum. E1A-induced hypoacetylation at histone H3 Lys27 and Lys18 (H3K27/18) was reversed. Most of the increase in H3K27/18ac was in enhancers near TEAD transcription factors bound by Hippo signaling-regulated coactivators YAP and TAZ. E1A causes YAP/TAZ cytoplasmic sequestration. After eliminating E1A, YAP/TAZ were transported into nuclei, where they associated with poised enhancers with DNA-bound TEAD4 and H3K4me1. This activation of YAP/TAZ required RHO family GTPase signaling and caused histone acetylation by p300/CBP, chromatin remodeling, and cohesin loading to establish MSC-associated enhancers and then superenhancers. Consistent results were also observed in primary rat embryo kidney cells, human fibroblasts, and human respiratory tract epithelial cells. These results together with earlier studies suggest that YAP/TAZ function in a developmental checkpoint controlled by signaling from the actin cytoskeleton that prevents differentiation of a progenitor cell until it is in the correct cellular and tissue environment.

Transcriptomic and ChIP-sequence interrogation of EGFR signaling in HER2+ breast cancer cells reveals a dynamic chromatin landscape and S100 genes as targets.

BACKGROUND: The Human Epidermal Growth Factor Receptor (EGFR/HER1) can be activated by several ligands including Transforming Growth Factor alpha (TGF-α) and Epidermal Growth Factor (EGF). Following ligand binding, EGFR heterodimerizes with other HER family members, such as HER2 (human epidermal growth factor receptor-2). Previously, we showed that the EGFR is upregulated in trastuzumab resistant HER2 positive (HER2+) breast cancer cells. This study is aimed to determine the downstream effects on transcription following EGFR upregulation in HER2+ breast cancer cells. METHODS: RNA-sequence and ChIP-sequence for H3K18ac and H3K27ac (Histone H3 lysine K18 and K27 acetylation) were conducted following an Epidermal Growth Factor (EGF) treatment time course in HER2+ breast cancer cells, SKBR3. The levels of several proteins of interest were confirmed by western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by western blot analysis. RESULTS: Over the course of 24 h, EGFR stimulation resulted in the modulation of over 4000 transcripts. Moreover, our data demonstrates that EGFR/HER2 signaling regulates the epigenome, with global H3K18ac and H3K27ac oscillating as a function of time following EGF treatment. RNA-sequence data demonstrates the activation of immediate early genes (IEGs) and delayed early genes (DEGs) within 1 h of EGF treatment. More importantly, we have identified members of the S100 (S100 Calcium Binding Protein) gene family as likely direct targets of EGFR signaling as H3K18ac, H3K27ac and pol2 (RNA polymerase II) increase near the transcription start sites of some of these genes. CONCLUSIONS: Our data suggests that S100 proteins, which act as Ca2+ sensors, could play a role in EGF induced tumor cell growth and metastasis, contribute to trastuzumab resistance and cell migration and that they are likely drug targets in HER2+ breast cancer.

Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters.

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters.

The Adenovirus E1A C Terminus Suppresses a Delayed Antiviral Response and Modulates RAS Signaling.

The N-terminal half of adenovirus e1a assembles multimeric complexes with host proteins that repress innate immune responses and force host cells into S-phase. In contrast, the functions of e1a's C-terminal interactions with FOXK, DCAF7, and CtBP are unknown. We found that these interactions modulate RAS signaling, and that a single e1a molecule must bind all three of these host proteins to suppress activation of a subset of IFN-stimulated genes (ISGs). These ISGs were otherwise induced in primary respiratory epithelial cells at 12 hr p.i. This delayed activation of ISGs required IRF3 and coincided with an ∼10-fold increase in IRF3 from protein stabilization. The induced IRF3 bound to chromatin and localized to the promoters of activated ISGs. While IRF3, STAT1/2, and IRF9 all greatly increased in concentration, there were no corresponding mRNA increases, suggesting that e1a regulates the stabilities of these key activators of innate immune responses, as shown directly for IRF3.

Adenovirus small E1A employs the lysine acetylases p300/CBP and tumor suppressor Rb to repress select host genes and promote productive virus infection.

Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGF-β, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication.

Caveolin isoform switching as a molecular, structural, and metabolic regulator of microglia.

Microglia are ramified cells that serve as central nervous system (CNS) guardians, capable of proliferation, migration, and generation of inflammatory cytokines. In non-pathological states, these cells exhibit ramified morphology with processes intermingling with neurons and astrocytes. Under pathological conditions, they acquire a rounded amoeboid morphology and proliferative and migratory capabilities. Such morphological changes require cytoskeleton rearrangements. The molecular control points for polymerization states of microtubules and actin are still under investigation. Caveolins (Cavs), membrane/lipid raft proteins, are expressed in inflammatory cells, yet the role of caveolin isoforms in microglia physiology is debatable. We propose that caveolins provide a necessary control point in the regulation of cytoskeletal dynamics, and thus investigated a role for caveolins in microglia biology. We detected mRNA and protein for both Cav-1 and Cav-3. Cav-1 protein was significantly less and localized to plasmalemma (PM) and cytoplasmic vesicles (CVs) in the microglial inactive state, while the active (amoeboid-shaped) microglia exhibited increased Cav-1 expression. In contrast, Cav-3 was highly expressed in the inactive state and localized with cellular processes and perinuclear regions and was detected in active amoeboid microglia. Pharmacological manipulation of the cytoskeleton in the active or non-active state altered caveolin expression. Additionally, increased Cav-1 expression also increased mitochondrial respiration, suggesting possible regulatory roles in cell metabolism necessary to facilitate the morphological changes. The present findings strongly suggest that regulation of microglial morphology and activity are in part due to caveolin isoforms, providing promising novel therapeutic targets in CNS injury or disease.