DNA-based ForceChrono probes for deciphering single-molecule force dynamics in living cells.

Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.

SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity.

Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFß and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFß-SMAD signaling axis.

Analysis of Genetically Diverse Macrophages Reveals Local and Domain-wide Mechanisms that Control Transcription Factor Binding and Function.

Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of ∼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.

The Three-Dimensional Organization of Mammalian Genomes.

Animal development depends on not only the linear genome sequence that embeds millions of cis-regulatory elements, but also the three-dimensional (3D) chromatin architecture that orchestrates the interplay between cis-regulatory elements and their target genes. Compared to our knowledge of the cis-regulatory sequences, the understanding of the 3D genome organization in human and other eukaryotes is still limited. Recent advances in technologies to map the 3D genome architecture have greatly accelerated the pace of discovery. Here, we review emerging concepts of chromatin organization in mammalian cells, discuss the dynamics of chromatin conformation during development, and highlight important roles for chromatin organization in cancer and other human diseases.

Stressing the role of a short linear motif in ataxin-2 condensation.

Ataxin-2, an RNA-binding protein that is conserved across eukaryotes, is involved in stress granule assembly and age-associated neurodegenerative diseases. In this issue of Molecular Cell, Boeynaems et al.1 identify a short linear motif in ataxin-2 as a condensation switch, providing molecular insights into its essential role in cellular stress response.

N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.

N(6)-methyldeoxyadenosine (6mA or m(6)A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here, we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms.

Visualizing the disordered nuclear transport machinery in situ.

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs-about 50 MDa-is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4-11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides-in the terminology of the Flory polymer theory12-a 'good solvent' environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder-function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos.

5-methylcytosine is a major epigenetic modification that is sometimes called "the fifth nucleotide." However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo's methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos.

Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.

The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.

Chromothripsis drives the evolution of gene amplification in cancer.

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.

Phase Separation Comes of Age: From Phenomenology to Single Molecules.

In this issue of Molecular Cell, Pitchiaya et al. (2019) use high-resolution single-molecule microscopy to dissect the localization of different types of RNAs with processing bodies (PBs) in cells, revealing novel insights about their dynamic recruitment to PBs.

Regulation of endogenous retrovirus-derived regulatory elements by GATA2/3 and MSX2 in human trophoblast stem cells.

The placenta is an organ with extraordinary phenotypic diversity in eutherian mammals. Recent evidence suggests that numerous human placental enhancers are evolved from lineage-specific insertions of endogenous retroviruses (ERVs), yet the transcription factors (TFs) underlying their regulation remain largely elusive. Here, by first focusing on MER41, a primate-specific ERV family previously linked to placenta and innate immunity, we uncover the binding motifs of multiple crucial trophoblast TFs (GATA2/3, MSX2, GRHL2) in addition to innate immunity TFs STAT1 and IRF1. Integration of ChIP-seq data confirms the binding of GATA2/3, MSX2, and their related factors on the majority of MER41-derived enhancers in human trophoblast stem cells (TSCs). MER41-derived enhancers that are constitutively active in human TSCs are distinct from those activated upon interferon stimulation, which is determined by the binding of relevant TFs and their subfamily compositions. We further demonstrate that GATA2/3 and MSX2 have prevalent binding to numerous other ERV families - indicating their broad impact on ERV-derived enhancers. Functionally, the derepression of many syncytiotrophoblast genes after MSX2 knockdown is likely to be mediated by regulatory elements derived from ERVs - suggesting ERVs are also important for mediating transcriptional repression. Overall, this study characterizes the regulation of ERV-derived regulatory elements by GATA2/3, MSX2, and their cofactors in human TSCs, and provides mechanistic insights into the importance of ERVs in human trophoblast regulatory network.

Reversible acetylation of HDAC8 regulates cell cycle.

HDAC8, a member of class I HDACs, plays a pivotal role in cell cycle regulation by deacetylating the cohesin subunit SMC3. While cyclins and CDKs are well-established cell cycle regulators, our knowledge of other regulators remains limited. Here we reveal the acetylation of K202 in HDAC8 as a key cell cycle regulator responsive to stress. K202 acetylation in HDAC8, primarily catalyzed by Tip60, restricts HDAC8 activity, leading to increased SMC3 acetylation and cell cycle arrest. Furthermore, cells expressing the mutant form of HDAC8 mimicking K202 acetylation display significant alterations in gene expression, potentially linked to changes in 3D genome structure, including enhanced chromatid loop interactions. K202 acetylation impairs cell cycle progression by disrupting the expression of cell cycle-related genes and sister chromatid cohesion, resulting in G2/M phase arrest. These findings indicate the reversible acetylation of HDAC8 as a cell cycle regulator, expanding our understanding of stress-responsive cell cycle dynamics.

Nasal delivery of polymeric nanoDisc mobilizes a synergy of central and peripheral amyloid-ß clearance to treat Alzheimer's disease.

The disequilibrium of amyloid ß-peptide (Aß) between the central and peripheral pools has been claimed as an initiating event in Alzheimer's disease (AD). In this study, we employ discoidal high-density lipoproteins (HDL-Disc) mimicking Aß antibody for directional flux of Aß from central to peripheral catabolism, with desirable safety and translation potential. Structurally, HDL-Disc assembly (polyDisc) is prepared with aid of chitosan derivative polymerization. After intranasal administration and response to slightly acidic nasal microenvironment, polyDisc depolymerizes into carrier-free HDL-Disc with chitosan derivatives that adhere to the mucosal layer to reversibly open tight junctions, helping HDL-Disc penetrate the olfactory pathway into brain. Thereafter, HDL-Disc captures Aß into microglia for central clearance or ferries Aß out of the brain for liver-mediated compensatory catabolism. For synergy therapy, intranasal administration of polyDisc can effectively reduce intracerebral Aß burden by 97.3% and vascular Aß burden by 73.5%, ameliorate neurologic damage, and rescue memory deficits in APPswe/PS1dE9 transgenic AD mice with improved safety, especially vascular safety. Collectively, this design provides a proof of concept for developing Aß antibody mimics to mobilize a synergy of central and peripheral Aß clearance for AD treatment.

Mapping chromatin loops in single cells.

Recent advances in high-throughput chromatin conformation capture (Hi-C) technologies at the single-cell level enable the identification of cell type-specific chromatin loops directly from complex tissues. This may help to interpret noncoding variants identified by genome-wide association studies (GWAS) in disease-relevant cell types. We briefly review current experimental and computational strategies for mapping chromatin loops in single cells.

Multiple E3 ligases act as antiviral factors against SARS-CoV-2 via inducing the ubiquitination and degradation of ORF9b.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.

SnapHiC-D: a computational pipeline to identify differential chromatin contacts from single-cell Hi-C data.

Single-cell high-throughput chromatin conformation capture technologies (scHi-C) has been used to map chromatin spatial organization in complex tissues. However, computational tools to detect differential chromatin contacts (DCCs) from scHi-C datasets in development and through disease pathogenesis are still lacking. Here, we present SnapHiC-D, a computational pipeline to identify DCCs between two scHi-C datasets. Compared to methods designed for bulk Hi-C data, SnapHiC-D detects DCCs with high sensitivity and accuracy. We used SnapHiC-D to identify cell-type-specific chromatin contacts at 10 Kb resolution in mouse hippocampal and human prefrontal cortical tissues, demonstrating that DCCs detected in the hippocampal and cortical cell types are generally associated with cell-type-specific gene expression patterns and epigenomic features. SnapHiC-D is freely available at https://github.com/HuMingLab/SnapHiC-D.

The sugar transporter ZmSUGCAR1 of the nitrate transporter 1/peptide transporter family is critical for maize grain filling.

Maternal-to-filial nutrition transfer is central to grain development and yield. nitrate transporter 1/peptide transporter (NRT1-PTR)-type transporters typically transport nitrate, peptides, and ions. Here, we report the identification of a maize (Zea mays) NRT1-PTR-type transporter that transports sucrose and glucose. The activity of this sugar transporter, named Sucrose and Glucose Carrier 1 (SUGCAR1), was systematically verified by tracer-labeled sugar uptake and serial electrophysiological studies including two-electrode voltage-clamp, non-invasive microelectrode ion flux estimation assays in Xenopus laevis oocytes and patch clamping in HEK293T cells. ZmSUGCAR1 is specifically expressed in the basal endosperm transfer layer and loss-of-function mutation of ZmSUGCAR1 caused significantly decreased sucrose and glucose contents and subsequent shrinkage of maize kernels. Notably, the ZmSUGCAR1 orthologs SbSUGCAR1 (from Sorghum bicolor) and TaSUGCAR1 (from Triticum aestivum) displayed similar sugar transport activities in oocytes, supporting the functional conservation of SUGCAR1 in closely related cereal species. Thus, the discovery of ZmSUGCAR1 uncovers a type of sugar transporter essential for grain development and opens potential avenues for genetic improvement of seed-filling and yield in maize and other grain crops.

Circular ecDNA promotes accessible chromatin and high oncogene expression.

Oncogenes are commonly amplified on particles of extrachromosomal DNA (ecDNA) in cancer1,2, but our understanding of the structure of ecDNA and its effect on gene regulation is limited. Here, by integrating ultrastructural imaging, long-range optical mapping and computational analysis of whole-genome sequencing, we demonstrate the structure of circular ecDNA. Pan-cancer analyses reveal that oncogenes encoded on ecDNA are among the most highly expressed genes in the transcriptome of the tumours, linking increased copy number with high transcription levels. Quantitative assessment of the chromatin state reveals that although ecDNA is packaged into chromatin with intact domain structure, it lacks higher-order compaction that is typical of chromosomes and displays significantly enhanced chromatin accessibility. Furthermore, ecDNA is shown to have a significantly greater number of ultra-long-range interactions with active chromatin, which provides insight into how the structure of circular ecDNA affects oncogene function, and connects ecDNA biology with modern cancer genomics and epigenetics.