The conserved histone variant H3.3 plays pivotal roles in heterochromatin formation and retrotransposon silencing.However,the molecular mechanism underlying H3.3-primed heterochromatin regulation remains elusive.Here,we demonstrate that H3.3-specific Ser31 phosphorylation and Lys27 trimethyla-tion synergistically promote H3K9me3-heterochromatin formation.Mechanistically,polycomb protein chromobox homolog 7(CBX7)preferentially binds Ser31-phosphorylated H3.3K27me3 nucleosomes and then recruits KRAB-associated protein 1(KAP1),which may further engage the histone lysine 9 methyltransferase to establish H3K9me3-associated heterochromatin.Remarkably,H3K9me3 is signifi-cantly impaired when the H3.3-CBX7 interaction is disrupted,accompanied by the activation of retro-transposons.Moreover,during X-chromosome inactivation(XCI),H3K9me2/3 fails to accumulate at the inactive X(Xi)when blocking the H3.3-CBX7-KAP1 axis.Taken together,our results reveal a novel molecular mechanism by which H3.3 Ser31 phosphorylation(H3.3Ser31p)facilitates H3K9me3-heterochromatin formation during retrotransposon silencing and XCI via the H3.3K27me3-CBX7-KAP1 axis.

The suppression of transposable elements in the germline is critical for safeguarding fertility. Here we identified that DAXX (death-associated protein 6), a potent transcription repressor and an H3.3 chaperone, was indispensable for silencing of transposable elements during spermatogenesis. Daxx germline conditional knockout mice exhibited delayed meiotic progression, spermiogenesis defects, and an age-dependent decline in fertility. The ablation of DAXX led to activation of evolutionarily young long interspersed nuclear element-1 (LINE1) and endogenous retrovirus (ERV) subfamilies. Further mechanistic study revealed that DAXX participated in the repression of young LINE1s by regulating H3K9me3 enrichment and maintaining DNA methylation. In summary, this study identifies DAXX as a previously unknown regulator of spermatogenesis and demonstrates that it functions as an epigenetic regulator to silence young LINE1s.

Double-stranded DNA cytosine deaminase DddA orthologs from multiple types of bacteria have been fused with the transcription-activator-like effector system for mitochondrial DNA (mtDNA) base editing, while the efficiencies remain limited and its nuclear off-targeting activity cannot be ignored yet. Here we identified a DddA ortholog from Burkholderia gladioli (BgDddA) and generated nuclear or mtDNA cytosine base editors (mitoCBEs), exhibiting higher C⋅G-to-T⋅A editing frequencies compared with canonical DdCBE, and fusion with transactivator Rta remarkably improved editing efficiencies by up to 6.4-fold at non-TC targets. Referring to DddA11, we further introduced six substitutions into BgDddA and generated mitoCBE3.2, which efficiently induced disease-associated mtDNA mutations in mouse and human cell lines at both TC and non-TC targets with efficiency reaching up to 99.2%. Using mitoCBE3.2, single clones containing homoplasmic mtDNA mutations or premature stop codons associated with human diseases were generated, and the functions of these mutations have been evaluated upon the treatment of reactive oxygen species inducers. Importantly, mitochondria harboring these homoplasmic mutations were transplanted into wild-type cells, enabling precise base conversions, without the risk of nuclear gene off-targets. Thus, we have engineered an efficient mitoCBE using BgDddA, facilitating mitochondrial disease modeling and potential mutation correction with the aid of mitochondrial transplantation.

The gut-liver axis plays a crucial role in maintaining metabolic balance and overall human health. It orchestrates various processes, such as blood flow, nutrient transfer, metabolite processing, and immune cell communication between the two organs. Traditional methods, such as animal models and two-dimensional (2D) cell cultures, are insufficient in fully replicating the intricate functions of the gut-liver axis. The emergence of microfluidic technology has revolutionized this field, facilitating the development of organ-on-a-chip (OOC) systems. These systems are capable of mimicking the complex structures and dynamic environments of the gut and liver in vitro and incorporating sensors for real-time monitoring. In this article, we review the latest progress in gut-on-a-chip (GOC) and liver-on-a-chip (LOC) systems, as well as the integrated gut-liver-on-a-chip (GLOC) models. Our focus lies in the simulation of physiological parameters, three-dimensional (3D) structural mimicry, microbiome integration, and multicellular co-culture. All these aspects are essential for constructing accurate in vitro models of the gut and liver. Furthermore, we explore the current applications of OOC technology in the study of the gut and liver, including its use in disease modeling, toxicity testing, and drug screening. Finally, we discuss the challenges that remain and outline potential future directions for advancing GOC and LOC development in vitro.

Eucommia ulmoides Oliver (E. ulmoides) is a tree species of considerable medicinal and economic value, while trans-zeatin (tZ) serves as a cytokinin (CTK) plant growth regulator. Our early results have shown that tZ significantly promotes the regeneration of E. ulmoides axillary buds. To uncover the underlying regulatory mechanisms, we conducted the first comprehensive metabolomic and transcriptomic analyses of axillary buds across five developmental stages (0-4 weeks post-initiation). Developmental progression was characterized by two distinct phases: an induction phase (W0-W1) and an elongation phase (W1-W4). Metabolomic profiling revealed dynamic phytohormone fluctuations, with a significant increase in tZR, dZ, and GA concentrations at the W1 stage, followed by a decline. In particular, tZR levels surged 16.4-fold from W0 to W1 stage. Conversely, IAA, ABA, and SA concentrations decreased at the W1 stage but subsequently increased in later stages. To investigate these changes, we comprehensive analyzed transcriptomes across five axillary bud development stages, revealing DEGs were significantly enriched in plant hormone signaling and zeatin biosynthesis pathways. Additionally, we employed weighted gene co-expression network analysis (WGCNA) to explore the phytohormone regulatory network during tZ-induced axillary bud development. By utilizing hormone concentration as a trait and jointly analyzing WGCNA with DEGs transcripts, we identified 11 hub genes. Among these, histidine phosphotransfer proteins (AHPs) in the CTK signaling pathway emerged as the central hub within the phytohormone crosstalk regulatory network. These studies provide a theoretical basis for further research on the regulatory mechanisms of CTKs in the growth and development of E. ulmoides, while also provide technical support for the in vitro rapid propagation of high-quality E. ulmoides seedlings.

Gut microbiota is linked to human metabolic diseases. The previous work showed that leucine deprivation improved metabolic dysfunction, but whether leucine deprivation alters certain specific species of bacterium that brings these benefits remains unclear. Here, this work finds that leucine deprivation alters gut microbiota composition, which is sufficient and necessary for the metabolic improvements induced by leucine deprivation. Among all the affected bacteria, B. coccoides is markedly increased in the feces of leucine-deprived mice. Moreover, gavage with B. coccoides improves insulin sensitivity and reduces body fat in high-fat diet (HFD) mice, and singly colonization of B. coccoides increases insulin sensitivity in gnotobiotic mice. The effects of B. coccoides are mediated by metabolizing tryptophan into indole-3-acetic acid (I3AA) that activates the aryl hydrocarbon receptor (AhR) in the liver. Finally, this work reveals that reduced fecal B. coccoides and I3AA levels are associated with the clinical metabolic syndrome. These findings suggest that B. coccoides is a newly identified bacterium increased by leucine deprivation, which improves metabolic disorders via metabolizing tryptophan into I3AA.

In 1928,Emil Heitz discovered that some regions of mitotic chromosomes are more densely stained than others,thereby first defining heterochromatin and euchromatin[1].In 1930,Hermann Joseph Muller observed position-effect variegation(PEV)in Droso-phila[2],a phenomenon in which heterochromatin can spread across the heterochromatin/euchromatin border and silence nearby euchromatic genes.

Nuclear architecture underlies the transcriptional programs within the cell to establish cell identity. As previously demonstrated, long-range chromatin interactions of the Oct4 distal enhancer (DE) are correlated with active transcription in naïve state embryonic stem cells. Here, we identify and characterize extreme long-range interactions of the Oct4 DE through a novel CRISPR labeling technique we developed and chromosome conformation capture to identify lethal giant larvae 2 (Llgl2) and growth factor receptor-bound protein 7 (Grb7) as putative functional interacting target genes in different chromosomes. We show that the Oct4 DE directly regulates expression of Llgl2 and Grb7 in addition to Oct4. Expression of Llgl2 and Grb7 closely correlates with the pluripotent state, where knock down of either result in loss of pluripotency, and overexpression enhances somatic cell reprogramming. We demonstrated that biologically important interactions of the Oct4 DE can occur at extreme distances that are necessary for the maintenance of the pluripotent state.

Histone H2B mono-ubiquitination at lysine 120 (ubH2B) has been found to regulate transcriptional elongation by collaborating with the histone chaperone FACT (Facilitates Chromatin Transcription) and plays essential roles in chromatin-based transcriptional processes. However, the mechanism of how ubH2B directly collaborates with FACT at the nucleosome level still remains elusive. In this study, we demonstrate that ubH2B impairs the mechanical stability of the nucleosome and helps to recruit FACT by enhancing the binding of FACT on the nucleosome. FACT prefers to bind and deposit H2A-ubH2B dimers to form an intact nucleosome. Strikingly, the preferable binding of FACT on ubH2B-nucleosome greatly enhances nucleosome stability and maintains its integrity. The stable altered nucleosome state obtained by ubH2B and FACT provides a key platform for gene transcription, as revealed by genome-wide and time-course ChIP-qPCR analyses. Our findings provide mechanistic insights of how ubH2B directly collaborates with FACT to regulate nucleosome dynamics for gene transcription.

Aims: We investigated the neuroprotective effects of Akkermansia muciniphila through the 'gut-brain' axis. Methods: Human colon cancer (Caco-2) cells treated with A. muciniphila metabolites were used to create the conditioned medium from Caco-2 cells treated with A. muciniphila metabolites (AC medium) medium, then treated human microglial clone 3 (HMC3) cells to simulate the gut-brain axis in vitro. Bioinformatics analyses were performed to investigate the molecular mechanisms by which the AC medium affected HMC3 cells. Results: The secretion of inflammatory cytokines IL-6 (0.37 ± 0.80-fold) and IL-17A (0.05 ± 0.18-fold) by HMC3 cells was inhibited by the AC medium. Differentially expressed genes were mainly enriched in immune-related signaling pathways, such as the cAMP and TGF-β signaling pathways. Conclusion: A. muciniphila might be a source of therapeutic approaches to alleviate microglia-mediated neuroinflammatory diseases.

Base editing tools enable precise genome modifications, disease modeling, and promising gene therapy. However, many human genetic diseases are elicited by multi-nucleotide variants (MNVs) with heterogeneous substitutions at the same genomic locus. Based on the adenine and cytosine base editors, dual base editors that can catalyze concurrent C-to-T and A-to-G editing have been developed, while simultaneous C&G-to-T&A and A&T-to-G&C conversions on the same allele have not been achieved at the desirable site. Here we propose a strategy of combining base editors with dual guide RNAs (gRNAs) that target two overlapped neighboring loci on the opposite strands, which can induce simultaneous C&G-to-T&A and A&T-to-G&C conversions within their overlapping targeting windows. Moreover, one of the paired gRNAs is mutated to perfectly match another gRNA-edited sequence, efficiently facilitating concurrent base conversions on the same allele. To further expand the targeting scopes, PAMless SpRY Cas9-mediated base editors are combined with our optimized dual gRNAs system to induce expected concurrent base editing and to install neighboring pathogenic MNVs in TP53 in cancer cells. In addition, more complex mutation types can be achieved by integrating dual base editors and our dual gRNAs strategy. Thus, we establish a general strategy to efficiently induce MNVs in human genome, helping to dissect the functions of pathogenic MNVs with multifarious types.

CRISPR/Cas9 system has been applied as an effective genome-targeting technology. By fusing deaminases with Cas9 nickase (nCas9), various cytosine and adenine base editors (CBEs and ABEs) have been successfully developed that can efficiently induce nucleotide conversions and install pathogenic single nucleotide variants (SNVs) in cultured cells and animal models. However, the applications of BEs are frequently limited by the specific protospacer adjacent motif (PAM) sequences and protein sizes. To expand the toolbox for BEs that can recognize novel PAM sequences, we cloned a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) with a smaller size and constructed it into APOBEC1- or APOBEC3A-composed CBEs and TadA or TadA*-composed ABEs, which yield high editing efficiencies, low off-targeting activities, and low indel rates in human cells. Compared to PAMless SpRY Cas9-composed BE4max, SsiCas9-mediated BE4max displayed higher editing efficiencies for targets with "NNAAAA" PAM sequences. Moreover, SsiCas9-mediated BE4max induced highly efficient C-to-T conversions in the mouse Ar gene (R841C) to introduce a human androgen resistance syndrome-related mutation (AR R820C) in early mouse embryos. Thus, we developed novel BEs mediated by SsiCas9, expanded the toolbox for base conversions, and broadened the range of editable genomes in vitro and in vivo.

During mammalian embryogenesis, spatial regulation of gene expression and cell signaling are functionally coupled with lineage specification, patterning of tissue progenitors, and germ layer morphogenesis. While the mouse model has been instrumental for understanding mammalian development, comparatively little is known about human and non-human primate gastrulation due to the restriction of both technical and ethical issues. Here, we present a spatial and temporal survey of the molecular dynamics of cell types populating the non-human primate embryos during gastrulation. We reconstructed three-dimensional digital models from serial sections of cynomolgus monkey (Macaca fascicularis) gastrulating embryos at 1-day temporal resolution from E17 to E21. Spatial transcriptomics identifies gene expression profiles unique to the germ layers. Cross-species comparison reveals a developmental coordinate of germ layer segregation between mouse and primates, and species-specific transcription programs during gastrulation. These findings offer insights into evolutionarily conserved and divergent processes during mammalian gastrulation.

Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer-enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

Histone variants have been implicated in regulating chromatin dynamics and genome functions. Previously, we have shown that histone variant H3.3 actively marks enhancers and cooperates with H2A.Z at promoters to prime the genes into a poised state in mouse embryonic stem cells (mESCs). However, how these two important histone variants collaboratively function in this process still remains elusive. In this study, we found that depletion of different components of HIRA complex, a specific chaperone of H3.3, results in significant decreases of H2A.Z enrichment at genome scale. In addition, CUT&Tag data revealed a genomic colocalization between HIRA complex and SRCAP complex. In vivo and in vitro biochemical assays verified that HIRA complex could interact with SRCAP complex through the Hira subunit. Furthermore, our chromatin accessibility and transcription analyses demonstrated that HIRA complex contributed to preset a defined chromatin feature around TSS region for poising gene transcription. In summary, our results unveiled that while regulating the H3.3 incorporation in the regulatory regions, HIRA complex also collaborates with SRCAP to deposit H2A.Z onto the promoters, which cooperatively determines the transcriptional potential of the poised genes in mESCs.

Acquired resistance to temozolomide (TMZ) is a major obstacle in glioblastoma treatment. MGMT, a DNA repair protein, and the methylation at its gene promoter, plays an important role in TMZ resistance. However, some evidences have suggested a MGMT-independent mechanisms underlying TMZ resistance. Here, we used MGMT enhancer as a model and discovered that its deletion in glioma cells of low MGMT expression induced increased sensitivity to temozolomide. Analysis of a combination of RNA-seq and Capture Hi-C further suggested multiple long-range target genes regulated by the MGMT enhancer and that interactions may play important roles in glioma cell sensitivity to TMZ. This study reveals a novel mechanism of regulation of TMZ sensitivity in glioma cells.

Signaling pathway-driven target gene transcription is critical for fate determination of embryonic stem cells (ESCs), but enhancer-dependent transcriptional regulation in these processes remains poorly understood. Here, we report enhancer architecture-dependent multilayered transcriptional regulation at the Halr1-Hoxa1 locus that orchestrates retinoic acid (RA) signaling-induced early lineage differentiation of ESCs. We show that both homeobox A1 (Hoxa1) and Hoxa adjacent long non-coding RNA 1 (Halr1) are identified as direct downstream targets of RA signaling and regulated by RARA/RXRA via RA response elements (RAREs). Chromosome conformation capture-based screens indicate that RA signaling promotes enhancer interactions essential for Hoxa1 and Halr1 expression and mesendoderm differentiation of ESCs. Furthermore, the results also show that HOXA1 promotes expression of Halr1 through binding to enhancer; conversely, loss of Halr1 enhances interaction between Hoxa1 chromatin and four distal enhancers but weakens interaction with chromatin inside the HoxA cluster, leading to RA signaling-induced Hoxa1 overactivation and enhanced endoderm differentiation. These findings reveal complex transcriptional regulation involving synergistic regulation by enhancers, transcription factors and lncRNA. This work provides new insight into intrinsic molecular mechanisms underlying ESC fate determination during RA signaling-induced early differentiation.

The candidate anticancer drug curaxins can insert into DNA base pairs and efficiently inhibit the growth of various cancers. However, how curaxins alter the genomic DNA structure and affect the DNA binding property of key proteins remains to be clarified. Here, we first showed that curaxin CBL0137 strongly stabilizes the interaction between the double strands of DNA and reduces DNA bending and twist rigidity simultaneously, by single-molecule magnetic tweezers. More importantly, we found that CBL0137 greatly impairs the binding of CTCF but facilitates trapping FACT on DNA. We revealed that CBL0137 clamps the DNA double helix that may induce a huge barrier for DNA unzipping during replication and transcription and causes the distinct binding response of CTCF and FACT on DNA. Our work provides a novel mechanical insight into CBL0137's anticancer mechanisms at the nucleic acid level.

Extracellular vesicles (EVs) are thought to mediate intercellular communication through the delivery of cargo proteins and RNA to target cells. The uptake of EVs is often followed visually using lipophilic-dyes or fluorescently-tagged proteins to label membrane constituents that are then internalized into recipient cells ( Christianson et al., 2013 ; De Jong et al., 2019 ). However, these methods do not probe the exposure of EV cargo to intracellular compartments, such as the cytoplasm and nucleus, where protein or RNA molecules could elicit functional changes in recipient cells. In this protocol, we employ an EV cargo protein-APEX fusion to detect proximity interactions with recipient cell cytoplasmic/nuclear targets. This approach results in the biotinylation of proteins in close contact with the reporter fusion and thus permits profiling of biotinylated proteins affinity purified on immobilized streptavidin beads. Graphic abstract: Schematic showing three steps of APEX-mediated proximity labeling of proteins in cells targeted by EVs.

The long noncoding RNA HOTAIRM1 reportedly plays important roles in acute myeloid leukemia, gastric cancer and colorectal cancer. Here, we analyzed potential function of HOTAIRM1 in glioma and asked whether it participates in long-range chromatin interactions. We monitored expression of HOTAIRM1 in glioma tissues and correlated levels with patient survival using the TCGA dataset. HOTAIRM1 was highly expressed in glioma tissue, with high levels associated with shortened patient survival time. We then suppressed HOTAIRM1 activity in the human glioblastoma U251 line using CRISPR-cas9 to knock in a truncating polyA fragment. Reporter analysis of these and control cells confirmed that the HOTAIRM1 locus serves as an active enhancer. We then performed Capture-C analysis to identify target genes of that locus and applied RNA antisense purification to assess chromatin interactions between the HOTAIRM1 locus and HOXA cluster genes. HOTAIRM1 knockdown in glioma cells decreased proliferation and reduced expression of HOXA cluster genes. HOTAIRM1 regulates long-range interactions between the HOTAIRM1 locus and HOXA genes. Our work suggests a new mechanism by which HOTAIRM1 regulates glioma progression by regulating high-order chromatin structure and could suggest novel therapeutic targets to treat an intractable cancer.