WNT signalling control by KDM5C during development affects cognition.

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability1, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function.

In vitro generation of mouse morula-like cells.

Generating cells with the molecular and functional properties of embryo cells and with full developmental potential is an aim with fundamental biological significance. Here we report the in vitro generation of mouse transient morula-like cells (MLCs) via the manipulation of signaling pathways. MLCs are molecularly distinct from embryonic stem cells (ESCs) and cluster instead with embryo 8- to 16-cell stage cells. A single MLC can generate a blastoid, and the efficiency increases to 80% when 8-10 MLCs are used. MLCs make embryoids directly, efficiently, and within 4 days. Transcriptomic analysis shows that day 4-5 MLC-derived embryoids contain the cell types found in natural embryos at early gastrulation. Furthermore, MLCs introduced into morulae segregate into epiblast (EPI), primitive endoderm (PrE), and trophectoderm (TE) fates in blastocyst chimeras and have a molecular signature indistinguishable from that of host embryo cells. These findings represent the generation of cells that are molecularly and functionally similar to the precursors of the first three cell lineages of the embryo.

c-Jun as a one-way valve at the naive to primed interface.

BACKGROUND: c-Jun is a proto-oncogene functioning as a transcription factor to activate gene expression under many physiological and pathological conditions, particularly in somatic cells. However, its role in early embryonic development remains unknown. RESULTS: Here, we show that c-Jun acts as a one-way valve to preserve the primed state and impair reversion to the naïve state. c-Jun is induced during the naive to primed transition, and it works to stabilize the chromatin structure and inhibit the reverse transition. Loss of c-Jun has surprisingly little effect on the naïve to primed transition, and no phenotypic effect on primed cells, however, in primed cells the loss of c-Jun leads to a failure to correctly close naïve-specific enhancers. When the primed cells are induced to reprogram to a naïve state, these enhancers are more rapidly activated when c-Jun is lost or impaired, and the conversion is more efficient. CONCLUSIONS: The results of this study indicate that c-Jun can function as a chromatin stabilizer in primed EpiSCs, to maintain the epigenetic cell type state and act as a one-way valve for cell fate conversions.

Loss of PHF8 induces a viral mimicry response by activating endogenous retrotransposons.

Immunotherapy has become established as major treatment modality for multiple types of solid tumors, including colorectal cancer. Identifying novel immunotherapeutic targets to enhance anti-tumor immunity and sensitize current immune checkpoint blockade (ICB) in colorectal cancer is needed. Here we report the histone demethylase PHD finger protein 8 (PHF8, KDM7B), a Jumonji C domain-containing protein that erases repressive histone methyl marks, as an essential mediator of immune escape. Ablation the function of PHF8 abrogates tumor growth, activates anti-tumor immune memory, and augments sensitivity to ICB therapy in mouse models of colorectal cancer. Strikingly, tumor PHF8 deletion stimulates a viral mimicry response in colorectal cancer cells, where the depletion of key components of endogenous nucleic acid sensing diminishes PHF8 loss-meditated antiviral immune responses and anti-tumor effects in vivo. Mechanistically, PHF8 inhibition elicits H3K9me3-dependent retrotransposon activation by promoting proteasomal degradation of the H3K9 methyltransferase SETDB1 in a demethylase-independent manner. Moreover, PHF8 expression is anti-correlated with canonical immune signatures and antiviral immune responses in human colorectal adenocarcinoma. Overall, our study establishes PHF8 as an epigenetic checkpoint, and targeting PHF8 is a promising viral mimicry-inducing approach to enhance intrinsic anti-tumor immunity or to conquer immune resistance.

Three-dimensional molecular architecture of mouse organogenesis.

Mammalian embryos exhibit sophisticated cellular patterning that is intricately orchestrated at both molecular and cellular level. It has recently become apparent that cells within the animal body display significant heterogeneity, both in terms of their cellular properties and spatial distributions. However, current spatial transcriptomic profiling either lacks three-dimensional representation or is limited in its ability to capture the complexity of embryonic tissues and organs. Here, we present a spatial transcriptomic atlas of all major organs at embryonic day 13.5 in the mouse embryo, and provide a three-dimensional rendering of molecular regulation for embryonic patterning with stacked sections. By integrating the spatial atlas with corresponding single-cell transcriptomic data, we offer a detailed molecular annotation of the dynamic nature of organ development, spatial cellular interactions, embryonic axes, and divergence of cell fates that underlie mammalian development, which would pave the way for precise organ engineering and stem cell-based regenerative medicine.

The NuRD complex cooperates with SALL4 to orchestrate reprogramming.

Cell fate decision involves rewiring of the genome, but remains poorly understood at the chromatin level. Here, we report that chromatin remodeling complex NuRD participates in closing open chromatin in the early phase of somatic reprogramming. Sall4, Jdp2, Glis1 and Esrrb can reprogram MEFs to iPSCs efficiently, but only Sall4 is indispensable capable of recruiting endogenous components of NuRD. Yet knocking down NuRD components only reduces reprogramming modestly, in contrast to disrupting the known Sall4-NuRD interaction by mutating or deleting the NuRD interacting motif at its N-terminus that renders Sall4 inept to reprogram. Remarkably, these defects can be partially rescured by grafting NuRD interacting motif onto Jdp2. Further analysis of chromatin accessibility dynamics demonstrates that the Sall4-NuRD axis plays a critical role in closing the open chromatin in the early phase of reprogramming. Among the chromatin loci closed by Sall4-NuRD encode genes resistant to reprogramming. These results identify a previously unrecognized role of NuRD in reprogramming, and may further illuminate chromatin closing as a critical step in cell fate control.

Single-cell RNA sequencing reveals the developmental program underlying proximal-distal patterning of the human lung at the embryonic stage.

The lung is the primary respiratory organ in human, in which the proximal airway and the distal alveoli are responsible for air conduction and gas exchange, respectively. However, the regulation of proximal-distal patterning at the embryonic stage of human lung development is largely unknown. Here we investigated the early lung development of human embryos at weeks 4-8 post fertilization (Carnegie stages 12-21) using single-cell RNA sequencing, and obtained a transcriptomic atlas of 169,686 cells. We observed discernible gene expression patterns of proximal and distal epithelia at week 4, upon the initiation of lung organogenesis. Moreover, we identified novel transcriptional regulators of the patterning of proximal (e.g., THRB and EGR3) and distal (e.g., ETV1 and SOX6) epithelia. Further dissection revealed various stromal cell populations, including an early-embryonic BDNF+ population, providing a proximal-distal patterning niche with spatial specificity. In addition, we elucidated the cell fate bifurcation and maturation of airway and vascular smooth muscle progenitor cells at the early stage of lung development. Together, our study expands the scope of human lung developmental biology at early embryonic stages. The discovery of intrinsic transcriptional regulators and novel niche providers deepens the understanding of epithelial proximal-distal patterning in human lung development, opening up new avenues for regenerative medicine.

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing.

N6-methyladenosine (m6A) has been increasingly recognized as a new and important regulator of gene expression. To date, transcriptome-wide m6A detection primarily relies on well-established methods using next-generation sequencing (NGS) platform. However, direct RNA sequencing (DRS) using the Oxford Nanopore Technologies (ONT) platform has recently emerged as a promising alternative method to study m6A. While multiple computational tools are being developed to facilitate the direct detection of nucleotide modifications, little is known about the capabilities and limitations of these tools. Here, we systematically compare ten tools used for mapping m6A from ONT DRS data. We find that most tools present a trade-off between precision and recall, and integrating results from multiple tools greatly improve performance. Using a negative control could improve precision by subtracting certain intrinsic bias. We also observed variation in detection capabilities and quantitative information among motifs, and identified sequencing depth and m6A stoichiometry as potential factors affecting performance. Our study provides insight into the computational tools currently used for mapping m6A based on ONT DRS data and highlights the potential for further improving these tools, which may serve as the basis for future research.

Transposable elements activation triggers necroptosis in mouse embryonic stem cells.

Deficiency of the histone H3K9 methyltransferase SETDB1 induces RIPK3-dependent necroptosis in mouse embryonic stem cells (mESCs). However, how necroptosis pathway is activated in this process remains elusive. Here we report that the reactivation of transposable elements (TEs) upon SETDB1 knockout is responsible for the RIPK3 regulation through both cis and trans mechanisms. IAPLTR2_Mm and MMERVK10c-int, both of which are suppressed by SETDB1-dependent H3K9me3, act as enhancer-like cis-regulatory elements and their RIPK3 nearby members enhance RIPK3 expression when SETDB1 is knockout. Moreover, reactivated endogenous retroviruses generate excessive viral mimicry, which promotes necroptosis mainly through Z-DNA-binding protein 1 (ZBP1). These results indicate TEs play an important role in regulating necroptosis.

Species-specific rewiring of definitive endoderm developmental gene activation via endogenous retroviruses through TET1-mediated demethylation.

Transposable elements (TEs) are the major sources of lineage-specific genomic innovation and comprise nearly half of the human genome, but most of their functions remain unclear. Here, we identify that a series of endogenous retroviruses (ERVs), a TE subclass, regulate the transcriptome at the definitive endoderm stage with in vitro differentiation model from human embryonic stem cell. Notably, these ERVs perform as enhancers containing binding sites for critical transcription factors for endoderm lineage specification. Genome-wide methylation analysis shows most of these ERVs are derepressed by TET1-mediated DNA demethylation. LTR6B, a representative definitive endoderm activating ERV, contains binding sites for FOXA2 and GATA4 and governs the primate-specific expression of its neighboring developmental genes such as ERBB4 in definitive endoderm. Together, our study proposes evidence that recently evolved ERVs represent potent de novo developmental regulatory elements, which, in turn, fine-tune species-specific transcriptomes during endoderm and embryonic development.

KCNQ1OT1 promotes genome-wide transposon repression by guiding RNA-DNA triplexes and HP1 binding.

Transposon (de)repression and heterochromatin reorganization are dynamically regulated during cell fate determination and are hallmarks of cellular senescence. However, whether they are sequence specifically regulated remains unknown. Here we uncover that the KCNQ1OT1 lncRNA, by sequence-specific Hoogsteen base pairing with double-stranded genomic DNA via its repeat-rich region and binding to the heterochromatin protein HP1α, guides, induces and maintains epigenetic silencing at specific repetitive DNA elements. Repressing KCNQ1OT1 or deleting its repeat-rich region reduces DNA methylation and H3K9me3 on KCNQ1OT1-targeted transposons. Engineering a fusion KCNQ1OT1 with an ectopically targeting guiding triplex sequence induces de novo DNA methylation at the target site. Phenotypically, repressing KCNQ1OT1 induces senescence-associated heterochromatin foci, transposon activation and retrotransposition as well as cellular senescence, demonstrating an essential role of KCNQ1OT1 to safeguard against genome instability and senescence.

BMP4 drives primed to naïve transition through PGC-like state.

Multiple pluripotent states have been described in mouse and human stem cells. Here, we apply single-cell RNA-seq to a newly established BMP4 induced mouse primed to naïve transition (BiPNT) system and show that the reset is not a direct reversal of cell fate but goes through a primordial germ cell-like cells (PGCLCs) state. We first show that epiblast stem cells bifurcate into c-Kit+ naïve and c-Kit- trophoblast-like cells, among which, the naïve branch undergoes further transition through a PGCLCs intermediate capable of spermatogenesis in vivo. Mechanistically, we show that DOT1L inhibition permits the transition from primed pluripotency to PGCLCs in part by facilitating the loss of H3K79me2 from Gata3/6. In addition, Prdm1/Blimp1 is required for PGCLCs and naïve cells, while Gata2 inhibits PGC-like state by promoting trophoblast-like fate. Our work not only reveals an alternative route for primed to naïve transition, but also gains insight into germ cell development.

Fast and Efficient Mouse Pluripotency Reprogramming Using a Chemically-Defined Medium.

The reprogramming of somatic cells to obtain induced pluripotent stem cells (iPSCs) is an important biological and medical breakthrough, providing important applications for fields such as regenerative medicine and disease modeling. However, this promising technology is damped due to its low efficiency and slow kinetics. Therefore, we generated a practical workflow to rapidly and efficiently induce iPSCs from mouse embryonic fibroblasts (MEFs) using iCD1 (iPS chemically-defined medium 1). This protocol can easily be implemented in a standard cell culture laboratory and be applied to cell fate research.

Mitolysosome exocytosis, a mitophagy-independent mitochondrial quality control in flunarizine-induced parkinsonism-like symptoms.

Mitochondrial quality control plays an important role in maintaining mitochondrial homeostasis and function. Disruption of mitochondrial quality control degrades brain function. We found that flunarizine (FNZ), a drug whose chronic use causes parkinsonism, led to a parkinsonism-like motor dysfunction in mice. FNZ induced mitochondrial dysfunction and decreased mitochondrial mass specifically in the brain. FNZ decreased mitochondrial content in both neurons and astrocytes, without affecting the number of nigral dopaminergic neurons. In human neural progenitor cells, FNZ also induced mitochondrial depletion. Mechanistically, independent of ATG5- or RAB9-mediated mitophagy, mitochondria were engulfed by lysosomes, followed by a vesicle-associated membrane protein 2- and syntaxin-4-dependent extracellular secretion. A genome-wide CRISPR knockout screen identified genes required for FNZ-induced mitochondrial elimination. These results reveal not only a previously unidentified lysosome-associated exocytosis process of mitochondrial quality control that may participate in the FNZ-induced parkinsonism but also a drug-based method for generating mitochondria-depleted mammal cells.

Human induced-T-to-natural killer cells have potent anti-tumour activities.

BACKGROUND: Adoptive cell therapy (ACT) is a particularly promising area of cancer immunotherapy, engineered T and NK cells that express chimeric antigen receptors (CAR) are being explored for treating hematopoietic malignancies but exhibit limited clinical benefits for solid tumour patients, successful cellular immunotherapy of solid tumors demands new strategies. METHODS: Inactivation of BCL11B were performed by CRISPR/Cas9 in human T cells. Immunophenotypic and transcriptional profiles of sgBCL11B T cells were characterized by cytometer and transcriptomics, respectively. sgBCL11B T cells are further engineered with chimeric antigen receptor. Anti-tumor activity of ITNK or CAR-ITNK cells were evaluated in preclinical and clinical studies. RESULTS: We report that inactivation of BCL11B in human CD8+ and CD4+ T cells induced their reprogramming into induced T-to-natural killer cells (ITNKs). ITNKs contained a diverse TCR repertoire; downregulated T cell-associated genes such as TCF7 and LEF1; and expressed high levels of NK cell lineage-associated genes. ITNKs and chimeric antigen receptor (CAR)-transduced ITNKs selectively lysed a variety of cancer cells in culture and suppressed the growth of solid tumors in xenograft models. In a preliminary clinical study, autologous administration of ITNKs in patients with advanced solid tumors was well tolerated, and tumor stabilization was seen in six out nine patients, with one partial remission. CONCLUSIONS: The novel ITNKs thus may be a promising novel cell source for cancer immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03882840 . Registered 20 March 2019-Retrospectively registered.

scDIOR: single cell RNA-seq data IO software.

BACKGROUND: Single-cell RNA sequencing is becoming a powerful tool to identify cell states, reconstruct developmental trajectories, and deconvolute spatial expression. The rapid development of computational methods promotes the insight of heterogeneous single-cell data. An increasing number of tools have been provided for biological analysts, of which two programming languages- R and Python are widely used among researchers. R and Python are complementary, as many methods are implemented specifically in R or Python. However, the different platforms immediately caused the data sharing and transformation problem, especially for Scanpy, Seurat, and SingleCellExperiemnt. Currently, there is no efficient and user-friendly software to perform data transformation of single-cell omics between platforms, which makes users spend unbearable time on data Input and Output (IO), significantly reducing the efficiency of data analysis. RESULTS: We developed scDIOR for single-cell data transformation between platforms of R and Python based on Hierarchical Data Format Version 5 (HDF5). We have created a data IO ecosystem between three R packages (Seurat, SingleCellExperiment, Monocle) and a Python package (Scanpy). Importantly, scDIOR accommodates a variety of data types across programming languages and platforms in an ultrafast way, including single-cell RNA-seq and spatial resolved transcriptomics data, using only a few codes in IDE or command line interface. For large scale datasets, users can partially load the needed information, e.g., cell annotation without the gene expression matrices. scDIOR connects the analytical tasks of different platforms, which makes it easy to compare the performance of algorithms between them. CONCLUSIONS: scDIOR contains two modules, dior in R and diopy in Python. scDIOR is a versatile and user-friendly tool that implements single-cell data transformation between R and Python rapidly and stably. The software is freely accessible at https://github.com/JiekaiLab/scDIOR .

Practical bioinformatics pipelines for single-cell RNA-seq data analysis.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool to explore cells. With an increasing number of scRNA-seq data analysis tools that have been developed, it is challenging for users to choose and compare their performance. Here, we present an overview of the workflow for computational analysis of scRNA-seq data. We detail the steps of a typical scRNA-seq analysis, including experimental design, pre-processing and quality control, feature selection, dimensionality reduction, cell clustering and annotation, and downstream analysis including batch correction, trajectory inference and cell-cell communication. We provide guidelines according to our best practice. This review will be helpful for the experimentalists interested in analyzing their data, and will aid the users seeking to update their analysis pipelines.

MAP2K6 remodels chromatin and facilitates reprogramming by activating Gatad2b-phosphorylation dependent heterochromatin loosening.

Somatic cell reprogramming is an ideal model for studying epigenetic regulation as it undergoes dramatic chromatin remodeling. However, a role for phosphorylation signaling in chromatin protein modifications for reprogramming remains unclear. Here, we identified mitogen-activated protein kinase kinase 6 (Mkk6) as a chromatin relaxer and found that it could significantly enhance reprogramming. The function of Mkk6 in heterochromatin loosening and reprogramming requires its kinase activity but does not depend on its best-known target, P38. We identified Gatad2b as a novel target of Mkk6 phosphorylation that acts downstream to elevate histone acetylation levels and loosen heterochromatin. As a result, Mkk6 over-expression facilitates binding of Sox2 and Klf4 to their targets and promotes pluripotency gene expression during reprogramming. Our studies not only reveal an Mkk phosphorylation mediated modulation of chromatin status in reprogramming, but also provide new rationales to further investigate and improve the cell fate determination processes.