STAG2 mutations reshape the cohesin-structured spatial chromatin architecture to drive gene regulation in acute myeloid leukemia.

Cohesin shapes the chromatin architecture, including enhancer-promoter interactions. Its components, especially STAG2, but not its paralog STAG1, are frequently mutated in myeloid malignancies. To elucidate the underlying mechanisms of leukemogenesis, we comprehensively characterized genetic, transcriptional, and chromatin conformational changes in acute myeloid leukemia (AML) patient samples. Specific loci displayed altered cohesin occupancy, gene expression, and local chromatin activation, which were not compensated by the remaining STAG1-cohesin. These changes could be linked to disrupted spatial chromatin looping in cohesin-mutated AMLs. Complementary depletion of STAG2 or STAG1 in primary human hematopoietic progenitors (HSPCs) revealed effects resembling STAG2-mutant AML-specific changes following STAG2 knockdown, not invoked by the depletion of STAG1. STAG2-deficient HSPCs displayed impaired differentiation capacity and maintained HSPC-like gene expression. This work establishes STAG2 as a key regulator of chromatin contacts, gene expression, and differentiation in the hematopoietic system and identifies candidate target genes that may be implicated in human leukemogenesis.

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia.

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.

Genome-wide DNA methylation changes in human spermatogenesis.

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.

Specific photoreceptor cell fate pathways are differentially altered in NR2E3-associated diseases.

Mutations in NR2E3, a gene encoding an orphan nuclear transcription factor, cause two retinal dystrophies with a distinct phenotype, but the precise role of NR2E3 in rod and cone transcriptional networks remains unclear. To dissect NR2E3 function, we performed scRNA-seq in the retinas of wildtype and two different Nr2e3 mouse models that show phenotypes similar to patients carrying NR2E3 mutations. Our results reveal that rod and cone populations are not homogeneous and can be separated into different sub-classes. We identify a previously unreported cone pathway that generates hybrid cones co-expressing both cone- and rod-related genes. In mutant retinas, this hybrid cone subpopulation is more abundant and includes a subpopulation of rods transitioning towards a cone cell fate. Hybrid photoreceptors with high misexpression of cone- and rod-related genes are prone to regulated necrosis. Overall, our results shed light on the role of NR2E3 in modulating photoreceptor differentiation towards cone and rod fates and explain how different mutations in NR2E3 lead to distinct visual disorders in humans.

Doxorubicin Changes the Spatial Organization of the Genome around Active Promoters.

In this study, we delve into the impact of genotoxic anticancer drug treatment on the chromatin structure of human cells, with a particular focus on the effects of doxorubicin. Using Hi-C, ChIP-seq, and RNA-seq, we explore the changes in chromatin architecture brought about by doxorubicin and ICRF193. Our results indicate that physiologically relevant doses of doxorubicin lead to a local reduction in Hi-C interactions in certain genomic regions that contain active promoters, with changes in chromatin architecture occurring independently of Top2 inhibition, cell cycle arrest, and differential gene expression. Inside the regions with decreased interactions, we detected redistribution of RAD21 around the peaks of H3K27 acetylation. Our study also revealed a common structural pattern in the regions with altered architecture, characterized by two large domains separated from each other. Additionally, doxorubicin was found to increase CTCF binding in H3K27 acetylated regions. Furthermore, we discovered that Top2-dependent chemotherapy causes changes in the distance decay of Hi-C contacts, which are driven by direct and indirect inhibitors. Our proposed model suggests that doxorubicin-induced DSBs cause cohesin redistribution, which leads to increased insulation on actively transcribed TAD boundaries. Our findings underscore the significant impact of genotoxic anticancer treatment on the chromatin structure of the human genome.

Early developmental plasticity enables the induction of an intermediate extraembryonic cell state.

Two fundamental elements of pre-implantation embryogenesis are cells' intrinsic self-organization program and their developmental plasticity, which allows embryos to compensate for alterations in cell position and number; yet, these elements are still poorly understood. To be able to decipher these features, we established culture conditions that enable the two fates of blastocysts' extraembryonic lineages-the primitive endoderm and the trophectoderm-to coexist. This plasticity emerges following the mechanisms of the first lineage segregation in the mouse embryo, and it manifests as an extended potential for extraembryonic chimerism during the pre-implantation embryogenesis. Moreover, this shared state enables robust assembly into higher-order blastocyst-like structures, thus combining both the cell fate plasticity and self-organization features of the early extraembryonic lineages.

Multiomic atlas with functional stratification and developmental dynamics of zebrafish cis-regulatory elements.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

Shear stress switches the association of endothelial enhancers from ETV/ETS to KLF transcription factor binding sites.

Endothelial cells (ECs) lining blood vessels are exposed to mechanical forces, such as shear stress. These forces control many aspects of EC biology, including vascular tone, cell migration and proliferation. Despite a good understanding of the genes responding to shear stress, our insight into the transcriptional regulation of these genes is much more limited. Here, we set out to study alterations in the chromatin landscape of human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress. To do so, we performed ChIP-Seq for H3K27 acetylation, indicative of active enhancer elements and ATAC-Seq to mark regions of open chromatin in addition to RNA-Seq on HUVEC exposed to 6 h of laminar shear stress. Our results show a correlation of gained and lost enhancers with up and downregulated genes, respectively. DNA motif analysis revealed an over-representation of KLF transcription factor (TF) binding sites in gained enhancers, while lost enhancers contained more ETV/ETS motifs. We validated a subset of flow responsive enhancers using luciferase-based reporter constructs and CRISPR-Cas9 mediated genome editing. Lastly, we characterized the shear stress response in ECs of zebrafish embryos using RNA-Seq. Our results lay the groundwork for the exploration of shear stress responsive elements in controlling EC biology.

Lima1 mediates the pluripotency control of membrane dynamics and cellular metabolism.

Lima1 is an extensively studied prognostic marker of malignancy and is also considered to be a tumour suppressor, but its role in a developmental context of non-transformed cells is poorly understood. Here, we characterise the expression pattern and examined the function of Lima1 in mouse embryos and pluripotent stem cell lines. We identify that Lima1 expression is controlled by the naïve pluripotency circuit and is required for the suppression of membrane blebbing, as well as for proper mitochondrial energetics in embryonic stem cells. Moreover, forcing Lima1 expression enables primed mouse and human pluripotent stem cells to be incorporated into murine pre-implantation embryos. Thus, Lima1 is a key effector molecule that mediates the pluripotency control of membrane dynamics and cellular metabolism.

Emerging mechanisms and dynamics of three-dimensional genome organisation at zygotic genome activation.

The genome of an early embryo undergoes significant remodelling at the epigenetic, transcriptional, and structural levels. New technological developments have made it possible to study 3D genome organisation in the zygote and early embryo of many different species. Recent studies in human embryos, zebrafish, medaka, and Xenopus have revealed that, similar to previous results in mouse and Drosophila, the zygotic genome is unstructured prior to zygotic genome activation. While these studies show that topologically associating domains are established coincident with zygotic genome activation across species, other 3D genome structures have more varied timing. Here, we review recent studies examining the timing and mechanisms of establishment of 3D genome organisation in the early embryo, and discuss similarities and differences between species. Investigating the establishment of 3D chromatin conformation in early embryos has the potential to reveal novel mechanisms of 3D genome organisation.

Zebrafish transposable elements show extensive diversification in age, genomic distribution, and developmental expression.

There is considerable interest in understanding the effect of transposable elements (TEs) on embryonic development. Studies in humans and mice are limited by the difficulty of working with mammalian embryos and by the relative scarcity of active TEs in these organisms. The zebrafish is an outstanding model for the study of vertebrate development, and over half of its genome consists of diverse TEs. However, zebrafish TEs remain poorly characterized. Here we describe the demography and genomic distribution of zebrafish TEs and their expression throughout embryogenesis using bulk and single-cell RNA sequencing data. These results reveal a highly dynamic genomic ecosystem comprising nearly 2000 distinct TE families, which vary in copy number by four orders of magnitude and span a wide range of ages. Longer retroelements tend to be retained in intergenic regions, whereas short interspersed nuclear elements (SINEs) and DNA transposons are more frequently found nearby or within genes. Locus-specific mapping of TE expression reveals extensive TE transcription during development. Although two-thirds of TE transcripts are likely driven by nearby gene promoters, we still observe stage- and tissue-specific expression patterns in self-regulated TEs. Long terminal repeat (LTR) retroelements are most transcriptionally active immediately following zygotic genome activation, whereas DNA transposons are enriched among transcripts expressed in later stages of development. Single-cell analysis reveals several endogenous retroviruses expressed in specific somatic cell lineages. Overall, our study provides a valuable resource for using zebrafish as a model to study the impact of TEs on vertebrate development.

Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development.

During implantation, the murine embryo transitions from a "quiet" into an active metabolic/proliferative state, which kick-starts the growth and morphogenesis of the post-implantation conceptus. Such transition is also required for embryonic stem cells to be established from mouse blastocysts, but the factors regulating this process are poorly understood. Here, we show that Ronin plays a critical role in the process by enabling active energy production, and the loss of Ronin results in the establishment of a reversible quiescent state in which naïve pluripotency is promoted. In addition, Ronin fine-tunes the expression of genes that encode ribosomal proteins and is required for proper tissue-scale organisation of the pluripotent lineage during the transition from blastocyst to egg cylinder stage. Thus, Ronin function is essential for governing the metabolic capacity so that it can support the pluripotent lineage's high-energy demands for cell proliferation and morphogenesis.

Chromatin architecture transitions from zebrafish sperm through early embryogenesis.

Chromatin architecture mapping in 3D formats has increased our understanding of how regulatory sequences and gene expression are connected and regulated in a genome. The 3D chromatin genome shows extensive remodeling during embryonic development, and although the cleavage-stage embryos of most species lack structure before zygotic genome activation (pre-ZGA), zebrafish has been reported to have structure. Here, we aimed to determine the chromosomal architecture in paternal/sperm zebrafish gamete cells to discern whether it either resembles or informs early pre-ZGA zebrafish embryo chromatin architecture. First, we assessed the higher-order architecture through advanced low-cell in situ Hi-C. The structure of zebrafish sperm, packaged by histones, lacks topological associated domains and instead displays "hinge-like" domains of ∼150 kb that repeat every 1-2 Mbs, suggesting a condensed repeating structure resembling mitotic chromosomes. The pre-ZGA embryos lacked chromosomal structure, in contrast to prior work, and only developed structure post-ZGA. During post-ZGA, we find chromatin architecture beginning to form at small contact domains of a median length of ∼90 kb. These small contact domains are established at enhancers, including super-enhancers, and chemical inhibition of Ep300a (p300) and Crebbpa (CBP) activity, lowering histone H3K27ac, but not transcription inhibition, diminishes these contacts. Together, this study reveals hinge-like domains in histone-packaged zebrafish sperm chromatin and determines that the initial formation of high-order chromatin architecture in zebrafish embryos occurs after ZGA primarily at enhancers bearing high H3K27ac.

Repression of endogenous retroviruses prevents antiviral immune response and is required for mammary gland development.

The role of heterochromatin in cell fate specification during development is unclear. We demonstrate that loss of the lysine 9 of histone H3 (H3K9) methyltransferase G9a in the mammary epithelium results in de novo chromatin opening, aberrant formation of the mammary ductal tree, impaired stem cell potential, disrupted intraductal polarity, and loss of tissue function. G9a loss derepresses long terminal repeat (LTR) retroviral sequences (predominantly the ERVK family). Transcriptionally activated endogenous retroviruses generate double-stranded DNA (dsDNA) that triggers an antiviral innate immune response, and knockdown of the cytosolic dsDNA sensor Aim2 in G9a knockout (G9acKO) mammary epithelium rescues mammary ductal invasion. Mammary stem cell transplantation into immunocompromised or G9acKO-conditioned hosts shows partial dependence of the G9acKO mammary morphological defects on the inflammatory milieu of the host mammary fat pad. Thus, altering the chromatin accessibility of retroviral elements disrupts mammary gland development and stem cell activity through both cell-autonomous and non-autonomous mechanisms.

Independence of chromatin conformation and gene regulation during Drosophila dorsoventral patterning.

The relationship between chromatin organization and gene regulation remains unclear. While disruption of chromatin domains and domain boundaries can lead to misexpression of developmental genes, acute depletion of regulators of genome organization has a relatively small effect on gene expression. It is therefore uncertain whether gene expression and chromatin state drive chromatin organization or whether changes in chromatin organization facilitate cell-type-specific activation of gene expression. Here, using the dorsoventral patterning of the Drosophila melanogaster embryo as a model system, we provide evidence for the independence of chromatin organization and dorsoventral gene expression. We define tissue-specific enhancers and link them to expression patterns using single-cell RNA-seq. Surprisingly, despite tissue-specific chromatin states and gene expression, chromatin organization is largely maintained across tissues. Our results indicate that tissue-specific chromatin conformation is not necessary for tissue-specific gene expression but rather acts as a scaffold facilitating gene expression when enhancers become active.

One-step Reprogramming of Human Fibroblasts into Oligodendrocyte-like Cells by SOX10, OLIG2, and NKX6.2.

Limited access to human oligodendrocytes impairs better understanding of oligodendrocyte pathology in myelin diseases. Here, we describe a method to robustly convert human fibroblasts directly into oligodendrocyte-like cells (dc-hiOLs), which allows evaluation of remyelination-promoting compounds and disease modeling. Ectopic expression of SOX10, OLIG2, and NKX6.2 in human fibroblasts results in rapid generation of O4+ cells, which further differentiate into MBP+ mature oligodendrocyte-like cells within 16 days. dc-hiOLs undergo chromatin remodeling to express oligodendrocyte markers, ensheath axons, and nanofibers in vitro, respond to promyelination compound treatment, and recapitulate in vitro oligodendroglial pathologies associated with Pelizaeus-Merzbacher leukodystrophy related to PLP1 mutations. Furthermore, DNA methylome analysis provides evidence that the CpG methylation pattern significantly differs between dc-hiOLs derived from fibroblasts of young and old donors, indicating the maintenance of the source cells' "age." In summary, dc-hiOLs represent a reproducible technology that could contribute to personalized medicine in the field of myelin diseases.

Germ cell differentiation requires Tdrd7-dependent chromatin and transcriptome reprogramming marked by germ plasm relocalization.

In many animal models, primordial germ cell (PGC) development depends on maternally deposited germ plasm, which prevents somatic cell fate. Here, we show that PGCs respond to regulatory information from the germ plasm in two distinct phases using two distinct mechanisms in zebrafish. We demonstrate that PGCs commence zygotic genome activation together with the somatic blastocysts with no demonstrable differences in transcriptional and chromatin opening. Unexpectedly, both PGC and somatic blastocysts activate germ-cell-specific genes, which are only stabilized in PGCs by cytoplasmic germ plasm determinants. Disaggregated perinuclear relocalization of germ plasm during PGC migration is regulated by the germ plasm determinant Tdrd7 and is coupled to dramatic divergence between PGC and somatic transcriptomes. This transcriptional divergence relies on PGC-specific cis-regulatory elements characterized by promoter-proximal distribution. We show that Tdrd7-dependent reconfiguration of chromatin accessibility is required for elaboration of PGC fate but not for PGC migration.

The Transition from Quiescent to Activated States in Human Hematopoietic Stem Cells Is Governed by Dynamic 3D Genome Reorganization.

Lifelong blood production requires long-term hematopoietic stem cells (LT-HSCs), marked by stemness states involving quiescence and self-renewal, to transition into activated short-term HSCs (ST-HSCs) with reduced stemness. As few transcriptional changes underlie this transition, we used single-cell and bulk assay for transposase-accessible chromatin sequencing (ATAC-seq) on human HSCs and hematopoietic stem and progenitor cell (HSPC) subsets to uncover chromatin accessibility signatures, one including LT-HSCs (LT/HSPC signature) and another excluding LT-HSCs (activated HSPC [Act/HSPC] signature). These signatures inversely correlated during early hematopoietic commitment and differentiation. The Act/HSPC signature contains CCCTC-binding factor (CTCF) binding sites mediating 351 chromatin interactions engaged in ST-HSCs, but not LT-HSCs, enclosing multiple stemness pathway genes active in LT-HSCs and repressed in ST-HSCs. CTCF silencing derepressed stemness genes, restraining quiescent LT-HSCs from transitioning to activated ST-HSCs. Hence, 3D chromatin interactions centrally mediated by CTCF endow a gatekeeper function that governs the earliest fate transitions HSCs make by coordinating disparate stemness pathways linked to quiescence and self-renewal.