Cdt1 overexpression drives colorectal carcinogenesis through origin overlicensing and DNA damage.

Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI). © 2022 The Pathological Society of Great Britain and Ireland.

RIP2-deficiency induces inflammation in response to SV40 Large T induced genotoxic stress through altered ROS homeostasis.

Deciphering signaling pathways that regulate the complex interplay between inflammation and cell death is a key challenge in understanding innate immune responses. Over recent years, receptor interacting protein (RIP) kinases have been described to regulate the interplay between inflammation and cell death. Whereas RIP1 and 3, the most well described members of the RIP kinase family, play important roles in necroptosis, RIP2's involvement in regulating inflammation, cell death processes and cancer is less well described and controversially discussed. Here, we demonstrate that RIP2 exerts immune regulatory functions by regulating mitochondrial damage and mitochondrial superoxide production in response to SV40 LT-induced genotoxic stress by the induction of ULK1-phosphorylation, therefore regulating the expression of interferon stimulated genes (ISGs) and NLRP3-inflammasome dependent IL-1ß release. Because RIP2 is upregulated and/or activated in autoimmune/inflammatory disease and cancer, observations from this study promise implications of RIP kinases in human disease.

N6-methyladenosine (m6A) depletion regulates pluripotency exit by activating signaling pathways in embryonic stem cells.

N6-methyladenosine (m6A) deposition on messenger RNA (mRNA) controls embryonic stem cell (ESC) fate by regulating the mRNA stabilities of pluripotency and lineage transcription factors (TFs) [P. J. Batista et al., Cell Stem Cell 15, 707-719 (2014); Y. Wang et al., Nat. Cell Biol. 16, 191-198 (2014); and S. Geula et al., Science 347, 1002-1006 (2015)]. If the mRNAs of these two TF groups become stabilized, it remains unclear how the pluripotency or lineage commitment decision is implemented. We performed noninvasive quantification of Nanog and Oct4 TF protein levels in reporter ESCs to define cell-state dynamics at single-cell resolution. Long-term single-cell tracking shows that immediate m6A depletion by Mettl3 knock-down in serum/leukemia inhibitory factor supports both pluripotency maintenance and its departure. This is mediated by differential and opposing signaling pathways. Increased FGF5 mRNA stability activates pErk, leading to Nanog down-regulation. FGF5-mediated coactivation of pAkt reenforces Nanog expression. In formative stem cells poised toward differentiation, m6A depletion activates both pErk and pAkt, increasing the propensity for mesendodermal lineage induction. Stable m6A depletion by Mettl3 knock-out also promotes pErk activation. Higher pErk counteracts the pluripotency exit delay exhibited by stably m6A-depleted cells upon differentiation. At single-cell resolution, we illustrate that decreasing m6A abundances activates pErk and pAkt-signaling, regulating pluripotency departure.

MLL1 is required for maintenance of intestinal stem cells.

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.

Reduced purine biosynthesis in humans after their divergence from Neandertals.

We analyze the metabolomes of humans, chimpanzees, and macaques in muscle, kidney and three different regions of the brain. Although several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution contributes to much or all of the reduction of de novo synthesis of purines in humans.

Tsga8 is required for spermatid morphogenesis and male fertility in mice.

During spermatogenesis, intricate gene expression is coordinately regulated by epigenetic modifiers, which are required for differentiation of spermatogonial stem cells (SSCs) contained among undifferentiated spermatogonia. We have previously found that KMT2B conveys H3K4me3 at bivalent and monovalent promoters in undifferentiated spermatogonia. Because these genes are expressed late in spermatogenesis or during embryogenesis, we expect that many of them are potentially programmed by KMT2B for future expression. Here, we show that one of the genes targeted by KMT2B, Tsga8, plays an essential role in spermatid morphogenesis. Loss of Tsga8 in mice leads to male infertility associated with abnormal chromosomal distribution in round spermatids, malformation of elongating spermatid heads and spermiation failure. Tsga8 depletion leads to dysregulation of thousands of genes, including the X-chromosome genes that are reactivated in spermatids, and insufficient nuclear condensation accompanied by reductions of TNP1 and PRM1, key factors for histone-to-protamine transition. Intracytoplasmic sperm injection (ICSI) of spermatids rescued the infertility phenotype, suggesting competency of the spermatid genome for fertilization. Thus, Tsga8 is a KMT2B target that is vitally necessary for spermiogenesis and fertility.

Enhancers are activated by p300/CBP activity-dependent PIC assembly, RNAPII recruitment, and pause release.

The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.

A Novel GATA2 Protein Reporter Mouse Reveals Hematopoietic Progenitor Cell Types.

The transcription factor (TF) GATA2 plays a key role in organ development and cell fate control in the central nervous, urogenital, respiratory, and reproductive systems, and in primitive and definitive hematopoiesis. Here, we generate a knockin protein reporter mouse line expressing a GATA2VENUS fusion from the endogenous Gata2 genomic locus, with correct expression and localization of GATA2VENUS in different organs. GATA2VENUS expression is heterogeneous in different hematopoietic stem and progenitor cell populations (HSPCs), identifies functionally distinct subsets, and suggests a novel monocyte and mast cell lineage bifurcation point. GATA2 levels further correlate with proliferation and lineage outcome of hematopoietic progenitors. The GATA2VENUS mouse line improves the identification of specific live cell types during embryonic and adult development and will be crucial for analyzing GATA2 protein dynamics in TF networks.

The role of SETD1A and SETD1B in development and disease.

The Trithorax-related Set1 H3K4 methyltransferases are conserved from yeast to human. In yeast loss of Set1 causes pleiotropic effects but is compatible with life. In contrast, both mammalian Set1 orthologs: SETD1A and SETD1B are essential for embryonic development, however they have distinct functions. SETD1A is required shortly after epiblast formation whereas SETD1B becomes indispensible during early organogenesis. In adult mice both SETD1A and SETD1B regulate hematopoiesis differently: SETD1A is required for the establishment of definitive hematopoiesis whereas SETD1B is important for the maintenance of long-term hematopoietic stem cells. Both are implicated in different diseases with accumulating evidence for the association of SETD1A variants in neurological disorders and SETD1B variants with cancer. Why the two paralogs cannot or only partially compensate for the loss of each other is part of the puzzle that we try to sort out in this review.

MLL4 is required after implantation, whereas MLL3 becomes essential during late gestation.

Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of MLL3 and MLL4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sex-distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors.

Aldh1b1 expression defines progenitor cells in the adult pancreas and is required for Kras-induced pancreatic cancer.

The presence of progenitor or stem cells in the adult pancreas and their potential involvement in homeostasis and cancer development remain unresolved issues. Here, we show that mouse centroacinar cells can be identified and isolated by virtue of the mitochondrial enzyme Aldh1b1 that they uniquely express. These cells are necessary and sufficient for the formation of self-renewing adult pancreatic organoids in an Aldh1b1-dependent manner. Aldh1b1-expressing centroacinar cells are largely quiescent, self-renew, and, as shown by genetic lineage tracing, contribute to all 3 pancreatic lineages in the adult organ under homeostatic conditions. Single-cell RNA sequencing analysis of these cells identified a progenitor cell population, established its molecular signature, and determined distinct differentiation pathways to early progenitors. A distinct feature of these progenitor cells is the preferential expression of small GTPases, including Kras, suggesting that they might be susceptible to Kras-driven oncogenic transformation. This finding and the overexpression of Aldh1b1 in human and mouse pancreatic cancers, driven by activated Kras, prompted us to examine the involvement of Aldh1b1 in oncogenesis. We demonstrated genetically that ablation of Aldh1b1 completely abrogates tumor development in a mouse model of KrasG12D-induced pancreatic cancer.

Prospective isolation of nonhematopoietic cells of the niche and their differential molecular interactions with HSCs.

A major limitation preventing in vivo modulation of hematopoietic stem cells (HSCs) is the incomplete understanding of the cellular and molecular support of the microenvironment in regulating HSC fate decisions. Consequently, murine HSCs cannot be generated, maintained, or expanded in culture over extended periods of time. A significantly improved understanding of the bone marrow niche environment and its molecular interactions with HSCs is pivotal to overcoming this challenge. We here prospectively isolated all major nonhematopoietic cellular niche components and cross-correlate them in detail with niche cells defined by lineage marking or tracing. Compiling an extensive database of soluble and membrane-bound ligand-receptor interactions, we developed a computational method to infer potential cell-to-cell interactions based on transcriptome data of sorter-purified niche cells and hematopoietic stem and progenitor cell subpopulations. Thus, we establish a compendium of the molecular communication between defined niche components and HSCs. Our analysis suggests an important role for cytokine antagonists in the regulation of HSC functions.

Inducible IL-7 Hyperexpression Influences Lymphocyte Homeostasis and Function and Increases Allograft Rejection.

The IL-7/IL-7R pathway is essential for lymphocyte development and disturbances in the pathway can lead to immune deficiency or T cell mediated destruction. Here, the effect of transient hyperexpression of IL-7 was investigated on immune regulation and allograft rejection under immunosuppression. An experimental in vivo immunosuppressive mouse model of IL-7 hyperexpression was developed using transgenic mice (C57BL/6 background) carrying a tetracycline inducible IL-7 expression cassette, which allowed the temporally controlled induction of IL-7 hyperexpression by Dexamethasone and Doxycycline treatment. Upon induction of IL-7, the B220+ c-kit+ Pro/Pre-B I compartment in the bone marrow increased as compared to control mice in a serum IL-7 concentration-correlated manner. IL-7 hyperexpression also preferentially increased the population size of memory CD8+ T cells in secondary lymphoid organs, and reduced the proportion of CD4+Foxp3+ T regulatory cells. Of relevance to disease, conventional CD4+ T cells from an IL-7-rich milieu escaped T regulatory cell-mediated suppression in vitro and in a model of autoimmune diabetes in vivo. These findings were validated using an IL-7/anti-IL7 complex treatment mouse model to create an IL-7 rich environment. To study the effect of IL-7 on islet graft survival in a mismatched allograft model, BALB/c mice were rendered diabetic by streptozotocin und transplanted with IL-7-inducible or control islets from C57BL/6 mice. As expected, Dexamethasone and Doxycycline treatment prolonged graft median survival as compared to the untreated control group in this transplantation mouse model. However, upon induction of local IL-7 hyperexpression in the transplanted islets, graft survival time was decreased and this was accompanied by an increased CD4+ and CD8+ T cell infiltration in the islets. Altogether, the findings show that transient elevations of IL-7 can impair immune regulation and lead to graft loss also under immune suppression.

Hox Genes Polymorphism Depicts Developmental Disruption of Common Sole Eggs.

In sole aquaculture production, consistency in the quality of produced eggs throughout the year is unpredictable. Hox genes have a crucial role in controlling embryonic development and their genetic variation could alter the phenotype dramatically. In teleosts genome duplication led paralog hox genes to become diverged. Direct association of polymorphism in hoxa1a, hoxa2a & hoxa2b of Solea solea with egg viability indicates hoxa2b as a potential genetic marker. High Resolution Melt (HRM) analysis was carried out in 52 viable and 61 non-viable eggs collected at 54±6 hours post fertilization (hpf). Allelic and genotypic frequencies of polymorphism were analyzed and results illustrated a significantly increased risk for non-viability for minor alleles and their homozygous genotypes. Haplotype analysis demonstrated a significant recessive effect on the risk of non-viability, by increasing the odds of disrupting embryonic development up to three-fold. Phylogenetic analysis showed that the paralog genes hoxa2a and hoxa2b, are separated distinctly in two clades and presented a significant ω variation, revealing their diverged evolutionary rate.

Distinct pathways affected by menin versus MLL1/MLL2 in MLL-rearranged acute myeloid leukemia.

Disrupting the protein-protein interaction for molecularly targeted cancer therapeutics can be a challenging but promising strategy. Compounds that disrupt the interaction between menin, a chromatin-binding protein, and oncogenic mixed lineage leukemia fusion proteins (MLL-FPs) have shown significant promise in preclinical models of leukemia and have a high degree of selectivity for leukemia versus normal hematopoietic cells. Biochemical and structural studies demonstrate that, in addition to disrupting the menin-MLL-FP interaction, such compounds also inhibit menin-MLL1, menin-MLL2, and other menin-interacting proteins. Here, we address the degree to which disruption of menin-MLL-FP interactions or menin-MLL1/MLL2 interactions contribute to the antileukemia effect of menin inhibition. We show that Men1 deletion in MLL-AF9-transformed leukemia cells produces distinct cellular and molecular consequences compared with Mll1;Mll2 co-deletion and that compounds disrupting menin-MLL N-terminal interactions largely phenocopy menin loss. Moreover, we show that Mll1;Mll2-deficient leukemia cells exhibit enhanced sensitivity to menin interaction inhibitors, which is consistent with each regulating complementary genetic pathways. These data illustrate the heightened dependency of MLL-FPs on menin compared with wild-type MLL1/MLL2 for regulation of downstream target genes and argue that the predominant action of menin inhibitory compounds is through direct inhibition of MLL-FPs without significant contribution from MLL1/MLL2 inhibition.

Kmt2b conveys monovalent and bivalent H3K4me3 in mouse spermatogonial stem cells at germline and embryonic promoters.

The mammalian male germline is sustained by a pool of spermatogonial stem cells (SSCs) that can transmit both genetic and epigenetic information to offspring. However, the mechanisms underlying epigenetic transmission remain unclear. The histone methyltransferase Kmt2b is highly expressed in SSCs and is required for the SSC-to-progenitor transition. At the stem-cell stage, Kmt2b catalyzes H3K4me3 at bivalent H3K27me3-marked promoters as well as at promoters of a new class of genes lacking H3K27me3, which we call monovalent. Monovalent genes are mainly activated in late spermatogenesis, whereas most bivalent genes are mainly not expressed until embryonic development. These data suggest that SSCs are epigenetically primed by Kmt2b in two distinguishable ways for the upregulation of gene expression both during the spermatogenic program and through the male germline into the embryo. Because Kmt2b is also the major H3K4 methyltransferase for bivalent promoters in embryonic stem cells, we also propose that Kmt2b has the capacity to prime stem cells epigenetically.

A single reporter mouse line for Vika, Flp, Dre, and Cre-recombination.

Site-specific recombinases (SSR) are utilized as important genome engineering tools to precisely modify the genome of mice and other model organisms. Reporter mice that mark cells that at any given time had expressed the enzyme are frequently used for lineage tracing and to characterize newly generated mice expressing a recombinase from a chosen promoter. With increasing sophistication of genome alteration strategies, the demand for novel SSR systems that efficiently and specifically recombine their targets is rising and several SSR-systems are now used in combination to address complex biological questions in vivo. Generation of reporter mice for each one of these recombinases is cumbersome and increases the number of mouse lines that need to be maintained in animal facilities. Here we present a multi-reporter mouse line for loci-of-recombination (X) (MuX) that streamlines the characterization of mice expressing prominent recombinases. MuX mice constitutively express nuclear green fluorescent protein after recombination by either Cre, Flp, Dre or Vika recombinase, rationalizing the number of animal lines that need to be maintained. We also pioneer the use of the Vika/vox system in mice, illustrating its high efficacy and specificity, thereby facilitating future designs of sophisticated recombinase-based in vivo genome engineering strategies.

Human axial progenitors generate trunk neural crest cells in vitro.

The neural crest (NC) is a multipotent embryonic cell population that generates distinct cell types in an axial position-dependent manner. The production of NC cells from human pluripotent stem cells (hPSCs) is a valuable approach to study human NC biology. However, the origin of human trunk NC remains undefined and current in vitro differentiation strategies induce only a modest yield of trunk NC cells. Here we show that hPSC-derived axial progenitors, the posteriorly-located drivers of embryonic axis elongation, give rise to trunk NC cells and their derivatives. Moreover, we define the molecular signatures associated with the emergence of human NC cells of distinct axial identities in vitro. Collectively, our findings indicate that there are two routes toward a human post-cranial NC state: the birth of cardiac and vagal NC is facilitated by retinoic acid-induced posteriorisation of an anterior precursor whereas trunk NC arises within a pool of posterior axial progenitors.

The H3K4 methyltransferase Setd1b is essential for hematopoietic stem and progenitor cell homeostasis in mice.

Hematopoietic stem cells require MLL1, which is one of six Set1/Trithorax-type histone 3 lysine 4 (H3K4) methyltransferases in mammals and clinically the most important leukemia gene. Here, we add to emerging evidence that all six H3K4 methyltransferases play essential roles in the hematopoietic system by showing that conditional mutagenesis of Setd1b in adult mice provoked aberrant homeostasis of hematopoietic stem and progenitor cells (HSPCs). Using both ubiquitous and hematopoietic-specific deletion strategies, the loss of Setd1b resulted in peripheral thrombo- and lymphocytopenia, multilineage dysplasia, myeloid-biased extramedullary hematopoiesis in the spleen, and lethality. By transplantation experiments and expression profiling, we determined that Setd1b is autonomously required in the hematopoietic lineages where it regulates key lineage specification components, including Cebpa, Gata1, and Klf1. Altogether, these data imply that the Set1/Trithorax-type epigenetic machinery sustains different aspects of hematopoiesis and constitutes a second framework additional to the transcription factor hierarchy of hematopoietic homeostasis.

Clonal Analysis Delineates Transcriptional Programs of Osteogenic and Adipogenic Lineages of Adult Mouse Skeletal Progenitors.

Bone, cartilage, and marrow adipocytes are generated by skeletal progenitors, but the relationships between lineages and mechanisms controlling their differentiation are poorly understood. We established mouse clonal skeletal progenitors with distinct differentiation properties and analyzed their transcriptome. Unipotent osteogenic and adipogenic cells expressed specific transcriptional programs, whereas bipotent clones combined expression of those genes and did not show a unique signature. We tested potential regulators of lineage commitment and found that in the presence of interferon-γ (IFNγ) adipogenic clones can be induced to osteogenesis and that their adipogenic capacity is inhibited. Analysis of IFNγ-regulated genes showed that lineage signatures and fate commitment of skeletal progenitors were controlled by EGR1 and EGR2. Knockdown experiments revealed that EGR1 is a positive regulator of the adipogenic transcriptional program and differentiation capacity, whereas EGR2 inhibits the osteogenic program and potency. Therefore, our work revealed transcriptional signatures of osteogenic and adipogenic lineages and mechanism triggering cell fate.