Histone H3 trimethylation at lysine 36 guides m6A RNA modification co-transcriptionally.

DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2-4 and has crucial roles in various normal and pathological processes5-12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.

Tilt in Place Microscopy: a Simple, Low-Cost Solution to Image Neural Responses to Body Rotations.

Animals use information about gravity and other destabilizing forces to balance and navigate through their environment. Measuring how brains respond to these forces requires considerable technical knowledge and/or financial resources. We present a simple alternative-Tilt In Place Microscopy (TIPM), a low-cost and noninvasive way to measure neural activity following rapid changes in body orientation. Here, we used TIPM to study vestibulospinal neurons in larval zebrafish during and immediately after roll tilts. Vestibulospinal neurons responded with reliable increases in activity that varied as a function of ipsilateral tilt amplitude. TIPM differentiated tonic (i.e., sustained tilt) from phasic responses, revealing coarse topography of stimulus sensitivity in the lateral vestibular nucleus. Neuronal variability across repeated sessions was minor relative to trial-to-trial variability, allowing us to use TIPM for longitudinal studies of the same neurons across two developmental time points. There, we observed global increases in response strength and systematic changes in the neural representation of stimulus direction. Our data extend classical characterization of the body tilt representation by vestibulospinal neurons and establish the utility of TIPM to study the neural basis of balance, especially in developing animals.SIGNIFICANCE STATEMENT Vestibular sensation influences everything from navigation to interoception. Here, we detail a straightforward, validated, and nearly universal approach to image how the nervous system senses and responds to body tilts. We use our new method to replicate and expand on past findings of tilt sensing by a conserved population of spinal-projecting vestibular neurons. The simplicity and broad compatibility of our approach will democratize the study of the response of the brain to destabilization, particularly across development.

The CHK2 kinase is recurrently mutated and functionally impaired in the germline of pediatric cancer patients.

Predisposing CHEK2 germline variants are associated with various adult-type malignancies, whereas their impact on cancer susceptibility in childhood cancer is unclear. To understand the frequency of germline variants in the CHEK2 gene and their impact on pediatric malignancies, we used whole-exome sequencing to search for CHEK2 variants in the germlines of 418 children diagnosed with cancer in our clinics. Moreover, we performed functional analysis of the pathogenic CHEK2 variants to analyze the effect of the alterations on CHK2 protein function. We detected a CHEK2 germline variant in 32/418 (7.7%) pediatric cancer patients and 46.8% of them had leukemia. Functional analysis of the pathogenic variants revealed that 5 pathogenic variants impaired CHK2 protein function. 6/32 patients carried one of these clearly damaging CHEK2 variants and two of them harbored a matching family history of cancer. In conclusion, we detected germline CHEK2 variants in 7.7% of all pediatric cancer patients, of which a minority but still relevant fraction of approximately 20% had a profound impact on protein expression or its phosphorylation after irradiation-induced DNA damage. Accordingly, we conclude that CHEK2 variants increase the risk for not only adult-onset but also pediatric cancer.

A clinical screening tool to detect genetic cancer predisposition in pediatric oncology shows high sensitivity but can miss a substantial percentage of affected children.

PURPOSE: Clinical checklists are the standard of care to determine whether a child with cancer shows indications for genetic testing. Nevertheless, the efficacy of these tests to reliably detect genetic cancer predisposition in children with cancer is still insufficiently investigated. METHODS: We assessed the validity of clinically recognizable signs to identify cancer predisposition by correlating a state-of-the-art clinical checklist to the corresponding exome sequencing analysis in an unselected single-center cohort of 139 child-parent data sets. RESULTS: In total, one-third of patients had a clinical indication for genetic testing according to current recommendations, and 10.1% (14 of 139) of children harbored a cancer predisposition. Of these, 71.4% (10 of 14) were identified through the clinical checklist. In addition, >2 clinical findings in the checklist increased the likelihood to identifying genetic predisposition from 12.5% to 50%. Furthermore, our data revealed a high rate of genetic predisposition (40%, 4 of 10) in myelodysplastic syndrome cases, while no (likely) pathogenic variants were identified in the sarcoma and lymphoma group. CONCLUSION: In summary, our data show high checklist sensitivity, particularly in identifying childhood cancer predisposition syndromes. Nevertheless, the checklist used here also missed 29% of children with a cancer predisposition, highlighting the drawbacks of sole clinical evaluation and underlining the need for routine germline sequencing in pediatric oncology.

An intact gut microbiome protects genetically predisposed mice against leukemia.

The majority of childhood leukemias are precursor B-cell acute lymphoblastic leukemias (pB-ALLs) caused by a combination of prenatal genetic predispositions and oncogenic events occurring after birth. Although genetic predispositions are frequent in children (>1% to 5%), fewer than 1% of genetically predisposed carriers will develop pB-ALL. Although infectious stimuli are believed to play a major role in leukemogenesis, the critical determinants are not well defined. Here, by using murine models of pB-ALL, we show that microbiome disturbances incurred by antibiotic treatment early in life were sufficient to induce leukemia in genetically predisposed mice, even in the absence of infectious stimuli and independent of T cells. By using V4 and full-length 16S ribosomal RNA sequencing of a series of fecal samples, we found that genetic predisposition to pB-ALL (Pax5 heterozygosity or ETV6-RUNX1 fusion) shaped a distinct gut microbiome. Machine learning accurately (96.8%) predicted genetic predisposition using 40 of 3983 amplicon sequence variants as proxies for bacterial species. Transplantation of either wild-type (WT) or Pax5+/- hematopoietic bone marrow cells into WT recipient mice revealed that the microbiome is shaped and determined in a donor genotype-specific manner. Gas chromatography-mass spectrometry (GC-MS) analyses of sera from WT and Pax5+/- mice demonstrated the presence of a genotype-specific distinct metabolomic profile. Taken together, our data indicate that it is a lack of commensal microbiota rather than the presence of specific bacteria that promotes leukemia in genetically predisposed mice. Future large-scale longitudinal studies are required to determine whether targeted microbiome modification in children predisposed to pB-ALL could become a successful prevention strategy.

Recurrent Germline Variant in RAD21 Predisposes Children to Lymphoblastic Leukemia or Lymphoma.

Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.

Germline POT1 Deregulation Can Predispose to Myeloid Malignancies in Childhood.

While the shelterin complex guards and coordinates the mechanism of telomere regulation, deregulation of this process is tightly linked to malignant transformation and cancer. Here, we present the novel finding of a germline stop-gain variant (p.Q199*) in the shelterin complex gene POT1, which was identified in a child with acute myeloid leukemia. We show that the cells overexpressing the mutated POT1 display increased DNA damage and chromosomal instabilities compared to the wildtype counterpart. Protein and mRNA expression analyses in the primary patient cells further confirm that, physiologically, the variant leads to a nonfunctional POT1 allele in the patient. Subsequent telomere length measurements in the primary cells carrying heterozygous POT1 p.Q199* as well as POT1 knockdown AML cells revealed telomeric elongation as the main functional effect. These results show a connection between POT1 p.Q199* and telomeric dysregulation and highlight POT1 germline deficiency as a predisposition to myeloid malignancies in childhood.

The homeobox transcription factor HB9 induces senescence and blocks differentiation in hematopoietic stem and progenitor cells.

The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiological expression pattern, aberrant HB9 expression has been observed in several neoplasias. Especially in infant translocation t(7;12) acute myeloid leukemia, aberrant HB9 expression is the only known molecular hallmark and is assumed to be a key factor in leukemic transformation. However, so far, only poor functional data exist addressing the oncogenic potential of HB9 or its influence on hematopoiesis. We investigated the influence of HB9 on cell proliferation and cell cycle in vitro, as well as on hematopoietic stem cell differentiation in vivo using murine and human model systems. In vitro, HB9 expression led to premature senescence in human HT1080 and murine NIH3T3 cells, providing for the first time evidence for an oncogenic potential of HB9. Onset of senescence was characterized by induction of the p53-p21 tumor suppressor network, resulting in growth arrest, accompanied by morphological transformation and expression of senescence-associated ß-galactosidase. In vivo, HB9-transduced primary murine hematopoietic stem and progenitor cells underwent a profound differentiation arrest and accumulated at the megakaryocyte/erythrocyte progenitor stage. In line, gene expression analyses revealed de novo expression of erythropoiesis-related genes in human CD34+hematopoietic stem and progenitor cells upon HB9 expression. In summary, the novel findings of HB9-dependent premature senescence and myeloid-biased perturbed hematopoietic differentiation, for the first time shed light on the oncogenic properties of HB9 in translocation t(7;12) acute myeloid leukemia.

STAT3 gain-of-function mutations associated with autoimmune lymphoproliferative syndrome like disease deregulate lymphocyte apoptosis and can be targeted by BH3 mimetic compounds.

Autoimmune lymphoproliferative syndrome (ALPS) is typically caused by mutations in genes of the extrinsic FAS mediated apoptotic pathway, but for about 30% of ALPS-like patients the genetic diagnosis is lacking. We analyzed 30 children with ALPS-like disease of unknown cause and identified two dominant gain-of-function mutations of the Signal Transducer And Activator Of Transcription 3 (STAT3, p.R278H, p.M394T) leading to increased transcriptional activity. Hyperactivity of STAT3, a known repressor of FAS, was associated with decreased FAS-mediated apoptosis, mimicking ALPS caused by FAS mutations. Expression of BCL2 family proteins, further targets of STAT3 and regulators of the intrinsic apoptotic pathway, was disturbed. Cells with hyperactive STAT3 were consequently more resistant to intrinsic apoptotic stimuli and STAT3 inhibition alleviated this effect. Importantly, STAT3-mutant cells were more sensitive to death induced by the BCL2-inhibitor ABT-737 indicating a dependence on anti-apoptotic BCL2 proteins and potential novel therapeutic options.

Tumoral stem cell reprogramming as a driver of cancer: Theory, biological models, implications in cancer therapy.

Cancer is a clonal malignant disease originated in a single cell and characterized by the accumulation of partially differentiated cells that are phenotypically reminiscent of normal stages of differentiation. According to current models, therapeutic strategies that block oncogene activity are likely to selectively target tumor cells. However, recent evidences have revealed that cancer stem cells could arise through a tumor stem cell reprogramming mechanism, suggesting that genetic lesions that initiate the cancer process might be dispensable for tumor progression and maintenance. This review addresses the impact of these results toward a better understanding of cancer development and proposes new approaches to treat cancer in the future.

Sick sinus syndrome in HCN1-deficient mice.

BACKGROUND: Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and requires surgical implantation of electric pacemaker devices. Frequently, SND occurs in heart failure and hypertension, conditions that lead to electric instability of the heart. Although the pathologies of acquired SND have been studied extensively, little is known about the molecular and cellular mechanisms that cause congenital SND. METHODS AND RESULTS: Here, we show that the HCN1 protein is highly expressed in the sinoatrial node and is colocalized with HCN4, the main sinoatrial pacemaker channel isoform. To characterize the cardiac phenotype of HCN1-deficient mice, a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparation, telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology was performed. On the basis of these experiments we demonstrate that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output. CONCLUSIONS: We propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.

Cerebellar Purkinje Cells Control Posture in Larval Zebrafish (Danio rerio).

Cerebellar dysfunction leads to postural instability. Recent work in freely moving rodents has transformed investigations of cerebellar contributions to posture. However, the combined complexity of terrestrial locomotion and the rodent cerebellum motivate development of new approaches to perturb cerebellar function in simpler vertebrates. Here, we used a powerful chemogenetic tool (TRPV1/capsaicin) to define the role of Purkinje cells - the output neurons of the cerebellar cortex - as larval zebrafish swam freely in depth. We achieved both bidirectional control (activation and ablation) of Purkinje cells while performing quantitative high-throughput assessment of posture and locomotion. Activation disrupted postural control in the pitch (nose-up/nose-down) axis. Similarly, ablations disrupted pitch-axis posture and fin-body coordination responsible for climbs. Postural disruption was more widespread in older larvae, offering a window into emergent roles for the developing cerebellum in the control of posture. Finally, we found that activity in Purkinje cells could individually and collectively encode tilt direction, a key feature of postural control neurons. Our findings delineate an expected role for the cerebellum in postural control and vestibular sensation in larval zebrafish, establishing the validity of TRPV1/capsaicin-mediated perturbations in a simple, genetically-tractable vertebrate. Moreover, by comparing the contributions of Purkinje cell ablations to posture in time, we uncover signatures of emerging cerebellar control of posture across early development. This work takes a major step towards understanding an ancestral role of the cerebellum in regulating postural maturation.

A brainstem circuit for gravity-guided vertical navigation.

The sensation of gravity anchors our perception of the environment and is crucial for navigation. However, the neural circuits that transform gravity into commands for navigation are undefined. We first determined that larval zebrafish (Danio rerio) navigate vertically by maintaining a consistent heading across a series of upward climb or downward dive bouts. Gravity-blind mutant fish swim with more variable heading and excessive veering, leading to inefficient vertical navigation. After targeted photoablation of ascending vestibular neurons and spinal projecting midbrain neurons, but not vestibulospinal neurons, vertical navigation was impaired. These data define a sensorimotor circuit that uses evolutionarily-conserved brainstem architecture to transform gravitational signals into persistent heading for vertical navigation. The work lays a foundation to understand how vestibular inputs allow animals to move efficiently through their environment.

Astrocyte growth is driven by the Tre1/S1pr1 phospholipid-binding G protein-coupled receptor.

Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways regulating astrocyte morphogenesis remain poorly defined. Here, we show the Drosophila lipid-binding G protein-coupled receptor (GPCR) Tre1 is required for astrocytes to establish their intricate morphology in vivo. The lipid phosphate phosphatases Wunen/Wunen2 also regulate astrocyte morphology and, via Tre1, mediate astrocyte-astrocyte competition for growth-promoting lipids. Loss of s1pr1, the functional analog of Tre1 in zebrafish, disrupts astrocyte process elaboration, and live imaging and pharmacology demonstrate that S1pr1 balances proper astrocyte process extension/retraction dynamics during growth. Loss of Tre1 in flies or S1pr1 in zebrafish results in defects in simple assays of motor behavior. Tre1 and S1pr1 are thus potent evolutionarily conserved regulators of the elaboration of astrocyte morphological complexity and, ultimately, astrocyte control of behavior.

The Eµ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia.

The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eµ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eµ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eµ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

Scalable Apparatus to Measure Posture and Locomotion (SAMPL): a high-throughput solution to study unconstrained vertical behavior in small animals.

Balance and movement are impaired in a wide variety of neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics, but without the throughput and scalability necessary to screen candidate genes / potential therapeutics. We present a powerful solution: a Scalable Apparatus to Measure Posture and Locomotion (SAMPL). SAMPL includes extensible imaging hardware and low-cost open-source acquisition software with real-time processing. We first demonstrate that SAMPL's hardware and acquisition software can acquire data from from D. melanogaster, C. elegans, and D. rerio as they move vertically. Next, we leverage SAMPL's throughput to rapidly (two weeks) gather a new zebrafish dataset. We use SAMPL's analysis and visualization tools to replicate and extend our current understanding of how zebrafish balance as they navigate through a vertical environment. Next, we discover (1) that key kinematic parameters vary systematically with genetic background, and (2) that such background variation is small relative to the changes that accompany early development. Finally, we simulate SAMPL's ability to resolve differences in posture or vertical navigation as a function of affect size and data gathered -- key data for screens. Taken together, our apparatus, data, and analysis provide a powerful solution for labs using small animals to investigate balance and locomotor disorders at scale. More broadly, SAMPL is both an adaptable resource for labs looking process videographic measures of behavior in real-time, and an exemplar of how to scale hardware to enable the throughput necessary for screening.

SAMPL is a high-throughput solution to study unconstrained vertical behavior in small animals.

Balance and movement are impaired in many neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics but without the throughput and scalability necessary to screen candidate genes/potential therapeutics. Here, we present a scalable apparatus to measure posture and locomotion (SAMPL). SAMPL includes extensible hardware and open-source software with real-time processing and can acquire data from D. melanogaster, C. elegans, and D. rerio as they move vertically. Using SAMPL, we define how zebrafish balance as they navigate vertically and discover small but systematic variations among kinematic parameters between genetic backgrounds. We demonstrate SAMPL's ability to resolve differences in posture and navigation as a function of effect size and data gathered, providing key data for screens. SAMPL is therefore both a tool to model balance and locomotor disorders and an exemplar of how to scale apparatus to support screens.

The Larval Zebrafish Vestibular System Is a Promising Model to Understand the Role of Myelin in Neural Circuits.

Myelin is classically known for its role in facilitating nerve conduction. However, recent work casts myelin as a key player in both proper neuronal circuit development and function. With this expanding role comes a demand for new approaches to characterize and perturb myelin in the context of tractable neural circuits as they mature. Here we argue that the simplicity, strong conservation, and clinical relevance of the vestibular system offer a way forward. Further, the tractability of the larval zebrafish affords a uniquely powerful means to test open hypotheses of myelin's role in normal development and disordered vestibular circuits. We end by identifying key open questions in myelin neurobiology that the zebrafish vestibular system is particularly well-suited to address.

Clusters of neuronal neurofascin prefigure the position of a subset of nodes of Ranvier along individual central nervous system axons in vivo.

The spacing of nodes of Ranvier crucially affects conduction properties along myelinated axons. It is assumed that node position is primarily driven by growing myelin sheaths. Here, we reveal an additional mechanism of node positioning that is driven by the axon. Through longitudinal live imaging of node formation dynamics in the zebrafish central nervous system, we show that stable clusters of the cell adhesion molecule neurofascin a can accumulate at specific sites along axons prior to myelination. While some of these clusters are pushed into future node position by extending myelin sheaths, others are not and thus prefigure the position of where a mature node forms. Animals that lack full-length neurofascin a show increased internodal distances and less regular nodal spacing along single axons. Together, our data reveal the existence of an axonal mechanism to position nodes of Ranvier that does not depend on regulation by myelin sheath growth.

A rare PALB2 germline variant causing G2/M cell cycle arrest is associated with isolated myelosarcoma in infancy.

BACKGROUND: Isolated myelosarcoma of infancy is a rare presentation of acute myelogenous leukaemia (AML). Because of its rarity and early onset in infancy underlying genetic predisposition is potentially relevant in disease initiation. METHODS AND RESULTS: We report an oncologic emergency in an infant with thoracic and intraspinal aleukaemic myeloid sarcoma causing acute myelon compression and lower leg palsy. Whole-exome sequencing of the patient's germline DNA identified a rare PALB2 (OMIM 610355) variant (p.A1079S), which is located in a domain critical for the gene's proper function within the homology-directed repair pathway. In line with potential DNA damage repair defects mediated by the PALB2 deregulation, the patient's fibroblasts showed increased sensitivity towards radiation and DNA intercalating agents. CONCLUSION: Therefore, we suggest PALB2 p.A1079S as a pathogenic variant potentially contributing to the here observed patient phenotype.