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MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes , Proteínas Proto-Oncogênicas c-myc , Animais , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Transcrição Gênica , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Feminino , MasculinoRESUMO
Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5' truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation.
Assuntos
Adenilil Ciclases , Tecido Adiposo Marrom , Temperatura Baixa , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genética , Tecido Adiposo Marrom/metabolismo , Animais , Camundongos , Masculino , Humanos , Termogênese/genética , Metabolismo Energético , AMP Cíclico/metabolismo , Camundongos KnockoutRESUMO
Metastasis arises from disseminated tumour cells (DTCs) that are characterized by intrinsic phenotypic plasticity and the capability of seeding to secondary organs. DTCs can remain latent for years before giving rise to symptomatic overt metastasis. In this context, DTCs fluctuate between a quiescent and proliferative state in response to systemic and microenvironmental signals including immune-mediated surveillance. Despite its relevance, how intrinsic mechanisms sustain DTCs plasticity has not been addressed. By interrogating the epigenetic state of metastatic cells, we find that tumour progression is coupled with the activation of oncogenic enhancers that are organized in variable interconnected chromatin domains. This spatial chromatin context leads to the activation of a robust transcriptional response upon repeated exposure to retinoic acid (RA). We show that this adaptive mechanism sustains the quiescence of DTCs through the activation of the master regulator SOX9. Finally, we determine that RA-stimulated transcriptional memory increases the fitness of metastatic cells by supporting the escape of quiescent DTCs from NK-mediated immune surveillance. Overall, these findings highlight the contribution of oncogenic enhancers in establishing transcriptional memories as an adaptive mechanism to reinforce cancer dormancy and immune escape, thus amenable for therapeutic intervention.
Assuntos
Vigilância Imunológica , Sequências Reguladoras de Ácido Nucleico , Divisão Celular , Linhagem Celular Tumoral , CromatinaRESUMO
Myeloperoxidase (MPO) is an enzyme that functions in host defense. MPO is released into the vascular lumen by neutrophils during inflammation and may adhere and subsequently penetrate endothelial cells (ECs) coating vascular walls. We show that MPO enters the nucleus of ECs and binds chromatin independently of its enzymatic activity. MPO drives chromatin decondensation at its binding sites and enhances condensation at neighboring regions. It binds loci relevant for endothelial-to-mesenchymal transition (EndMT) and affects the migratory potential of ECs. Finally, MPO interacts with the RNA-binding factor ILF3 thereby affecting its relative abundance between cytoplasm and nucleus. This interaction leads to change in stability of ILF3-bound transcripts. MPO-knockout mice exhibit reduced number of ECs at scar sites following myocardial infarction, indicating reduced neovascularization. In summary, we describe a non-enzymatic role for MPO in coordinating EndMT and controlling the fate of endothelial cells through direct chromatin binding and association with co-factors.
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The interactions between genetic and environmental risk factors contribute to the aetiology of complex human diseases. Genome-wide association studies (GWAS) have revealed that most of the genetic variants associated with complex diseases are located in the non-coding part of the genome, preferentially within enhancers. Enhancers are distal cis-regulatory elements composed of clusters of transcription factors binding sites that positively regulate the expression of their target genes. The generation of genome-wide maps for histone marks (e.g., H3K27ac), chromatin accessibility and transcription factor and coactivator (e.g., p300) binding profiles have enabled the identification of enhancers across many human cell types and tissues. Nonetheless, the functional and pathological consequences of the majority of disease-associated genetic variants located within enhancers seem to be rather minor under normal conditions, thus questioning their medical relevance. Here we propose that, due to the prevalence of enhancer redundancy, the pathological effects of many disease-associated non-coding genetic variants might be preferentially (or even only) manifested under environmental stress.
Assuntos
Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla , Humanos , Elementos Facilitadores Genéticos/genética , Cromatina/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão GênicaRESUMO
Understanding the pathological impact of non-coding genetic variation is a major challenge in medical genetics. Accumulating evidences indicate that a significant fraction of genetic alterations, including structural variants (SVs), can cause human disease by altering the function of non-coding regulatory elements, such as enhancers. In the case of SVs, described pathomechanisms include changes in enhancer dosage and long-range enhancer-gene communication. However, there is still a clear gap between the need to predict and interpret the medical impact of non-coding variants, and the existence of tools to properly perform these tasks. To reduce this gap, we have developed POSTRE (Prediction Of STRuctural variant Effects), a computational tool to predict the pathogenicity of SVs implicated in a broad range of human congenital disorders. By considering disease-relevant cellular contexts, POSTRE identifies SVs with either coding or long-range pathological consequences with high specificity and sensitivity. Furthermore, POSTRE not only identifies pathogenic SVs, but also predicts the disease-causative genes and the underlying pathological mechanism (e.g, gene deletion, enhancer disconnection, enhancer adoption, etc.). POSTRE is available at https://github.com/vicsanga/Postre.
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Biologia Computacional , Doenças Genéticas Inatas , Mutação , Software , Humanos , Biologia Computacional/métodos , Doenças Genéticas Inatas/genéticaRESUMO
The potential of pluripotent cells to respond to developmental cues and trigger cell differentiation is enhanced during the G1 phase of the cell cycle, but the molecular mechanisms involved are poorly understood. Variations in polycomb activity during interphase progression have been hypothesized to regulate the cell-cycle-phase-dependent transcriptional activation of differentiation genes during lineage transition in pluripotent cells. Here, we show that recruitment of Polycomb Repressive Complex 1 (PRC1) and associated molecular functions, ubiquitination of H2AK119 and three-dimensional chromatin interactions, are enhanced during S and G2 phases compared to the G1 phase. In agreement with the accumulation of PRC1 at target promoters upon G1 phase exit, cells in S and G2 phases show firmer transcriptional repression of developmental regulator genes that is drastically perturbed upon genetic ablation of the PRC1 catalytic subunit RING1B. Importantly, depletion of RING1B during retinoic acid stimulation interferes with the preference of mouse embryonic stem cells (mESCs) to induce the transcriptional activation of differentiation genes in G1 phase. We propose that incremental enrolment of polycomb repressive activity during interphase progression reduces the tendency of cells to respond to developmental cues during S and G2 phases, facilitating activation of cell differentiation in the G1 phase of the pluripotent cell cycle.
Assuntos
Histonas , Células-Tronco Pluripotentes , Complexo Repressor Polycomb 1 , Animais , Camundongos , Diferenciação Celular/genética , Cromatina/genética , Histonas/metabolismo , Interfase , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Células-Tronco Pluripotentes/citologiaRESUMO
BACKGROUND: Nonsyndromic cleft lip with/without cleft palate (nsCL/P) is a congenital malformation of multifactorial etiology. Research has identified >40 genome-wide significant risk loci, which explain less than 40% of nsCL/P heritability. Studies show that some of the hidden heritability is explained by rare penetrant variants. METHODS: To identify new candidate genes, we searched for highly penetrant de novo variants (DNVs) in 50 nsCL/P patient/parent-trios with a low polygenic risk for the phenotype (discovery). We prioritized DNV-carrying candidate genes from the discovery for resequencing in independent cohorts of 1010 nsCL/P patients of diverse ethnicities and 1574 population-matched controls (replication). Segregation analyses and rare variant association in the replication cohort, in combination with additional data (genome-wide association data, expression, protein-protein-interactions), were used for final prioritization. CONCLUSION: In the discovery step, 60 DNVs were identified in 60 genes, including a variant in the established nsCL/P risk gene CDH1. Re-sequencing of 32 prioritized genes led to the identification of 373 rare, likely pathogenic variants. Finally, MDN1 and PAXIP1 were prioritized as top candidates. Our findings demonstrate that DNV detection, including polygenic risk score analysis, is a powerful tool for identifying nsCL/P candidate genes, which can also be applied to other multifactorial congenital malformations.
Assuntos
Fenda Labial , Fissura Palatina , Humanos , Fissura Palatina/genética , Fenda Labial/genética , Estudo de Associação Genômica Ampla , Proteínas de Ligação a DNA/genética , Fatores de RiscoRESUMO
Enhancers play a central role in the spatiotemporal control of gene expression and tend to work in a cell-type-specific manner. In addition, they are suggested to be major contributors to phenotypic variation, evolution and disease. There is growing evidence that enhancer dysfunction due to genetic, structural or epigenetic mechanisms contributes to a broad range of human diseases referred to as enhanceropathies. Such mechanisms often underlie the susceptibility to common diseases, but can also play a direct causal role in cancer or Mendelian diseases. Despite the recent gain of insights into enhancer biology and function, we still have a limited ability to predict how enhancer dysfunction impacts gene expression. Here we discuss the major challenges that need to be overcome when studying the role of enhancers in disease etiology and highlight opportunities and directions for future studies, aiming to disentangle the molecular basis of enhanceropathies.
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Elementos Facilitadores Genéticos , Epigênese Genética , Humanos , Elementos Facilitadores Genéticos/genéticaRESUMO
Significant evidence points to Strip2 being a key regulator of the differentiation processes of pluripotent embryonic stem cells. However, Strip2 mediated epigenetic regulation of embryonic differentiation and development is quite unknown. Here, we identified several interaction partners of Strip2, importantly the co-repressor molecular protein complex nucleosome remodeling deacetylase/Tripartite motif-containing 28/Histone deacetylases/Histone-lysine N-methyltransferase SETDB1 (NuRD/TRIM28/HDACs/SETDB1) histone methyltransferase, which is primarily involved in regulation of the pluripotency of embryonic stem cells and its differentiation. The complex is normally activated by binding of Krueppel-associated box zinc-finger proteins (KRAB-ZFPs) to specific DNA motifs, causing methylation of H3 to Lysin-9 residues (H3K9). Our data showed that Strip2 binds to a DNA motif (20 base pairs), like the KRAB-ZFPs. We establish that Strip2 is an epigenetic regulator of pluripotency and differentiation by modulating DNA KRAB-ZFPs as well as the NuRD/TRIM28/HDACs/SETDB1 histone methyltransferase complex.
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cis-regulatory elements (CREs) orchestrate the spatiotemporal control of gene expression. The regulatory activity of CREs is typically assessed by reporter assays, in which CREs are studied outside their endogenous context. To circumvent this problem, we developed a CRISPR-Cas9 knockin approach to study CREs in a scar-free genomic context. Here, we describe the design, transfection, and screening protocol to insert CREs in mouse embryonic stem cells. Our strategy can provide important insights into the sufficiency of CREs for gene expression control. For complete details on the use and execution of this protocol, please refer to Pachano et al. (2021).
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Células-Tronco Embrionárias Murinas , Sequências Reguladoras de Ácido Nucleico , Animais , Genômica , Camundongos , TransfecçãoRESUMO
Enhancers control the establishment of spatiotemporal gene expression patterns throughout development. Over the past decade, the development of new technologies has improved our capacity to link enhancers with their target genes based on their colocalization within the same topological domains. However, the mechanisms that regulate how enhancers specifically activate some genes but not others within a given domain remain unclear. In this Review, we discuss recent insights into the factors controlling enhancer specificity, including the genetic composition of enhancers and promoters, the linear and 3D distance between enhancers and their target genes, and cell-type specific chromatin landscapes. We also discuss how elucidating the molecular principles of enhancer specificity might help us to better understand and predict the pathological consequences of human genetic, epigenetic and structural variants.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Epigenômica , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
Enhancers are key regulatory elements that govern gene expression programs in response to developmental signals. However, how multiple enhancers arrange in the 3D-space to control the activation of a specific promoter remains unclear. To address this question, we exploited our previously characterized TGFß-response model, the neural stem cells, focusing on a ~374 kb locus where enhancers abound. Our 4C-seq experiments reveal that the TGFß pathway drives the assembly of an enhancer-cluster and precise gene activation. We discover that the TGFß pathway coactivator JMJD3 is essential to maintain these structures. Using live-cell imaging techniques, we demonstrate that an intrinsically disordered region contained in JMJD3 is involved in the formation of phase-separated biomolecular condensates, which are found in the enhancer-cluster. Overall, in this work we uncover novel functions for the coactivator JMJD3, and we shed light on the relationships between the 3D-conformation of the chromatin and the TGFß-driven response during mammalian neurogenesis.
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Células-Tronco Neurais , Fator de Crescimento Transformador beta , Animais , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Expressão Gênica , Genoma , Mamíferos/genética , Células-Tronco Neurais/metabolismo , Ativação Transcricional/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
Assuntos
Elementos Facilitadores Genéticos , Células Germinativas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Diferenciação Celular , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica , Células Germinativas/citologia , Camadas Germinativas/citologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Mutação/genética , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , RNA-Seq , Análise de Célula Única , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Tissue turnover requires activation and lineage commitment of tissue-resident stem cells (SCs). These processes are impacted by ageing, but the mechanisms remain unclear. Here, we addressed the mechanisms of ageing in murine hair follicle SCs (HFSCs) and observed a widespread reduction in chromatin accessibility in aged HFSCs, particularly at key self-renewal and differentiation genes, characterized by bivalent promoters occupied by active and repressive chromatin marks. Consistent with this, aged HFSCs showed reduced ability to activate bivalent genes for efficient self-renewal and differentiation. These defects were niche dependent as the transplantation of aged HFSCs into young recipients or synthetic niches restored SC functions. Mechanistically, the aged HFSC niche displayed widespread alterations in extracellular matrix composition and mechanics, resulting in mechanical stress and concomitant transcriptional repression to silence promoters. As a consequence, increasing basement membrane stiffness recapitulated age-related SC changes. These data identify niche mechanics as a central regulator of chromatin state, which, when altered, leads to age-dependent SC exhaustion.
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Diferenciação Celular , Autorrenovação Celular , Senescência Celular , Montagem e Desmontagem da Cromatina , Folículo Piloso/fisiologia , Regiões Promotoras Genéticas , Nicho de Células-Tronco , Células-Tronco/fisiologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Autorrenovação Celular/genética , Células Cultivadas , Senescência Celular/genética , Matriz Extracelular/fisiologia , Inativação Gênica , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Masculino , Mecanotransdução Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Envelhecimento da Pele , Células-Tronco/metabolismo , Estresse Mecânico , Transcrição GênicaRESUMO
Poised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that the interactions between PEs and their target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo.
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Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Embrião de Galinha , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Ilhas de CpG , Células-Tronco Embrionárias/efeitos dos fármacos , Epigênese Genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camadas Germinativas/metabolismo , Homozigoto , Camundongos , Filogenia , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Fatores de Transcrição/genéticaRESUMO
CpG islands (CGIs) represent a widespread feature of vertebrate genomes, being associated with ~70% of all gene promoters. CGIs control transcription initiation by conferring nearby promoters with unique chromatin properties. In addition, there are thousands of distal or orphan CGIs (oCGIs) whose functional relevance is barely known. Here we show that oCGIs are an essential component of poised enhancers that augment their long-range regulatory activity and control the responsiveness of their target genes. Using a knock-in strategy in mouse embryonic stem cells, we introduced poised enhancers with or without oCGIs within topologically associating domains harboring genes with different types of promoters. Analysis of the resulting cell lines revealed that oCGIs act as tethering elements that promote the physical and functional communication between poised enhancers and distally located genes, particularly those with large CGI clusters in their promoters. Therefore, by acting as genetic determinants of gene-enhancer compatibility, CGIs can contribute to gene expression control under both physiological and potentially pathological conditions.
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Ilhas de CpG , Metilação de DNA , Elementos Facilitadores Genéticos , Epigênese Genética , Regulação da Expressão Gênica , Animais , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Técnicas de Introdução de Genes , Camundongos , Regiões Promotoras GenéticasRESUMO
Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.
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Autofagia/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Autofagia/genética , Cromatina , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Epigenômica , Edição de Genes , Expressão Gênica , Síndrome de Hallermann/genética , Humanos , Mutação , FenótipoRESUMO
Circumferential skin creases (CSC-KT) is a rare polymalformative syndrome characterised by intellectual disability associated with skin creases on the limbs, and very characteristic craniofacial malformations. Previously, heterozygous and homozygous mutations in MAPRE2 were found to be causal for this disease. MAPRE2 encodes for a member of evolutionary conserved microtubule plus end tracking proteins, the end binding (EB) family. Unlike MAPRE1 and MAPRE3, MAPRE2 is not required for the persistent growth and stabilization of microtubules, but plays a role in other cellular processes such as mitotic progression and regulation of cell adhesion. The mutations identified in MAPRE2 all reside within the calponin homology domain, responsible to track and interact with the plus-end tip of growing microtubules, and previous data showed that altered dosage of MAPRE2 resulted in abnormal branchial arch patterning in zebrafish. In this study, we developed patient derived induced pluripotent stem cell lines for MAPRE2, together with isogenic controls, using CRISPR/Cas9 technology, and differentiated them towards neural crest cells with cranial identity. We show that changes in MAPRE2 lead to alterations in neural crest migration in vitro but also in vivo, following xenotransplantation of neural crest progenitors into developing chicken embryos. In addition, we provide evidence that changes in focal adhesion might underlie the altered cell motility of the MAPRE2 mutant cranial neural crest cells. Our data provide evidence that MAPRE2 is involved in cellular migration of cranial neural crest and offers critical insights into the mechanism underlying the craniofacial dysmorphisms and cleft palate present in CSC-KT patients. This adds the CSC-KT disorder to the growing list of neurocristopathies.