Analysis of Dominant Megacolon: Another Hirschsprung Disease Model

显性巨结肠分析：另一种先天性巨结肠模型

• 批准号: 10691103
• 负责人: William J Pavan
• 金额: $ 116.64万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10691103
• 关键词: Address; Animal Model; Apoptosis; BRAF gene; Bad protein; Basic Science; Binding; Cells; Cephalic; Chromatin; Chromatin Remodeling Factor; Cleft Lip; Clinical; Colon; Congenital Disorders; Congenital Megacolon; Critical Pathways; DNA Sequence Alteration; Data; Defect; Development; Diagnosis; Disease; Disease Management; Disease model; Distal; Enhancers; Evolution; Exhibits; Future; Ganglia; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Variation; Genome; Genomics; Goals; Growth; Guide RNA; Hair; Health; Human; Hypopigmentation; Impairment; In Vitro; Individual; Intervention; Investigation; Lead; Link; Malignant Neoplasms; Measures; Megacolon; Melanoma Cell; Messenger RNA; Metabolism; Methods; Mus; Mutagenesis; Mutation; Neoplasm Metastasis; Neural Crest; Nevus; Oncogenes; Organism; Pathology; Pathway interactions; Patients; Peripheral Nervous System; Phenotype; Pigments; Population; Process; Production; Regulator Genes; Regulatory Pathway; Role; SMARCA4 gene; Severities; Signal Pathway; Signal Transduction; Site; Skin; Stress; TYR gene; Therapeutic; Therapeutic Intervention; Transgenes; Untranslated RNA; Variant; Waardenburg syndrome; adult stem cell; chromatin remodeling; deafness; defined contribution; disease diagnosis; genetic manipulation; genetic variant; genome editing; genomic tools; human disease; in utero; in vitro Model; in vivo; inhibitor; innovation; melanoblast; melanocyte; melanoma; metaplastic cell transformation; mouse model; mutant; novel; nuclease; pro-apoptotic protein; recruit; response; stem cell biology; stem cell differentiation; stem cell function; stem cells; success; therapeutic target; therapy development; transcription factor; tumor

The ability to generate complete genomic sequences will provide unique opportunities to explore how variation impacts evolution and human health, to discover mechanisms regulating organism development, and to manage disease diagnosis and intervention. The effective utilization of this genomic information requires a detailed understanding of the function of encoded information. While tremendous progress has been made in defining individual components of genomic sequences, we still do not understand the function of most annotated genes, we have a limited understanding of the role of non-coding sequences in gene regulation, and we have just started to define the contribution of genomic alterations to human disease. Our section directly addresses these issues by using genomic tools and genetic manipulation of model organisms to unravel genome function and to dissect gene regulatory pathways in development and disease. We integrate data from basic science with clinical information to: 1) identify pathways that regulate mammalian development, 2) understand how alterations in these pathways lead to disease states, and 3) develop paradigms for therapeutic interventions. Our group has demonstrated that mice heterozygous for mutations in the transcription factor SOX10 exhibit multiple defects in neural crest development including reduced numbers of melanocytes in the skin and absence of myenteric ganglia in the colon. We have also shown that SOX10 homozygous mutants die in utero and also exhibit extensive defects in the entire peripheral nervous system. The human congenital disorder Hirschsprung disease can be caused by SOX10 mutations, and it also exhibits rectocolic aganglionosis and can be associated with hypopigmentation. Thus SOX10 mice serve as mouse models for human disease as well as broadly inform us about neural crest development and disease. Our goal is to understand the function of SOX10 in mammalian development and use this information to understand the pathology of and develop treatments for neural crest disorders. SOX10 and adult stem cell genetics. Hair graying in mouse is attributed to the loss of melanocyte stem cell function and the progressive depletion of the follicular melanocyte population. Single-gene, hair graying mouse models have pointed to a number of critical pathways involved in melanocyte stem cell biology; however, the broad range of phenotypic variation observed in human hair graying suggests that additional genetic variants involved in this process may yet be discovered. Using a sensitized approach, we ask here whether natural genetic variation influences a predominant cellular mechanism of hair graying in mouse, melanocyte stem cell differentiation. We developed an innovative method to quantify melanocyte stem cell differentiation by measuring ectopically pigmented melanocyte stem cells in response to the melanocyte-specific transgene Tg(Dct-Sox10). We make the novel observation that the production of ectopically pigmented melanocyte stem cells varies considerably across strains. The success of sensitizing for melanocyte stem cell differentiation by Tg(Dct-Sox10) sets the stage for future investigations into the genetic basis of strain-specific contributions to melanocyte stem cell biology. BRAF. Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors. BRG1 signaling. Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function. Genome editing. Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.

产生完整的基因组序列的能力将为探索变异影响进化和人类健康，发现调节生物体发展的机制以及管理疾病诊断和干预的机制。 该基因组信息的有效利用需要详细了解编码信息的功能。 尽管在定义基因组序列的个体组成部分方面取得了巨大进展，但我们仍然不了解大多数注释基因的功能，我们对非编码序列在基因调节中的作用的理解有限，我们刚刚开始定义基因组改变对人类疾病的贡献。 我们的部分通过使用基因组工具和模型生物的遗传操纵来揭示基因组功能并剖析基因调节途径在发育和疾病中的基因调节途径，从而直接解决了这些问题。我们将基础科学的数据与临床信息集成到：1）确定调节哺乳动物发育的途径，2）了解这些途径的变化如何导致疾病状态，3）为治疗干预发展范式。 我们的小组已经证明，转录因子SOX10突变的小鼠在神经rest发育中表现出多种缺陷，包括皮肤中黑素细胞数量减少和结肠中的肌肠神经节的缺失。我们还表明，Sox10纯合突变体在子宫内死亡，并且在整个周围神经系统中也表现出广泛的缺陷。人类先天性疾病Hirschsprung疾病可能是由Sox10突变引起的，它也表现出直肠凝血症状，并且可能与不形成性相关。因此，Sox10小鼠是人类疾病的小鼠模型，并广泛地告知我们神经rest的发育和疾病。 我们的目标是了解Sox10在哺乳动物发育中的功能，并使用此信息来了解和开发神经rest疾病的治疗方法。 Sox10和成年干细胞遗传学。小鼠中的头发呈灰色归因于黑素干细胞功能的丧失以及卵泡黑色素群体的逐渐消耗。单基因，灰色小鼠模型指出了黑色素细胞干细胞生物学涉及的许多关键途径。然而，在人毛灰色中观察到的广泛的表型变异表明，在此过程中涉及的其他遗传变异可能仍被发现。使用敏化方法，我们在这里询问自然遗传变异是否影响小鼠中毛发灰色的主要细胞机制，黑素细胞干细胞分化。我们开发了一种创新的方法来量化黑素细胞干细胞分化，通过测量对黑素细胞特异性转基因TG（DCT-SOX10）的异位色素黑素细胞干细胞。我们使新的观察结果是，异位色素黑素细胞干细胞的产生在各种菌株之间差异很大。 TG对黑素细胞干细胞分化的敏化成功奠定了阶段，以使未来的研究对菌株特异性贡献对黑素细胞干细胞生物学的遗传基础。 布拉夫。 允许细胞应对癌基因应激的基因和途径代表选择性癌症治疗靶标，这些靶标在很大程度上未被发现。在这项研究中，我们确定了RHOJ信号通路，这是BRAF突变细胞的选择性治疗靶标。 BRAF突变体黑素细胞中的Rhoj缺失调节了促凋亡蛋白不良的表达以及与细胞代谢有关的基因，障碍形成，细胞转化和转移。用PAK抑制剂对新生的黑色素瘤肿瘤进行短期治疗，该抑制剂阻断RhoJ信号传导在体内停止了BRAF突变体黑色素瘤肿瘤的生长，并通过不良依赖性机制在体外诱导黑色素瘤细胞中凋亡。由于多达50％的BRAF突变人类黑色素瘤表达了高水平的RhoJ，因此这些研究提名为Rhoj-Bad信号网络，作为刚刚刚起步的BRAF突变体人肿瘤的治疗脆弱性。 BRG1信号传导。 Sox10中的突变会导致神经科学病，表现出不同程度的不形成性。使用敏化的诱变筛选，我们将Smarca4鉴定为一种修饰基因，这种基因加剧了Sox10 Haplo不足的小鼠的表型严重程度。 Sox10表达细胞中SMARCA4的条件缺失导致颅骨和腹干黑素细胞数量减少。为了定义SWI/SNF染色质重塑复合物的SMARCA4编码的BRG1亚基的需求，我们采用了黑色素细胞分化的体外模型，其中诱导黑素细胞特异性基因表达的诱导与染色质的改变相关。我们发现，BRG1是表达DCT，Tyrp1和Tyr所必需的，这些基因由Sox10和Mitf调节，以及在远端和近端调节位点进行染色质重塑。发现Sox10在分化的黑色素细胞中与BRG1物理相互作用，并与Sox10与Tyrp1远端增强子的结合在时间上与募集BRG1相一致。我们的数据表明，Sox10与MITF合作，以促进BRG1结合与黑素细胞特异性基因的远端增强子。因此，BRG1是一种Sox10共激活剂，需要建立黑素细胞谱系并促进对黑素细胞功能重要的基因表达。 基因组编辑。 CPF1已成为CAS9 RNA引导的核酸酶的替代方法。在这里，我们表明，使用CPF1的小鼠的基因靶向率可以满足甚至超过CAS9的靶向率（接近100％靶向），但需要更高浓度的mRNA和指南。我们还证明，即使其中一个指南具有最小的切割活性，与近距离靶向位点的两个指南进行共同进行。