Elucidating the mechanisms by which ectopically expressed genes and piRNAs perturb somatic cell function when histone methylation is inappropriately regulated

阐明组蛋白甲基化调节不当时异位表达基因和 piRNA 扰乱体细胞功能的机制

• 批准号: 10730632
• 负责人: Brandon Scott Carpenter
• 金额: $ 43.2万
• 依托单位: KENNESAW STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-19 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730632
• 关键词: ATAC-seq; Affect; Binding; Caenorhabditis elegans; Cell physiology; Chromatin; Complex; Craniofacial Abnormalities; Data; Defect; Deposition; Development; Developmental Delay Disorders; Disease; Ectopic Expression; Enzymes; Epigenetic Process; Event; Fluorescent in Situ Hybridization; Gene Expression; Gene Silencing; Generations; Genes; Genetic Transcription; Genomics; Histones; Homeostasis; Human; Inherited; Intellectual functioning disability; KDM1A gene; Kabuki Make-Up Syndrome; Legal patent; Lysine; Metabolic; Methylation; Methyltransferase; Modeling; Molecular; Mus; Muscle; Mutate; Mutation; Neurodevelopmental Disorder; Orthologous Gene; Pathway interactions; Patients; Phenotype; Proteins; RNA; Somatic Cell; Sotos syndrome; Specificity; Syndrome; Techniques; Testing; Tissue-Specific Gene Expression; Tissues; Transcript; Transgenes; Untranslated RNA; Vulva; cell motility; experimental study; gene repression; histone methylation; insight; knock-down; mutant; neuron development; neuronal cell body; next generation; piRNA; programs; single molecule; stem cell self renewal; transcription factor; transcriptome sequencing

PROJECT SUMMARY Kabuki-like syndrome is a neurodevelopmental disorder caused by mutations in the H3K4 demethylase, LSD1, and is characterized by a range of abnormal somatic phenotypes including intellectual disabilities, craniofacial abnormalities, and developmental delay. Despite their implication in Kabuki-like syndrome, and other neurodevelopmental syndromes, how mutations in histone modifying enzymes like LSD1 contribute to complex developmental phenotypes is unclear. To better understand how mutations in histone modifying enzymes affect somatic development we have developed a C. elegans model lacking SPR-5, the C. elegans ortholog of LSD1, in addition to the histone 3 lysine 9 (H3K9) methyltransferase MET-2. Without SPR-5 and MET-2, progeny ectopically express germline genes in somatic tissues leading to phenotypes that resemble those observed in neurodevelopmental syndromes, including muscle defects and developmental delay. Strikingly, the developmental delay and ectopic expression of candidate germline genes in spr-5; met-2 mutants can be rescued by knocking down the H3K36 methyltransferase, MES-4, which bookmarks germline genes for reexpression in the germline of the next generation. These data suggest that the developmental delay that we observe in the absence of SPR-5 and MET-2 is caused by ectopic expression of germline genes in somatic tissues. Among the MES-4 regulated germline loci that are ectopically expressed in the soma of spr-5; met-2 mutants, we detect PIWI-interacting RNAs, or piRNAs, and the genes that regulate their function. piRNAs are a large class of small non-coding RNAs that regulate development by functioning in several different pathways, including epigenetic programming, germline transcript silencing, RNA turnover, and translational control. Ectopic expression of piRNA genes has recently been implicated in somatic development and diseases, but how ectopic piRNAs function in somatic cells or if they contribute to abnormal developmental phenotypes poorly understood. Thus, our new C. elegans model provides a unique opportunity to molecularly demonstrate how ectopic germline gene and piRNA expression affects normal development. By combining cutting-edge large genomic experiments, classical developmental techniques, and single molecule fluorescent in situ hybridization we will use our C. elegans model in AIM 1 to determine how ectopic germline genes alter somatic gene expression, 2) define tissue specificity of ectopic germline gene expression, and 3) examine how distinct chromatin states contribute to misexpression of germline genes in somatic tissues. In AIM 2, we will employ similar approaches to examine how ectopic expression of piRNAs contributes to tissue specific somatic defects. Together, the proposed aims will provide mechanistic insight into how these ectopic transcriptional events, that occur when histone modifying enzymes are mutated, contribute to phenotypes that overlap with those seen in human neurodevelopmental disorders.

项目摘要 Kabuki样综合征是由H3K4脱甲基酶LSD1的突变引起的神经发育障碍 并以一系列异常的躯体表型为特征，包括智力障碍，颅面 异常和发展延迟。尽管它们对像Kabuki类似综合症和其他 神经发育综合征，组蛋白中的突变如何修饰LSD1等酶有助于复杂 发展表型尚不清楚。更好地了解组蛋白中的突变如何修饰酶影响 我们已经开发了缺乏Spr-5的C.秀丽隐杆线虫模型，即LSD1的秀丽隐杆线虫直系同源物， 除了组蛋白3赖氨酸9（H3K9）甲基转移酶MET-2。没有SPR-5和MET-2，后代 在体细胞组织中的异位表达生殖基因，导致表型，类似于观察到的表型 神经发育综合征，包括肌肉缺陷和发育延迟。令人惊讶的是， SPR-5中候选种系基因的发育延迟和异位表达； Met-2突变体可以是 通过击倒H3K36甲基转移酶Mes-4来挽救，该甲基转移酶为书签添加了书签基因 在下一代的种系中重新表达。这些数据表明我们的发展延迟 在没有SPR-5和MET-2的情况下观察是由细菌基因的异位表达引起的 组织。在SPR-5的SOMA中以异位表达的Mes-4调节种系基因座中； Met-2 突变体，我们检测PIWI相互作用的RNA或PIRNA，以及调节其功能的基因。 pirnas是一个 大的小型非编码RNA通过在几种不同的途径中运行来调节发育， 包括表观遗传编程，种系笔录沉默，RNA更新和翻译控制。异位 PIRNA基因的表达最近与体细胞发育和疾病有关，但如何异位 PIRNA在体细胞中的功能，或者如果它们导致异常的发育表型，那么理解的。 因此，我们的新秀丽隐杆线虫模型提供了一个独特的机会，可以证明异位种系 基因和piRNA表达会影响正常发育。通过结合尖端的大基因组实验， 经典的发育技术和单分子荧光原位杂交我们将使用我们的C。 AIM 1中的秀丽隐杆线模型确定异位种系基因如何改变体细胞基因表达，2）定义组织 异位种系基因表达的特异性，3）检查不同的染色质状态如何促进 躯体组织中种系基因的表现。在AIM 2中，我们将采用类似的方法来检查 PIRNA的异位表达如何有助于组织特定的体细胞缺陷。拟议的目标在一起 将提供有关这些异位转录事件在组蛋白修饰时如何发生的机械洞察力 酶是突变的，有助于表型与人类神经发育中看到的表型重叠 疾病。