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.

项目概要 歌舞伎样综合征是一种由 H3K4 去甲基化酶 LSD1 突变引起的神经发育障碍， 其特征是一系列异常的躯体表型，包括智力障碍、颅面障碍 异常和发育迟缓。尽管它们与歌舞伎样综合症和其他 神经发育综合征，LSD1 等组蛋白修饰酶的突变如何导致复杂的 发育表型尚不清楚。为了更好地了解组蛋白修饰酶的突变如何影响 体细胞发育我们开发了一个缺乏SPR-5的线虫模型，SPR-5是LSD1的线虫直系同源物， 除组蛋白 3 赖氨酸 9 (H3K9) 甲基转移酶 MET-2 外。没有 SPR-5 和 MET-2，后代 在体细胞组织中异位表达种系基因，导致与在 神经发育综合征，包括肌肉缺陷和发育迟缓。引人注目的是， spr-5中候选种系基因的发育迟缓和异位表达； met-2突变体可以是 通过敲除 H3K36 甲基转移酶 MES-4 来拯救，该酶为种系基因添加书签 在下一代种系中重新表达。这些数据表明我们的发育迟缓 在缺乏 SPR-5 和 MET-2 的情况下观察到的现象是由体细胞种系基因的异位表达引起的 组织。 MES-4 调控的种系位点在 spr-5 的体细胞中异位表达；梅特-2 突变体中，我们检测 PIWI 相互作用的 RNA（或 piRNA）以及调节其功能的基因。 piRNA 是 一大类小非编码 RNA，通过在几种不同途径中发挥作用来调节发育， 包括表观遗传编程、种系转录物沉默、RNA 周转和翻译控制。异位 最近，piRNA 基因的表达与体细胞发育和疾病有关，但异位表达如何 piRNA 在体细胞中发挥作用，或者是否会导致异常发育表型，人们对此知之甚少。 因此，我们的新秀丽隐杆线虫模型提供了一个独特的机会来从分子角度展示异位种系如何 基因和piRNA的表达影响正常发育。通过结合尖端的大型基因组实验， 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 中的线虫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，以及 3) 检查不同的染色质状态如何有助于 体细胞组织中种系基因的错误表达。在 AIM 2 中，我们将采用类似的方法来检查 piRNA 的异位表达如何导致组织特异性体细胞缺陷。共同提出的目标 将提供有关组蛋白修饰时如何发生这些异位转录事件的机制见解 酶发生突变，导致与人类神经发育中所见的表型重叠 失调。