Xu Jingyue, Liu Han, Lan Yu, Jiang Rulang
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States.
Division of Plastic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States.
Front Cell Dev Biol. 2021 Apr 8;9:665109. doi: 10.3389/fcell.2021.665109. eCollection 2021.
Disruption of , encoding a member of the Forkhead family transcription factors, has been associated with cleft palate in humans and mice. is located in a conserved gene cluster containing , , and . We found that expression of is dramatically upregulated in the embryonic palatal mesenchyme in mouse embryos. We show here that the promoter-deletion mutation caused dramatically increased expression of the -linked allele but had little effect on the allele in . We analyzed effects of the mutation on the expression of other neighboring genes and compared those effects with the chromatin domain structure and recently identified enhancer-promoter associations as well as H3K27ac ChIP-seq data. We show that the mutation resulted in significantly increased expression of the and genes located in the same topologically associated domain with but not the expression of the and genes located in the adjacent chromatin domain. We inactivated the gene in mice homozygous for a conditional allele using CRISPR genome editing and generated mice with loss-of-function mutations in and in . Whereas the mice exhibited cleft palate at birth similar as in the mice, systematic expression analyses of a large number of Foxf2-dependent genes revealed that the (/ embryos exhibited distinct effects on the domain-specific expression of several important genes, including , , and , in the developing palatal shelves compared with embryos. These results identify a novel -regulatory effect of the mutation and demonstrate that regulation of contributed to alterations in palatal gene expression in embryos. These results have important implications for interpretation of results and mechanisms from studies of promoter- or gene-deletion alleles. In addition, the unique mouse lines generated in this study provide a valuable resource for understanding the cross-regulation and combinatorial functions of the and genes in development and disease.
编码叉头家族转录因子成员的基因的破坏已与人类和小鼠的腭裂相关联。该基因位于一个保守的基因簇中，该基因簇包含其他基因。我们发现，在特定小鼠胚胎的胚胎腭间充质中，该基因的表达显著上调。我们在此表明，该基因启动子缺失突变导致与该基因连锁的等位基因表达显著增加，但对另一等位基因的表达影响很小。我们分析了该突变对其他相邻基因表达的影响，并将这些影响与染色质结构域结构、最近确定的增强子 - 启动子关联以及H3K27ac ChIP - seq数据进行了比较。我们表明，该突变导致与该基因位于同一拓扑相关结构域中的其他两个基因的表达显著增加，但不影响位于相邻染色质结构域中的另外两个基因的表达。我们使用CRISPR基因组编辑技术在纯合特定条件等位基因的小鼠中使该基因失活，并在另一基因中产生功能丧失突变的小鼠。虽然该基因敲除小鼠出生时表现出与特定基因敲除小鼠相似的腭裂，但对大量依赖该基因的基因进行的系统表达分析表明，与特定基因敲除胚胎相比，该双基因敲除胚胎对发育中的腭板中几个重要基因（包括其他基因）的结构域特异性表达表现出不同的影响。这些结果确定了该突变的一种新的基因调控作用，并证明该基因的调控促成了特定基因敲除胚胎中腭部基因表达的改变。这些结果对于解释启动子或基因缺失等位基因研究的结果和机制具有重要意义。此外，本研究中产生的独特小鼠品系为理解该基因和其他基因在发育和疾病中的交叉调控及组合功能提供了宝贵资源。
