Evolutionary and functional relationships between Lgr proteins and ubiquitin-specific proteases

我们之前已经探索了脱泛素酶的进化史。我们确定编码USP4和USP15的基因是由古代的全基因组复制事件产生的(因此是同源的)，而USP11产生于USP4的小片段复制，然后迅速在序列上分化。为了确定功能冗余的水平，我们与Usp4和Usp15的失活突变的小鼠进行了杂交，发现虽然小鼠在任何一个基因都为空的情况下是存活的，但不能产生两个基因都为空的后代。这表明有必需的蛋白质底物可以被这两种底物中的任何一种去泛素化，但在没有这两种底物的情况下，这种底物被泛素化并被蛋白酶体降解。我们发现Wnt/β连环蛋白途径在复合零细胞中是缺失的，并已证实该基因的中期胚胎的肝脏中不存在LGR5(Wnt信号的调节器)。这将解释为什么我们认为造血失败是胚胎死亡的原因。为了加速这项研究，我们建立了一个合作伙伴来培育斑马鱼，其中Usp4和Usp15的同源基因已经被删除，目前正在进行遗传杂交，以确定我们的小鼠的发现是否在这个物种中重复。在透明斑马鱼胚胎中研究造血失败要容易得多。将对不同基因类型的鱼的血细胞进行单细胞RNAseq，以确定哪些细胞类型和分子途径是缺陷的。我们的下一个目标将是分析硬骨鱼中的LGR基因及其在造血中的潜在作用。鉴于Lgr5是小鼠的必需基因，我们惊讶地发现，Lgr5并不存在于斑马鱼中，但Ohnolog基因LGR4和LGR6存在于斑马鱼基因组中。将招募一名研究生来建立硬骨鱼LGR基因的进化史，并确定脱泛素酶基因进化的时间关系。我建议使用原位杂交来研究斑马鱼的基因表达，以确定LGR4和/或LGR6是否在造血中发挥任何作用。同时，将在青冈鱼身上研究LGR基因，这是一种拥有所有三种LGR基因的鱼类。斑马鱼和青竹的LGR基因将使用CRISPR/Cas9方法删除，表型分析将特别关注造血。总而言之，这些实验将为我们提供对复制基因的进化和亚功能化的洞察，了解这些基因是我们的长期目标。

We have previously explored the evolutionary history of the deubiquitinating enzymes. We determined that the genes encoding USP4 and USP15 were generated by an ancient whole genome duplication event (and are therefore ohnologs), whereas USP11 arose from a small segmental duplication of USP4 and then rapidly diverged in sequence. To determine the level of functional redundancy we crossed mice with inactivating mutations of Usp4 and Usp15 and found that while mice were viable when null for either gene no progeny could be generated that were null for both. This suggests that there are essential protein substrates that can be deubiquitinated by either, but in the absence of both such substrates are ubiquitinated and degraded by the proteasome. We found that the Wnt/beta catenin pathway is deficient in compound null cells, and have established that LGR5 (a modulator of Wnt signaling) is absent in the liver of midgestation embryos of that genotype. This would explain the failure of hematopoiesis that we believe is the cause of embryonic lethality. To accelerate the research we established a collaboration to generate zebrafish in which the orthologs of Usp4 and Usp15 have been deleted and are currently performing genetic crosses to determine if our mouse findings are recapitulated in this species. A failure of hematopoiesis would be much easier to study in the transparent zebrafish embryo. Single cell RNAseq will be performed on blood cells from fish of various genotypes to determine which cell types and molecular pathways are deficient. Our next objective will be an analysis of lgr genes in teleost fish and their potential role in hematopoiesis. Given that Lgr5 is an essential gene in mouse we were surprised to discover that lgr5 does not exist in zebrafish, but that the ohnolog genes lgr4 and lgr6 were present in the zebrafish genome. A graduate student will be recruited to establish the evolutionary history of the teleost lgr genes and determine temporal relationships to evolution of the deubiquitinating enzyme genes. I propose to use in situ hybridization to study gene expression in the zebrafish to determine if lgr4 and/or lgr6 play any role in hematopoiesis. In parallel lgr genes will be studied in medaka, a fish species with all three lgr ohnologs. The lgr genes of both zebrafish and medaka will be deleted using CRISPR/Cas9 methodology and phenotypes will be analyzed with particular attention to hematopoiesis. Collectively these experiments will provide insight into the evolution and subfunctionalization of duplicated genes, the understanding of which is our long term objective.