DRE-1調控BLMP-1使線蟲遠端細胞進行向背運動

Abstract

時間以及空間上的調控，對於生物體的發育非常重要。雖然在生物體發育的過程中，空間上如何被調控的機制的研究已經相當多，但是對於時間上如何被準確調控卻知道的非常少。在線蟲當中，有一群異時調控的基因，能夠去控制特定的發育時間點。在這些異時調控的基因當中，dre-1、lin-29 以及daf-12 共同調控線蟲遠端細胞移動的時間。實驗室先前的研究當中發現，這三個異時調控基因，共同去調控了一個轉錄因子BLMP-1，進而去調控遠端細胞的移動。其中lin-29 以及daf-12，主要是藉由抑制了BLMP-1 的轉錄去調控調控遠端細胞。然而，另一個異時調控基因dre-1 是如何去調控BLMP-1 尚未清楚。因此在本篇研究當中，主要是去研究dre-1 是藉由何種機制來調控BLMP-1 的表現。已知DRE-1 是一種F-box protein，是SCF E3 ligase complex 的組成之一，因此BLMP-1 可能藉由被DRE-1 加上泛素而降解。利用免疫共沈澱的方法，發現BLMP-1 能夠和DRE-1 相互作用。接下來，利用HEK 293T 細胞表現這兩種蛋白之後，發現BLMP-1 以及DRE-1 都是藉由進行蛋白酶體降解，而且BLMP-1的降解是經由DRE-1 來達成的。因為在人類中有一個轉錄因子PRDI-BF1 和BLMP-1 非常相似，而且也參與在許多發育以及腫瘤的生成。另外，DRE-1 在 人類的蛋白當中也有一個非常相似的蛋白FBXO11。因為先前已經發現兩個線蟲的蛋白有交互作用，所以在免疫共沈澱的結果，PRDI-BF1 也和FBXO11 有交互作用，顯示這兩個蛋白之間調控的關係在演化上是有保守性的。在本篇研究當中，我們提供了一個參與在發育過程中新的調控關係，此調控關係從線蟲到人類都是有保守性的。

The spatiotemporal control of gene expression is crucial for the development of multicellular organisms. Although the studies in spatial patterning are extensive, temporal regulation of development is poorly understood. In C. elegans, several heterochronic genes, which function in the timing of specific developmental stages, have been identified and characterized. Among them, dre-1, lin-29 and daf-12 act redundantly to control the temporal migration pattern of somatic gonadal cells, distal tip cells (DTC). Our laboratory found that BLMP-1, a zinc finger transcription repressor orthologous to mammalian Blimp-1 (B lymphocyte-induced maturation protein 1), acts downstream of these heterochronic genes to regulate DTC migration path. Blmp-1 is transcriptionally down-regulated by lin-29 and daf-12. However, the regulation of BLMP-1 by DRE-1 is still unclear. My thesis project focused on determining whether BLMP-1 is also down-regulated by DRE-1. Because dre-1 encodes an F-box protein, which functions in SCF E3 ligase complexes, I hypothesize that DRE-1 regulates BLMP-1 protein stability through ubiquitin-mediated proteolysis. Using co-immunoprecipitation assays, I found that DRE-1 and BLMP-1 physically interact with each other. I next assessed the stability of BLMP-1 and DRE-1 by transiently expressing either proteins in HEK 293T cells and found that the turnover of both DRE-1 and BLMP-1 are dependent on proteasome. When co-expressed with DRE-1 in HEK293T cells, the stability of BLMP-1 decreases, indicating the degradation of BLMP-1 is dependent on DRE-1. Because the mammalian homolog of BLMP-1, PRDI-BF1, has been shown to participate in various cellular processes, I next tested whether PRDI-BF1 is also regulated through distinct F-box proteinmediated degradation. I found that PRDI-BF1 co-immunoprecipitated with FBXO11, the mammalian homolog of DRE-1. These results support an evolutionarily conserved DRE-1/FBXO11–dependent degradation of BLMP-1/PRDI-BF1 in both mammals and nematodes. Based on the discovery in C. elegans, we provide an evolutionary conserved regulation that may involve in organism development.