斑馬魚肌肉調控因數myf-5上游新型調控序列的雙重功能：強化體節專一性並抑制非專一性的表現

Abstract

Myf-5是一種basic helix-loop-helix轉錄因數，負責控制胚胎發育時期肌肉組織的分化，myf-5的表現具有組織專一性與時期專一性的特性。在哺乳類與魚類中對於myf-5基因的調控機制至今尚不清楚。Chen et al.(2001)證實斑馬魚myf-5上游-82／-1序列具有驅動GFP 在體節表現的能力，但-62／-1則無。本文將斑馬魚myf-5-82／-62序列接在CMV minimal promoter的pEGFPm上，將此線性化質體顯微注入斑馬魚一細胞期授精卵中，結果顯示：myf-5-82／-62序列可驅動GFP在體節表現。若將myf-5-82/-62序列接在CMV minimal promoter與enhancer的pCMVm上，則發現myf-5-82/-62序列會抑制CMVm 所造成的非專一性的表現，但GFP在體節表現不受影響。綜合上述結果得知，斑馬魚myf-5-82／-62 casette 為一段cis-acting element，具有2種功能：促進肌肉組織的專一性表現並抑制非專一性表現。經序列比對發現，斑馬魚myf-5-82/-62序列與老鼠myf-5-161/-144序列（轉譯起始點上游）相似性高，將myf-5上游-290/-1序列中的-82/-62區域(CTCTTAGCTCTGTCCTGGCCA)代換成老鼠的-161/-144(CACTGACCGACCCTGGCCA）序列，結果證實myf-5-82／-62序列的功能在哺乳類與魚類是具有共通性。進一步地，利用sequential PCR mutagenesis將myf-5-82/-62 序列作連續性突變，結果顯示：若突變位置在-70／-67及-66／-62序列，就會影響GFP在體節的表現，使得在體節專一性表現並有translocation的比率由62.1%（控制組, pZMYP- 290E）降至37.5(mutation at -70/-67)和7.7%(mutation at -66/-62)。有一GGCCA (CCAAT-like box）位於myf-5-66/-62序列中，若構築並轉殖(-66／-62)/GFP，則GFP的表現不具有體節專一性；相反地，轉殖(-70/-62)/GFP，則GFP就具有體節專一性的表現。因此我們認為myf -5 CCAAT-like box是提供轉錄基因所必需的序列，而主導控制myf-5在體節專一性表現的關鍵核甘酸極可能存在在-70/-67序列(TCCT）。

Myf-5, one of basic helix-loop-helix transcription factors, controls muscle differentiation and is expressed in somite during early embryogenesis. However the gene regulation of myf-5 is poorly understood. Functional analysis of the regulatory cis-elements in more detail is needed. In zebrafish, Chen et al.,(2001) found that the upstream sequence from -82 to–l(-82／-l) of myf-5 was sufficient to direct reporter gene expression specifically in the somite of the transgenic embryos, but the -62／-1 segment was not. When -82／-62 segment fused With CMV minimal promoter (TATA box only,（-82／-62)/mCMV/EGFP ) was constructed and transferred,-82／-62 casette could specifically drive GFP reporter gene expression in the somites. Moreover, GFP signals were exclusively detected in the somites of the 9-hpf embryos injected with 5×（-82／-62) /CMV/EGFP, which contained 5 copies of cassette -82／-62 inserted within CMV promoter/ enhancer. Based on these results, the -82／-62 casette has dual functions: to enhance the somite - specific expression and to repress non-specific expression during the early development of zebrafish embryos. In addition, a conserved sequence like zebrafishn myf-5-82／-62 was also found in mouse myf-5-161／-144 and results showed that they played the same function . The somite-expression pattern of GFP Were greatly abolished in embryos injected with a DNA fragment containing mutated sequences both at -70／-67 and at -66/-62, indicating that -70／-62 sequence was a crucial element for controlling the specific expression of zebrafish myf-5 gene. A -66/-62 fragment, which contained a putative CCAAT-like box was constructed. Transgenesis showed that this -66／-62 sequence was not capable of direct the somite-specific expression, but if plasmid containing -70／-62 sequence was transferred, the GFP was expressed specifically in the somites. Taken together, we strongly suggest that myf- 5 CCAAT-like box in -66／-62 is an essential element for transcribing a gene , and a novel -70／-67 motif is a critical element for the somite-specific expression.