LMBRD1基因在神經分化過程中的轉錄調控

Abstract

LMBRD1基因主要編碼兩個已知功能之蛋白質LMBD1和NESI。LMBD1為一膜蛋白質，主要分布於細胞膜及溶酶體上，參與維生素B12從溶酶體至細胞質之運輸、胰島素受體的內吞作用、胚胎和神經發育及細胞骨架之調控。在Lmbrd1+/- 小鼠模式中，發現其在學習記憶上有所缺陷，並從腦組織切片配合免疫組織化學染色結果發現神經網絡較稀疏，且將海馬迴神經元分離培養時，發現神經突上之樹狀突脊顯著減少，推測為學習記憶缺陷之原因。從即時定量PCR及組織蛋白質分析結果，發現LMBD1在小鼠腦部有大量表達，並從VEGF誘導小鼠神經瘤細胞株N2A分化實驗中，發現LMBD1蛋白質表現會上升，並與肌動蛋白有共位現象，推測其在神經分化過程中參與調控細胞骨架之形成。本實驗欲探討以維生素A酸誘導人類神經瘤母細胞株SH-SY5Y的分化過程，是否LMBD1會受到調控，並探討其轉錄調控機制。結果顯示，在細胞分化前期LMBD1表現會有些微下降，而在分化兩天後顯著上升，且在分化過程中會分布於神經突中，並有區域聚集的現象，猜測可能與樹狀突脊生成有關。以cDNA 5’端快速擴增技術確認LMBD1之轉錄起始有多個位點，可能受3個GC box分別調控。利用構築帶有不同長度之LMBRD1啟動子的luciferase報導質體，分析神經分化前後啟動子活性變化，並以軟體進行轉錄因子預測，發現有3個KLF4可能結合的DNA序列，推測KLF4可能會作為轉錄抑制因子抑制LMBD1之表現。以染色體免疫沉澱及定點突變實驗證實KLF4會結合至LMBRD1(nt -936~ -525) 內之第2及第3預測位點。以西方墨點法及即時聚合酶連鎖反應分析發現在分化過程中，KLF4可能會受轉錄後修飾而抑制其表現，使結合至LMBRD1(nt -936~ -525)之能力下降而促進LMBD1之表現。 關鍵字： LMBRD1基因、維生素A酸、神經細胞分化、轉錄調控機制、轉錄起始位點。

LMBRD1 gene is located on human chromosome 6 and encodes two known proteins, namely LMBD1 and NESI. LMBD1 is a membrane protein, which is mainly located at plasma membrane and lysosome. It regulates several biological functions, such as export of lysosomal vitamin B12, internalization of insulin receptor, embryonic and neural development, and actin polymerization. In Lmbrd1+/- mouse model, defects in behaviors were identified, such as learning memory. Immunohistochemistry staining showed sparse neural networks in the brain of the Lmbrd1+/- mice. In addition, primary neuron cells derived from the hippocampus of Lmbrd1+/- mice had less spine formation. This could be one of the reasons why Lmbrd1+/- mice have memory defects. Realtime PCR and immunoblot analysis of various mouse tissues revealed that brain had the highest expression level of LMBD1. when VEGF was used to induce differentiation of mouse neuroblastoma N2A cells, LMBD1 was up-regulated and was colocolized with actin in neurites. These results indicate that LMBD1 might regulate neural differentiation through cytockelatal organization. However, the mechanism involved in the regulation of LMBD1 expression during neural differentiation is still unclear. In this study, the transcriptional regulation of LMBD1 during retinoic acid (RA) induced differentiation of human neuroblastoma SH-SY5Y cells was investigated. The results showed an up-regulated expression of LMBD1 in the neurites of differentiated SH-SY5Y cells on the second day after RA treatment. In addition, multiple transcriptional initiation sites, possibly regulated through different GC-boxes, were identified by cDNA 5’ RACE amplication. In order to identify important cis-elements in the LMBRD1 promoter region, luciferase reporter plasmid carrying LMBRD1 gene 5’ upstream sequences from nucleotides -2357 to +1 [LMBRD1(nt -2357~ +1)] and a series deletion were constructed and analyzed for their promoter activities. The results showed that [LMBRD1(nt -1580~ -936)] and [LMBRD1(nt -936~ -525)] contain positive and negative regulatory elements, respectively. Since the expression level of LMBD1 was increased during neural differentiation, it is likely that the negative regulator associated with the [LMBRD1(nt -936~ -525)] was lost. By using software TFBIND, PROMO and JASPAR, transcription factors that have potential to bind to the region were examed. KLF4 was chosen as a candidate that down-regulates the expression of LMBRD1. Three potential binding sites of KLF4 were found. In chromatin precipitation and site-direct mutagenesis analysis, KLF4 was identified to bind to the predicted sites 2 and 3 in [LMBRD1(nt -936~ -525)] region. Overexpression and knockdown experiments, further confirmed that KLF4 can serve as a repressor that down-regulated the expression of LMBD1 during neural differentiation. Western blot and real time qPCR analysis indicated that KLF4 may be down-regulated by post-transcriptional mechanism, resulting in the up-regulation of LMBRD1 gene. Key words： LMBRD1gene、retinoic acid、neural differentiation、transcriptional regulation、transcription initiation site。