HLH GENE FUNCTION IN NEURONAL CELL DETERMINATION

螺旋-环螺旋（HLH）蛋白是一个进化上保守的蛋白家族， 具有复杂二聚化和DNA结合的转录因子 特色 对于果蝇和人HLH蛋白， DNA结合异二聚体在不同的HLH蛋白之间形成。 在果蝇中，已经发现10个相互作用的HLH基因的系统控制 神经元细胞测定。 这10个HLH基因由5个激活因子组成 和5个阻遏物，其控制神经元模式的不同子集。 这项拟议的研究将检验HLH激活剂 蛋白质的功能是形成DNA结合同源二聚体和异源二聚体， HLH阻遏蛋白通过与激活蛋白结合而起作用， 形成非DNA结合的异二聚体。 HLH蛋白也将被测试 因为它们能够在培养的细胞中激活报告基因的转录， 细胞 这种HLH蛋白功能的生化分析将是 通过分析HLH蛋白在体内对候选物的调节来补充 目的基因T5。 HLH蛋白对T5的转录调节将是 通过分析HLH突变体中的T5转录本表达进行测试。 在 此外，缺失T5 5'结构域的不同部分的突变体将被 检查T5转录物表达的模式。 假定函数 调节区域将通过果蝇的P元件转化来确认 报告基因在T5顺式DNA的那些区域的控制下。 还将测定HLH蛋白与T5调节DNA的结合。一个 最终的目标是对特定位点进行定点诱变， 在体外结合HLH蛋白，并可能在体内调节T5。 在人类中，至少一些HLH基因是原癌基因。 两个人HLH 在急性淋巴细胞白血病患者中， 白血病 这些严重的突变可能会扰乱正常的蛋白质- 这些转录因子的蛋白质和蛋白质-DNA相互作用。 的 拟议的研究将确定HLH系统的特性， 苍蝇;这可能会提供对人类系统的更好理解， 及其在发育和疾病中的作用。

Helix-loop helix (HLH) proteins are an evolutionarily-conserved family of transcription factors which have complex dimerization and DNA-binding characteristics. For both Drosophila and human HLH proteins, high affinity DNA-binding heterodimers form between different HLH proteins. In flies, a system of 10 interacting HLH genes has been found to control neuronal cell determination. These 10 HLH genes consist of 5 activators and 5 repressors, which control different subsets of the neuronal pattern. The proposed research will test the hypothesis that the HLH activator proteins function to form DNA-binding homodimers and heterodimers, and that the HLH repressor proteins function by binding to the activator proteins to form non-DNA-binding heterodimers. The HLH proteins also will be tested for their ability to activate transcription of reporter genes in cultured cells. This biochemical analysis of HLH protein function will be complemented by analysis of HLH protein regulation of a candidate in vivo target gene, T5. Transcriptional regulation of T5 by HLH proteins will be tested by analysis of T5 transcript expression in HLH mutants. In addition, mutants with different parts of the T5 5' domain deleted will be examined for pattern of T5 transcript expression. Function of putative regulatory regions will be confirmed by P element transformation of flies with reporter genes under control of those regions of the T5 cis DNA. Binding of HLH proteins to T5 regulatory DNA also will be assayed. An eventual goal will be site-directed mutagenesis of specific sites which bind HLH proteins in vitro and are likely to regulate T5 in vivo. In humans, at least some HLH genes are proto-oncogenes. Two human HLH genes are consistently translocated in patients with acute lymphoblastic leukemia. These severe mutations are likely to perturb the normal protein- protein and protein-DNA interactions of these transcription factors. The proposed research will determine the characteristics of the HLH system in flies; this will likely provide a better understanding of the human system, and its role in development and disease.