The regulatory protein GAL80 is a determinant of the chromatin structure of the yeast GAL1-10 control region

Chromatin in the regions between the upstream activator sequence and the 5' ends of the yeast GAL1 and GAL10 genes has been analyzed by DNase I chromosomal footprinting and micrococcal nuclease digestion using the indirect end-labeling approach. Comparison of wild type chromatin digests to naked DNA digests shows that there are specific regions of these upstream sequences which are strongly protected in chromatin. Comparison to chromatin digests from cells disrupted for the positive regulatory gene, GAL4, or the negative regulatory gene, GAL80, and thus lacking GAL4 or GAL80 function, shows that these regions of protection in wild type chromatin are GAL80-dependent but not GAL4-dependent. The protected regions include DNA lying on (GAL10) or near (GAL1) the respective TATA boxes. These protections are present in both noninduced and induced cells. Both DNA strands are equally protected. Upstream of GAL1 there is a second protected region. This protection shows considerable expression and strand dependence. These observations provide the first evidence that the GAL80 function influences chromatin structure and suggest possible mechanisms by which GAL80 modulates the GAL1 and 10 promoters in induced cells. Micrococcal nuclease digests also suggest a role for GAL80 in a distinctive higher order organization of the intergenic region, perhaps involving multiprotein complexes.     

The relationship of regulatory proteins and DNase I hypersensitive sites in the yeast GAL1-10 genes.

We have used yeast strains containing a disrupted positive (GAL4) and/or a disrupted negative (GAL80) regulatory gene to investigate the relationship of these regulatory proteins to the hypersensitive sites upstream of their target genes, GAL1-10. We find that neither of these regulatory proteins is required for the formation of the hypersensitive region. There is positive regulatory protein (dependent) binding to a portion of the hypersensitive region when GAL1 and 10 are expressed. However, similar binding can also occur under conditions in which the genes are not expressed. Thus, such binding is necessary but not sufficient for expression of GAL1 and 10 and control of GAL1-10 expression must also include processes which occur subsequent to GAL4/DNA binding. The negative regulatory protein GAL80 plays a significant role in these processes.     

Intrinsically bent DNA in the promoter regions of the yeast GAAL1-10 and GAL80 genes

Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1-10 and GAL80 genes. These bends lie in functionally suggestive locations. On the promoter of the GAL1-10 structural genes, strong bends bracket nucleosome B, which lies between the UAS(G) and the GAL1 TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1-10 expression is induced. The strongest intrinsic bend ( approximately 70 degrees ) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it to be involved in GAL1 activation in several ways, including the recruitment of a yeast HMG protein that is required for the normally robust level of GAL1 expression in the induced state (Paull, T., Carey, M., and Johnson, R. (1996) Genes Dev. 10, 2769-2781). On the regulatory gene GAL80, the single bend lies in the non-nucleosomal hypersensitive region, between a GAL80-specific far upstream promoter element and the more gene-proximal promoter elements. GAL80 promoter region nucleosomes contain no intrinsically bent DNA.     

Organization of the GAL1-GAL10 intergenic control region chromatin.

A defined, "far upstream" promoter element, the Upstream Activator Sequence (UAS), which mediates the galactose dependent induction of expression of the GAL10 gene in yeast, is the locus of an anomalous, mainly expression independent chromatin structure. The UAS chromatin shows three symmetrical DNase I hypersensitive sites in brief digests, a loss of the 10 bp DNase I ladder pattern in more extensive digests and an enhanced staphylococcal nuclease sensitivity. This anomalous structure is confined to a small region of the UAS. The surrounding chromatin, including the TATA box regions shows a more typical, but expression dependent nucleoprotein, probably nucleosomal, organization. Such an arrangement may be a common feature of eukaryotic genes.     

GAL4/UAS系统在转基因技术中的应用研究进展

GAL4/UAS系统是一种转基因技术体系,其原理是利用特定的启动子或增强子,以组织特异性的方式激活酵母转录激活子GAL4的表达,GAL4又以同样的方式引起GAL4反应元件(UAS)-靶基因的转录。GAL4/UAS系统的关键点在于:GAL4基因和UAS-靶基因分别存在于两个转基因系中。GAL4转基因系中有转录激活子,但没有靶基因;在UAS-靶基因系中,转录激活子不存在,因而靶基因处于沉默状态,只有将GAL4转基因系与UAS-靶基因系进行杂交,才可能产生表达靶基因的后代。本文综述了GAL4/UAS系统的建立及其研究应用。     

GAL4/UAS系统在果蝇Tis11基因RNA干扰中的应用

TTP在哺乳动物许多关键基因表达的转录后水平上起调控作用,Tis11是TTP蛋白在果蝇中的同源物.目前还没有现成的可用于研究Tis11功能的基因敲除或敲低的果蝇.为了获得肌动蛋白启动子或者热激蛋白启动子驱动表达Tis11 mRNA干扰序列的具有较高干扰效率的Tis11基因干扰果蝇,将肌动蛋白启动子或者热激启动子驱动表达的GAL4果蝇品系与融合有Tis11 mRNA干扰序列的UAS品系杂交,收集同时带有GAL4基因和UAS序列的子一代果蝇.提取所收集果蝇的总RNA,将其中的mRNA逆转录成cDNA,并设计检测Tis11基因的特异性引物,然后通过Real-time PCR检测Tis11 mRNA的表达情况.结果显示所收集的能表达Tis11基因干扰序列的子一代果蝇与不能表达Tis11基因干扰序列的对照果蝇相比,其体内Tis11 mRNA的表达水平下降明显.收集的果蝇其体内所表达的干扰序列对Tis11 mRNA干扰效果显著,我们成功获得了Tis11基因的RNA干扰果蝇.     

Pleiotropy and GAL pathway degeneration in yeast

Traits that do not contribute to fitness are expected to be lost during the course of evolution, either as a result of selection or drift. The Leloir pathway of galactose metabolism (GAL) is an extensively studied metabolic pathway that degenerated on at least three independent occasions during the evolutionary diversification of yeasts, suggesting that the pathway is costly to maintain in environments that lack galactose. Here I test this hypothesis by competing GAL pathway deletion mutants of Saccharomyces cerevisiae against an isogenic strain with an intact GAL pathway under conditions where expression of the pathway is normally induced, repressed, or uninduced. These experiments do not support the hypothesis that pleiotropy drives GAL pathway degeneration, because mutations that knock out individual GAL genes do not tend to increase fitness in the absence of galactose. At a molecular level, this result can be explained by the fact that yeast uses inexpensive regulatory proteins to tightly regulate the expression of structural genes that are costly to express. I argue that these results have general relevance for our understanding of the fitness consequences of gene disruption in yeast.     

Nucleosome structure and repair of N-methylpurines in the GAL1-10 genes of Saccharomyces cerevisiae

Nucleosome structure and repair of N-methylpurines were analyzed at nucleotide resolution in the divergent GAL1-10 genes of intact yeast cells, encompassing their common upstream-activating sequence. In glucose cultures where genes are repressed, nucleosomes with fixed positions exist in regions adjacent to the upstream-activating sequence, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence. Galactose induction causes nucleosome disruption throughout the region analyzed, with those nucleosomes close to the upstream-activating sequence being most striking. In glucose cultures, a strong correlation between N-methylpurine repair and nucleosome positioning was seen in nucleosomes with fixed positions, where slow and fast repair occurred in nucleosome core and linker DNA, respectively. Galactose induction enhanced N-methylpurine repair in both strands of nucleosome core DNA, being most dramatic in the clearly disrupted, fixed nucleosomes. Furthermore, N-methylpurines are repaired primarily by the Mag1-initiated base excision repair pathway, and nucleotide excision repair contributes little to repair of these lesions. Finally, N-methylpurine repair is significantly affected by nearest-neighbor nucleotides, where fast and slow repair occurred in sites between pyrimidines and purines, respectively. These results indicate that nucleosome positioning and DNA sequence significantly modulate Mag1-initiated base excision repair in intact yeast cells.     

Heterochromatin protein 1 binds to nucleosomes and DNA in vitro

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein primarily associated with the pericentric heterochromatin and telomeres in Drosophila. The molecular mechanism by which HP1 specifically recognizes and binds to chromatin is unknown. The purpose of this study was to test whether HP1 can bind directly to nucleosomes. HP1 binds nucleosome core particles and naked DNA. HP1-DNA complex formation is length-dependent and cooperative but relatively sequence-independent. We show that histone H4 amino-terminal peptides bind to monomeric and dimeric HP1 in vitro. Acetylation of lysine residues had no significant effect on in vitro binding. The C-terminal chromo shadow domain of HP1 specifically binds H4 N-terminal peptide. Neither the chromo domain nor chromo shadow domain alone binds DNA; intact native HP1 is required for such interactions. Together, these observations suggest that HP1 may serve as a cross-linker in chromatin, linking nucleosomal DNA and nonhistone protein complexes to form higher order chromatin structures.