Role of the actin-related protein, Act3p of Saccharomyces cerevisiae, in regulation of chromatin structure and transcription control

Research project P 14452 Role of Act3p in tanscription control Ulrike WINTERSBERGER 08.05.2000 Recently we discovered an essential gene, ACT3, coding for an actin-related protein of S.cerevisiae, which we called Act3p (synonymous to Arp4). The protein is located in the nucleus of the yeast cell, and thermosensitive mutants exist, which exhibit a partial suppression of the effect of a Ty-delta element insertion within the HIS4 promoter (his4912delta). Remarkably the effect is a variagated phenotype, as cells descending from one clone may express the HIS4 gene or not. Because Act3p interacts with core histones and can be immunoprecipitated together with other proteins, the identity of which is as yet unknown, we suspect that Act3p might be a component of one of the complexes affecting chromation structure and thus influencing transcription regulation. In a cooperation with Dr. Masahiko Harata (University of Sendai, Japan) we plan to acquire insight into the mechanism of that regulation. Because competition in the field of transcription control by chromatin structure is severe we find it appropriate for a small group to concentrate on a detailed study of the Act3 molecule itself. We will express it in E.coli and test the purified protein for a possible ability of binding and hydrolysis of ATP. We will carry out site directed mutagenesis in amino acids conserved in relation to actin which seem to play vital roles in the three-dimensional structure and the properties of actin. These mutations will be introduced within the ATP pocket, which, in actin, is responsible for ATP binding and ATPase activity. On the other hand we will mutate domains specific for Act3p and found neither in actin nor in any other known actin related protein. The effects of the mutations will be determined in vitro (properties of the purified Act3 protein) and in vivo on transcription of the genes, HIS4 and ADE2 under the his4-912delta promoter. Dr. Harata will study chromatin structure in wild type and mutated cells around this promoter. Furthermore we will try to complement mutations of yeast ACT3 with related human genes (two of which were already isolated by Dr. Harata) to find out whether they might play similar biological roles as the yeast gene. The ultimate aim, of course, will be finding the putative complex(es) regulating chromatin structure of which Act3p is thought to be a component, and the discovery of genes which under their own promoters are influenced by Act3p. As human diseases exist in which transcription control is disturbed, we hope to contribute with our planed project not only to the advancement of knowledge (our primary interest), but also to better understanding of certain diseases, like e.g. tumour development.

It is well known that all attributes of living things depend on their genes as well as on their environmental conditions ("nature and nurture"). Thus, the range of application of genes (their expression per se and the extent of their expression) is coupled to signals from outside of cells. Originally we believed that all gene regulation (the extent of trancription of their DNA sequences into RNAs) depended exclusively on regulatory protein factors which according to the signals bind to the so-called promoters of the relevant genes to activate or repress them. It was thought that the genes of eukaryotic cells reside within a relatively rigid chromatin (a structure consisting of DNA packed together with the histone proteins). In recent years it became, however, obvious that the structure of chromatin is a highly dynamic one, influenced by numerous chromatin modulating complexes, without which gene regulation cannot take place. As well tuned gene expression is very important for the health of all organisms the field of chromatin modulating complexes is now pursued by many research-groups. In our lab, we use a simple eukaryotic model organism, which is astonishingly closely related to higher organisms like humans, namely bakers yeast. Earlier we had discovered a protein in yeast which is essential for survival of the microorganism. This protein which we called Act3p (because of its relatedness to the well known muscle actin) is a subunit of several chromatin modulating complexes. Importantly, there exist two human proteins which closely resemble yeast Act3p. In the present study we found (by a modern methode called microarray analysis giving information about gene expression), that Act3p is involved in the regulation of stress-proteins. These proteins are expressed to protect cells during adverse conditions like heat, osmotic shock, acidic environment, toxic chemicals and irradiation, etc. . . During conditions pleasant to cells these stress genes should not be expressed, i.e. transcribed into mRNAs and finally translated into the protecting proteins, because these mechanisms are costly for cells and therefore do normally not take place if not necessary. We found that cells with somewhat crippled (we had mutated the gene) Act3p (which however still allows the survival of cells) express stress proteins for no reason and therefore are less fit under non-stress conditions. They proliferate slowlier than cells with a perfect Act3p and under natural conditions would therefore be quickly outrun by there healthy relatives. Also a number of other genes misregulated in the cells with the mutated Act3p were observed. Furthermore we asked for the possible role of Act3p within the complexes. From our experiments we conclude that Act3p can change its molecular structure depending on whether it possesses an ATP molecule within its center or not. ATP (adenosine triphosphate) is a small molecule involved in energy consuming chemical reactions within all living cells. In the case of Act3p we think that, perhaps depending on its concentration within the cell, ATP is bound or released and therefore responsible for the structural change of Act3p. We have indications that ATP is necessary for the release of Act3p from the complexes, thus regulating their dynamic binding to different locations on chromatin.