This competitive renewal application from Dr. Martin Gorovsky requests five years of support for a project to study the function of the histones and their roles in nuclear differentiation in the protozoan Tetrahymena thermophila. Though it is unicellular, Tetrahymena possesses two nuclei, a dipoid, transcriptionally quiescent micronucleus that is analogous to the germline of higher eukaryotes, and a polyploid, transcriptionally active macronucleus that is analogous to the soma of higher eukaryotes. Each of the nuclei has the same genotype, although the chromosomes of the marconucles are fragmented into small pieces, each present about 45 times; only the micronucleus undergoes mitosis. In both nuclei, the DNA is complexed with histones, including the major core histones H2A, H2B, H3 and H4, as well as their minor variants hv1 (H2A) and hv2 (H3), and linker histones--H1 in the macronucleus and MicLH in the micronucleus; MicLH is actually a polyprotein that is cleaved into four smaller polypeptides, a, b, g and d. Previous research has established that the chromatin of the micro and macronuclei in Tetrahymena is organized differently. Thus, for example, the macronuclear chromatin contains DNase I hypersensitive sites, whereas the micronulcear chromatin does not; furthermore, the minor core histone variants hv1 and hv2 are found only in the macronucleus. These and other observations have led to the hypothesis that the core histone variants are related to transcriptional activity, and that their existence and function is coupled with the existence and function of the macronuclear linker histone H1. Dr. Gorovsky has organized his project around five specific aims.
The first aim i s to develop better materials for the genetic transformation of Tetrahymena cells. Recently Dr. Gorovsky and others have succeeded in devising two different procedures to knock out genes in Tetrahymena by homologous recombination with a sequence that contains a selectable marker. One objective of the proposed research is to develop knockout cassettes with different selectable markers; another objective is to create a construct that could be used in gene replacement experiments, and a third objective is to identify inducible promoters that could be used to drive the expression of genes in replacement constructs.
The second aim of the project is to study the function of the linker histones, principally H1, in vivo. Previous work has established that the H1 and MicLH genes can separately be knocked out in the macronucleus without impairing cell viability. Thus, these histones are individually not essential for growth. The proposed research has several objectives: (1) Determine the biological significance of the phosphorylation sites in H1; Dr. Gorovsky hypothesizes that H1 phosphorylation, which occurs only in the macronucleus, may be necessary to allow transcription of macronuclear genes. (2) Using nuclease digestion techniques and density gradient ultracentrifugation, compare the structure of chromatin in wild type cells and in cells that have had the macronuclear H1 gene knocked out. (3) Characterize the structure of two macronuclear genes in different states of transcriptional activity in the presence and absence of H1, i.e., in wild type and H1 knockout strains. (4) Introduce a chimeric H1 gene encoding the Tetrahymena N and C termini and the mouse central globular domain into Tetrahymena cells that have had their native H1 gene knocked out and determine if the chimeric protein is present in nuclei, if it is phosphorylated, and if it is associated with a phenotype (e.g. condensation of chromatin). (5) By cytological analysis in wild type and knockout strains, quantitate the contribution of linker histones to chromatin compaction in vivo.
The third aim i s to analyze the function of the H2A variant hv1, which previous research has shown is essential for Tetrahymena survival. This histone cannot be completely knocked out in the macronucleus. One objective is predicated on the hypothesis that hv1 facilitates the loss or rearrangement of H1 to enable transcription in the macronucleus. On this hypothesis, a cell that has lost its H1 gene may no longer need its hv1 gene. Thus, Dr. Gorovosky proposes to make the double knockout and determine if it is viable. Four strategies for accomplishing this objective are discussed. Another objective under this aim is to determine if the essential function of hv1 is due to its protein sequence or to constitutive synthesis of an H2A-like histone in the macronucleus (major core histones are synthesized in step with DNA; minor variants are synthesized constitutively). Dr. Gorovsky proposes a set of promoter swapping experiments--core H2A promoter + hv1 coding sequence, hv1 promoter + core H2A coding sequence--to address this issue; the chimeric constructs will be transformed into cells to determine whether the sequence or regulation of hv1 (or both) is needed for surivival. Another objective is to determine if both core H2A genes can be knocked out without imparing cell viability. Dr. Gorovsky also proposes to study the significance of the phosphorylation sites in both hv1 and H2A by replacing the native genes encoding these proteins with genes that have been mutated in vitro. If inducible transgenes can be constructed, he will also investigate the effect of misexpressing hv1 in cells.
The fourth aim i s to analyze the function of the hv2 variant of core histone H3. One objective is to determine if hv2 is associated with transcriptionally active genes. An epitope tagged hv2 gene will be introduced into cells so that hv2-associated chromatin can be purified by immunoprecipitation from cells grown under different conditions. This chromatin will then be analyzed for DNA that is or isn't transcribed under the growth conditions used. A second objective under this aim is to purify hv2-containing nucleosomes and use 2D gels to characterize any proteins that are associated with them. The fifth aim is to determine if phosphorylation of serine 10 in H3 is important for chromosome condensation in the micronucleus. The serine codon will be mutated to alanine (to prevent phosphorylation) or to glutamic acid (to mimic phosphorylation) and the mutant gene will be inserted in place of the native macronuclear H3 gene by homologous recombination within cells; the other H3 gene in these cells will have been knocked out. The expectation is that complete gene replacement will not be possible because the mutations will be lethal to mitosis.
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