ABSTRACT
Chromatin affects many, if not all aspects, of nuclear organization and function. For this reason, we have focused our attention on elucidating some of the basic mechanisms regulating the formation and maintenance of chromatin, specifically concerning Polycomb repressive complex 2 (PRC2) and PR-Set7. PRC2 is responsible for catalyzing trimethylation of lysine 27 of histone H3 and thus has a critical role in the formation of facultative heterochromatin. PR-Set7 is responsible for catalyzing monomethylation of lysine 20 of histone H4 and is required for proper cell cycle progression and DNA damage response. We have also expanded our work to establish novel techniques and approaches to determine how chromatin is spatially regulated within the nuclear landscape.
Subject(s)
Chromatin/metabolism , Animals , Cell Cycle , DNA Damage , Heterochromatin/metabolism , Humans , Polycomb-Group Proteins , Protein Structure, Tertiary , Repressor Proteins/metabolismABSTRACT
We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.
Subject(s)
Ascomycota/enzymology , Histone Deacetylase Inhibitors , Histone Deacetylases/chemistry , Silent Information Regulator Proteins, Saccharomyces cerevisiae , Aspergillus nidulans/enzymology , Cell Nucleus/metabolism , Chromatography , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Histone Deacetylases/metabolism , Histones/metabolism , Immunoblotting , Precipitin Tests , Protein Binding , Recombinant Proteins/metabolism , Sirtuin 2 , Sirtuins , Time Factors , Trans-Activators/metabolismABSTRACT
In eukaryotic organisms, acetylation of core histones plays a key role in the regulation of transcription. Multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) maintain a dynamic equilibrium of histone acetylation. The latter form a highly conserved protein family in many eukaryotic species. In this paper, we report the cloning and sequencing of two putative histone deacetylase genes (rpdA, hosA) of Aspergillus nidulans, which are the first to be analyzed from filamentous fungi. Hybridization with a chromosome-specific cosmid library of A. nidulans allowed the localization of rpdA to chromosome III and hosA to chromosome II, respectively. PCR analyses and Southern hybridization experiments revealed that no further members of the RPD3 family are present in the genome of the fungus. Although sequence alignment displays significant amino acid similarity to other eukaryotic RPD3-type deacetylases, the deduced RPDA sequence reveals an unusual 200-amino acid extension at the C-terminus. Expression of both genes was determined by RNA blot analysis. Treatment of the cells with trichostatin A (TSA), a potent inhibitor of HDACs, was found to stimulate expression of rpdA of A. nidulans.
Subject(s)
Aspergillus nidulans/genetics , Histone Deacetylases/genetics , Amino Acid Sequence , Aspergillus nidulans/enzymology , Blotting, Southern , Chromosome Mapping , Chromosomes , Gene Dosage , Histone Deacetylases/isolation & purification , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Transcription Factors/chemistryABSTRACT
Human complement component C8gamma is an unusual complement factor since it shows no homology to other complement proteins but is a member of the lipocalin superfamily. So far, it has been found exclusively in plasma, covalently linked to C8alpha by disulfide bridging. We have used dot blot and Northern blot analyses of a large number of different human tissues to survey systematically the expression pattern of C8gamma. Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney. Renal expression of C8gamma is not dependent on C8alpha expression, since we could not detect C8alpha expression in kidney. Thus its physiological function is not restricted to a specific action in association with complement components. As a prerequisite for further characterization of the structure and binding activities of the uncomplexed C8gamma, we have expressed the encoding cDNA in Escherichia coli. To increase the probability for proper folding of the characteristic intramolecular disulfide bridge the recombinant protein was produced by secretion to the periplasm.
