Although variants have been identified for every class of histone, their functions remain unknown. We have been studying the histone H2A variant hv1 in the ciliated protozoan Tetrahymena thermophila. Sequence analysis indicates that hv1 belongs to the H2A.F/Z type of histone variants. On the basis of the high degree of evolutionary conservation of this class of histones, they are proposed to have one or more distinct and essential functions that cannot be performed by their major H2A counterparts. Considerable evidence supports the hypothesis that the hv1 protein in T. thermophila and hv1-like proteins in other eukaryotes are associated with active chromatin. In T. thermophila, simple mass transformation and gene replacement techniques have recently become available. In this report, we demonstrate that either the HTA1 gene or the HTA2 gene, encoding the major H2As, can be completely replaced by disrupted genes in the polyploid, transcriptionally active macronucleus, indicating that neither of the two genes is essential. However, only some of the HTA3 genes encoding hv1 can be replaced by disrupted genes, indicating that the H2A.F/Z type variants have an essential function that cannot be performed by the major H2A genes. Thus, an essential gene in T. thermophila can be defined by the fact that it can be partially, but not completely, eliminated from the polyploid macronucleus. To our knowledge, this study represents the first use of gene disruption technology to study core histone gene function in any organism other than yeast and the first demonstration of an essential gene in T. thermophila using these methods. When a rescuing plasmid carrying a wild-type HTA3 gene was introduced into the T. thermophila cells, the endogenous chromosomal HTA3 could be completely replaced, defining a gene replacement strategy that can be used to analyze the function of essential genes.

H2A.F/Z histones are conserved variants that diverged from major H2A proteins early in evolution, suggesting they perform an important function distinct from major H2A proteins. Antisera specific for hv1, the H2A.F/Z variant of the ciliated protozoan Tetrahymena thermophila, cross-react with proteins from Saccharomyces cerevisiae. However, no H2A.F/Z variant has been reported in this budding yeast species. We sought to distinguish among three explanations for these observations: (i) that S. cerevisiae has an undiscovered H2A.F/Z variant, (ii) that the major S. cerevisiae H2A proteins are functionally equivalent to H2A.F/Z variants, or (iii) that the conserved epitope is found on a non-H2A molecule. Repeated attempts to clone an S. cerevisiae hv1 homolog only resulted in the cloning of the known H2A genes yHTA1 and yHTA2. To test for functional relatedness, we attempted to rescue strains lacking the yeast H2A genes with either the Tetrahymena major H2A genes (tHTA1 or tHTA2) or the gene (tHTA3) encoding hv1. Although they differ considerably in sequence from the yeast H2A genes, the major Tetrahymena H2A genes can provide the essential functions of H2A in yeast cells, the first such case of trans-species complementation of histone function. The Tetrahymena H2A genes confer a cold-sensitive phenotype. Although expressed at high levels and transported to the nucleus, hv1 cannot replace yeast H2A proteins. Proteins from S. cerevisiae strains lacking yeast H2A genes fail to cross-react with anti-hv1 antibodies. These studies make it likely that S. cerevisiae differs from most other eukaryotes in that it does not have an H2A.F/Z homolog. A hypothesis is presented relating the absence of H2A.F/Z in S. cerevisiae to its function in other organisms.

Human dental pulp stem cells (hDPSCs) reside in postnatal dental pulp and exhibit the potential to differentiate into odontoblasts as well as neurons. However, the intercellular signaling niches necessary for hDPSC survival and self-renewal remain largely unknown. The objective of this study is to demonstrate the existence of intercellular purinergic signaling in hDPSCs and to assess the impact of purinergic signaling on hDPSC survival and proliferation. hDPSCs were isolated from extracted third molars and cultured in minimum essential medium. To demonstrate responsiveness to ATP application and inhibitions by purinergic receptor antagonists, whole cell patchclamp recordings of ATP-induced currents were recorded from cultured hDPSCs. Immunofluorescence and enzymatic histochemistry staining were performed to assess purinergic receptor expression and ectonucleotidase activity in hDPSCs, respectively. To determine the effects of purinergic signaling on hDPSC, purinergic receptor antagonists and an ectonucleotidase inhibitor were applied in culture medium, and hDPSC survival and proliferation were assessed with DAPI staining and Ki67 immunofluorescence staining, respectively. We demonstrated that ATP application induced inward currents in hDPSCs. P2X and P2Y receptors are involved in the generation of ATP-induced inward currents. We also detected expression of NTPDase3 and ectonucleotidase activity in hDPSCs. We further demonstrated that purinergic receptors were tonically activated in hDPSCs and that inhibition of ectonucleotidase activity enhanced ATP-induced inward currents. Furthermore, we found that blocking P2Y and P2X receptors reduced-and inhibition of ecto-ATPase activity enhanced-the survival and proliferation of hDPSCs, while blocking P2X receptors alone affected only hDPSC proliferation. Autocrine/paracrine purinergic signaling is essential for hDPSC survival and proliferation. These results reveal potential targets to manipulate hDPSCs to promote tooth/dental pulp repair and regeneration.