Comparative Analyses of SUV420H1 Isoforms and SUV420H2 Reveal Differences in Their Cellular Localization and Effects on Myogenic Differentiation

Methylation of histone H4 on lysine 20 plays critical roles in chromatin structure and function via mono- (H4K20me1), di- (H4K20me2), and trimethyl (H4K20me3) derivatives. In previous analyses of histone methylation dynamics in mid-gestation mouse embryos, we documented marked changes in H4K20 methylation during cell differentiation. These changes were particularly robust during myogenesis, both in vivo and in cell culture, where we observed a transition from H4K20me1 to H4K20me3. To assess the significance of this change, we used a gain-of-function strategy involving the lysine methyltransferases SUV420H1 and SUV420H2, which catalyze H4K20me2 and H4K20me3. At the same time, we characterized a second isoform of SUV420H1 (designated SUV420H1_i2) and compared the activity of all three SUV420H proteins with regard to localization and H4K20 methylation.Immunofluorescence revealed that exogenous SUV420H1_i2 was distributed throughout the cell, while a substantial portion of SUV420H1_i1 and SUV420H2 displayed the expected association with constitutive heterochromatin. Moreover, SUV420H1_i2 distribution was unaffected by co-expression of heterochromatin protein-1α, which increased the targeting of SUV420H1_i1 and SUV420H2 to regions of pericentromeric heterochromatin. Consistent with their distributions, SUV420H1_i2 caused an increase in H4K20me3 levels throughout the nucleus, whereas SUV420H1_i1 and SUV420H2 facilitated an increase in pericentric H4K20me3. Striking differences continued when the SUV420H proteins were tested in the C2C12 myogenic model system. Specifically, although SUV420H1_i2 induced precocious appearance of the differentiation marker Myogenin in the presence of mitogens, only SUV420H2 maintained a Myogenin-enriched population over the course of differentiation. Paradoxically, SUV420H1_i1 could not be expressed in C2C12 cells, which suggests it is under post-transcriptional or post-translational control.These data indicate that SUV420H proteins differ substantially in their localization and activity. Importantly, SUV420H2 can induce a transition from H4K20me1 to H4K20me3 in regions of constitutive heterochromatin that is sufficient to enhance myogenic differentiation, suggesting it can act an as epigenetic ‘switch’ in this process

Quantitative comparison of functional screening by measuring intracellular Ca2+ with radioligand binding at recombinant human dopamine receptors

The purpose of this study was to test whether screening at dopamine receptors performed with a recently described functional assay for G-protein coupled receptors (GPCRs) provides data that correlate significantly with radioligand binding data in the literature, thus possibly allowing researchers to replace radioligand binding with nonradioactive functional screening. Human dopamine receptors hD1 and hD2L (representing Gs [hD1] or G [hD2L] coupled GPCRs) were recombinantly expressed in human embryonic kidney (HEK293) cells. Cells were loaded with Oregon Green 488 BAPTA-1/AM and evenly distributed in 384 well plates. Seventeen test compounds were screened for agonistic activity by injection into the cell suspension and monitoringH of intracellular Ca2+ with a fluorescence microplate reader. Then, standard agonists (100nM SKF38393 for hD1, 30nM quinpirole for hD2L) were injected into wells preincubated with test compounds (screening for antagonism). Injection of various agonists resulted in a concentration-dependent increase in fluorescence. Further, preincubation of antagonists with dopamine receptor expressing cells inhibits concentration-dependent the agonist-induced increase in fluorescence. Calculated apparent functional Ki values correlate with radioligand binding data in the literature (r2=0.7796 for D1, r2=0.7743 for D2). The correlation between apparent functional K values and radioligand binding data for the 17 tested compounds suggests that screening of test compounds at dopamine receptors with the functional Ca2+ assay can replace radioligand binding studies. Furthermore, besides apparent K values, information about agonistic or antagonistic properties of a test compound can be obtained with the functional Ca2+ assay

Understanding mechanisms and the role of differentiation in pathogenesis of Toxoplasma gondii: a review

Parasite differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission of the intracellular protozoan pathogen Toxoplasma gondii. The presence of bradyzoite-containing cysts in human hosts and their subsequent rupture can cause life-threatening recrudescence of acute infection in the immunocompromised and cyst formation in other animals contributes to zoonotic transmission and widespread dissemination of the parasite. In this review, we discuss the evidence showing how the clinically relevant process of bradyzoite differentiation is regulated at both transcriptional and post-transcriptional levels. Specific regulatory factors implicated in modulating bradyzoite differentiation include promoter-based cis-elements, epigenetic modifications and protein translation control through eukaryotic initiation factor -2 (eIF2). In addition to a summary of the current state of knowledge in these areas we discuss the pharmacological ramifications and pose some questions for future research