Loss of H2A.Z Is Not Sufficient to Determine Transcriptional Activity of Snf2-Related CBP Activator Protein or p400 Complexes

The p400 and SRCAP (Snf2-related CBP activator protein) complexes remodel chromatin by catalyzing deposition of histone H2A.Z into nucleosomes. This remodeling activity has been proposed as a basis for regulation of transcription by these complexes. Transcript levels of p21 or Sp1 mRNAs after knockdown of p400 or SRCAP reveals that each regulates transcription of these promoters differently. In this study, we asked whether deposition of H2A.Z within specific nucleosomes by p400 or SRCAP dictates transcriptional activity. Our data indicates that nucleosome density at specific p21 or Sp1 promoter positions is not altered by the loss of either remodeling complex. However, knockdown of SRCAP or p400 reduces deposition of H2A.Z∼50% into all p21 and Sp1 promoter nucleosomes. Thus, H2A.Z deposition is not targeted to specific nucleosomes. These results indicate that the deposition of H2A.Z by the p400 or SRCAP complexes is not sufficient to determine how each regulates transcription. This conclusion is further supported by studies that demonstrate a SRCAPΔATP mutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP

GTP-Dependent Hydrolysis of Phosphatidylinositol-4,5-bisphosphate by Soluble Phospholipase C from Adult Human Epidermis

The regulation of soluble phosphoinositide-specific phospholipase C from adult human epidermis by guanine nucleotide was investigated. In the presence of physiologic concentrations of Ca++ (1 μM) and Mg++ (1.5 mM), neither phosphatidylinositol (PI) nor phosphatidylinositol-4,5-bis-phosphate (PIP2) were appreciably hydrolyzed. Addition of guanosine-5' -triphosphate (GTP) or guanosine-5'-O-(3-thiotriphosphate) (GTP-γ-S) significantly stimulated hydrolysis of PIP2, but not PI. Stimulation of PIP2 hydrolysis by GTP was does-dependent between 1–100 μM GTP. Other nucleoside triphosphates and nucleotide analogues were unable to substitute for GTP or GTP-γ-S. A GTP-γ-S-stimulated PIP2 hydrolysis was inhibited by guanosine-5'-O-(2-thiodiphosphate (GDP-β-S). The phospholipase C preparation specifically bound [35S]GTP-γ -S and this binding was also inhibited by GDP-β-S. In addition to a 41,000-dalton pertussis toxin substrate, the phospholipase C preparation contained 3–4 GTP binding proteins with molecular weights between 20,000–30,000. These data demonstrate that human epidermis contains a soluble GTP-dependent phospholipase C activity that specifically hydrolyzes PIP2 and suggest that this reaction is regulated by a GTP-binding protein(s)