The proposed research deals with the differences in composition and metabolism of proteins associated with DNA in transcribing and nontranscribing regions of chromatin. Particular emphasis is placed on the post-synthetic modifications of histones, HMG proteins and other chromosomal proteins which are believed to modulate their interactions with DNA and influence its conformation and template function in chromatin. A primary aim is to characterize the enzymes involved, the substrate specificities and the control of reactions, such as acetylation, phosphorylation and methylation, that modify histones, high mobility group proteins and other components of chromatin. The temporal and spatial relationships between specific modifications of DNA-binding proteins and the transcriptional competence of the associated DNA sequences will be investigated in systems offering the potential for separating active and inactive segments of the same DNA molecule (e.g., coding and 'spacer' regions of ribosomal genes), and in tumor cells during the reprogramming of transcription induced by agents, such as butyrate and cAMP analogues, that suppress the malignant phenotype - and directly affect the acetylation and phosphorylation of DNA-associated proteins. New techniques for the recovery of proteins which have just been phosphorylated in vivo and in vitro will be applied to the study of site-specific phosphorylations in nuclear proteins and to the recovery of peptides containing modified serine, threonine, tyrosine, and lysine residues in normal, carcinogen-treated, and virally-transformed cells.
