Identification of inhibitors of chromatin modifying enzymes using the yeast phenotypic screens.

A multitude of enzymes that modify histones and remodel nucleosomes are required for the formation, maintenance, and propagation of the transcriptionally repressed chromatin state in eukaryotes. Robust phenotypic screens in yeast S. cerevisiae have proved instrumental in identifying these activities and for providing mechanistic insights into epigenetic regulation. These phenotypic assays, amenable for high throughput small molecule screening, enable identification and characterization of inhibitors of chromatin modifying enzymes largely bypassing traditional biochemical approaches.

Unlocking the mechanisms of transcription factor YY1: are chromatin modifying enzymes the key?

The transcription factor YY1 is a complex protein that is involved in repressing and activating a diverse number of promoters. Numerous studies have attempted to understand how this one factor can act both as a repressor and an activator in such a wide set of different contexts. The fact that YY1 interacts with a number of key regulatory proteins (e.g. TBP, TFIIB, TAFII55, Sp1, and E1A) has suggested that these interactions are important for determining which particular function of YY1 is displayed at a specific promoter. Two groups of proteins, previously known to function as corepressors and coactivators, that now seem likely to modulate YY1's functions, are the histone deacetylases (HDAC) and histone acetyltransferases (HAT). These two groups of enzymes modify histones, and this modification is proposed to alter chromatin structure. Acetylated histones are typically localized to active chromatin while deacetylated histones colocalize with transcriptionally inactive chromatin. When these enzymes are directed to a promoter through a DNA binding factor such as YY1, that promoter can be activated or repressed. This review will discuss the recent work dealing with the different proteins that interact with YY1, with particular emphasis on ones that modify chromatin, and how they could be involved in regulating YY1's activities.

Purification and characterization of chicken erythrocyte histone deacetylase 1.

Histone acetylation is involved in nuclear processes requiring chromatin remodeling. In chicken erythrocytes, DNA replication has ceased, and active reversible histone acetylation is restricted to transcriptionally active/competent chromatin domains. In this study, we set out to identify and purify the erythroid histone deacetylase responsible for catalyzing dynamic acetylation of transcriptionally active chromatin. Histone deacetylase purified from chicken erythrocytes had a molecular mass of 66 kDa. Complementary DNA encoding the chicken histone deacetylase was cloned from erythrocytes, and analysis of the derived amino acid sequence showed the chicken histone deacetylase to be the chicken homologue of mammalian HDAC1. Purified chicken erythrocyte HDAC1 deacetylated the four core histones, with a preference for H3. We present evidence that chicken HDAC1 is a metalloenzyme, the activity of which is lost when incubated with zinc chelators. In Western blot analysis with anti-HDAC1 antibodies, we found that most erythrocyte HDAC1 is associated with the low-salt insoluble chromatin fraction and, to a lesser extent, with 150 mM NaCl-soluble oligo- and polynucleosomes. The distribution of HDAC1 in erythrocyte chromatin parallels that of dynamically acetylated class 1 histones. Further, we show that HDAC1 is associated with the erythroid nuclear matrix and that the enzyme is bound to nuclear DNA in situ. We propose that in addition to catalyzing dynamic acetylation of transcribed chromatin, the enzyme has a role in the organization of nuclear DNA.

DNaseY: a rat DNaseI-like gene coding for a constitutively expressed chromatin-bound endonuclease.

A rat gene, designated DNaseY, encoding a 36 kDa endonuclease was identified and cloned. Sequence analysis of the cDNA showed it to be the rat homologue of human DNAS1L3. The DNaseY gene product had 42% identity to DNaseI, including conserved critical active site residues, the essential disulfide bridge, the calcium binding domain, and a signal peptide, as well as 2 of the 3 signature boxes. Significantly, DNaseY had 2 nuclear localization signals and was more basic (pI 9.5) than DNaseI (pI 4.8). The DNaseY gene contained a number of exons similar to that of DNaseI, separated by much larger introns, resulting in a gene of >17 kb compared to <4 kb gene of DNaseI. The 36 kDa DNaseY gene product was catalytically inactive but was converted to an active 33 kDa endonuclease following processing of the hydrophobic signal peptide. Antibody generated against peptides representing the predicted amino acid sequence of DNaseY cross-reacted with a 33 kDa nuclear protein which possessed endonucleolytic activity. The enzyme was active over a broad pH range (optimum pH 7-8), was Ca2+/Mg2+-dependent, was inhibited by Zn2+, and was capable of both single- and double-stranded DNA cleavage, producing DNA fragments with 3'-OH ends. Furthermore, the DNaseY gene was expressed constitutively in all cells and tissues tested, but it was not transcriptionally up-regulated in apoptotic cells. All these features were consistent with a role in the early stages of apoptotic DNA fragmentation.

Molecular weights of the major DNA polymerases in a higher plant, Pisum sativum L. (PEA).

The molecular weights of the soluble (alpha-like) and chromatin-bound (beta-like) DNA polymerases of pea have been determined. The bulk of the soluble activity consists of molecular species of ca 101,500 and ca 140,000 molecular weights. Smaller (49,000) and larger (182,000 and 234,000) species are also observed in some preparations. The chromatin-bound DNA polymerase exhibits a molecular weight of ca 50,000, although a larger (ca 88,000) species is also detected under conditions which favour aggregate formation.