Requirement of an E1A-sensitive coactivator for long-range transactivation by the beta-globin locus control region.

Four erythroid-specific DNase I-hypersensitive sites at the 5'-end of the beta-globin locus confer high-level transcription to the beta-globin genes. To identify coactivators that mediate long-range transactivation by this locus control region (LCR), we assessed the influence of E1A, an inhibitor of the CBP/p300 histone acetylase, on LCR function. E1A strongly inhibited transactivation of Agamma- and beta-globin promoters by the HS2, HS2-HS3, and HS1-HS4 subregions of the LCR in human K562 and mouse erythroleukemia cells. Short- and long-range transactivation mediated by the LCR were equally sensitive to E1A. The E1A sensitivity was apparent in transient and stable transfection assays, and E1A inhibited expression of the endogenous gamma-globin genes. Only sites for NF-E2 within HS2 were required for E1A sensitivity in K562 cells, and E1A abolished transactivation mediated by the activation domain of NF-E2. E1A mutants defective in CBP/p300 binding only weakly inhibited HS2-mediated transactivation, whereas a mutant defective in retinoblastoma protein binding strongly inhibited transactivation. Expression of CBP/p300 potentiated HS2-mediated transactivation. Moreover, expression of GAL4-CBP strongly increased transactivation of a reporter containing HS2 with a GAL4 site substituted for the NF-E2 sites. Thus, we propose that a CBP/p300-containing coactivator complex is the E1A-sensitive factor important for LCR function.

Enhancement of beta-globin locus control region-mediated transactivation by mitogen-activated protein kinases through stochastic and graded mechanisms.

Activation of the mitogen-activated protein kinase (MAPK) pathway enhances long-range transactivation by the beta-globin locus control region (LCR) (W. K. Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sci. USA 95:8756-8760, 1998). The enhancement requires tandem recognition sites for the hematopoietic transcription factor NF-E2 within the hypersensitive site 2 (HS2) subregion of the LCR. To distinguish between mechanisms of induction involving the activation of silent promoters or the increased efficacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhancer activity in single, living cells. K562 erythroleukemia cells stably transfected with constructs containing the human Agamma-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without HS2, were analyzed for EGFP expression by flow cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoters. However, under conditions of silencing, in which cells reverted to a state with no measurable EGFP expression, MAPK activated silent promoters. Furthermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted nonexpressing cells into expressing cells and increased expression in expressing cells. These results support a model in which MAPK elicits both graded and stochastic responses to increase HS2-mediated transactivation from single chromatin templates.

[Effect of diets with different carbohydrate/fat ratios on the cytochrome P-450 content and monooxygenase activity of rat liver]. / Vliianie ratsionov s razlichnym sootnosheniem uglevodov i zhirov na soderzhanie tsitokhroma P-450 i aktivnost' monooksigenaznoi sistemy pecheni krysy.

Wistar male rats kept on high-protein (36% protein) diets for 30 days showed an increase in the content of cytochrome P-450 and b5 in liver microsomes, which was accompanied by an increase in the rate of amidopyrine and aniline oxidation. The degree of induction of the monooxygenase system of the liver endoplasmic reticulum with phenobarbital (100 mg/kg bw daily for 4 days) in rats which received high-protein diets was lower than in control and depended on the carbohydrate/fat ratio in the diet.

[Effect of diets containing plant protein on liver mono-oxygenase activity in rats]. / Vliianie ratsionov s rastitel'nym belkom na aktivnost' monooksigenaznoi sistemy pecheni krys.

Male Wistar rats kept for 30 days on the diet containing vegetative (wheat) protein demonstrate an increase in cytochrome P-450 concentration in liver microsomes and an increase in the rate of amidopyrine N-demethylation and aniline p-hydroxylation. Administration of phenobarbital leads to cytochrome P-450 induction and activation of monooxygenase of rat liver microsomes, the quality of food protein not affecting cytochrome induction. It is assumed that high content of cytochrome P-450 is linked with prolongation of the time of cytochrome P-450 renewal at the expense of the decreased constant of its protein part degradation or with activation of cytochrome synthesis de novo (based on the preliminary data).

[Effect of protein on the induction and renewal of a cytochrome P-450 complex in the rat liver]. / Vliianie belka na induktsiiu i obnovlenie kompleksa tsitokhroma P450 pecheni krys.

It was disclosed that the effect on the content of cytochrome P450 and renewal of the protein portion of the cytochrome P450 complex in rat liver microsomes is determined by the protein amount in the diet. Protein deficiency in the diet leads to the reduced rate of protein fraction renewal in the cytochrome P450 complex. The degree of cytochrome P450 induction with phenobarbital was found to be dependent on the diet. In the animals fed low-protein diet (4.5%), the degree of cytochrome P450 induction was decreased which was likely to be a consequence of the increased semi-renewal period.

[Isolation and characteristics of 2 transcribing families of pigeon-genome repetitive sequences]. / Vydelenie i kharakteristika dvukh transkribiruiushchikhsia semeistv povtoriaiushchikhsia posledovatel'nostei genoma golubia.

From pigeon DNA two families of repeats have been isolated: one (frequency of repetitions 30--40 times per haploid genome) actively transcribing and the second (frequency of repetitions 2000--2800 times) weakly transcribing in erythroid cells. The reassociation kinetics, GC-content and size of isolated repeats were investigated. Actively transcribing repeats (average length of 350 nucleotides) contain about 55% GC-nucleotides and alternate in the genome with AT-rich sequences of the unique type. Comparison of isolated families of repeats revealed differences in the structure and posttranscriptional fate of RNA transcribed from them.