The role of H3K4me3 and H3K9/14ac in the induction by dexamethasone of Per1 and Sgk1, two glucocorticoid [corrected] early response genes that mediate the effects of acute stress in mammals.

Glucocorticoids are known to induce or repress the expression of a wide variety of genes with roles in various biological processes such as the circadian clock and the stress response. We studied the changes in the levels of two histone H3 post-translational modifications associated with active chromatin, H3 trimethylated at lysine 4 (H3K4me3) and H3 acetylated at lysines 9/14 (H3K9/14ac), that take place in the promoters of two glucocorticoid early response genes, Per1 and Sgk1, during their induction by the synthetic glucocorticoid, dexamethasone. Sgk1 mediates the effects of acute and chronic stress on the prefrontal cortex and other parts of the brain, while Per1 is a core circadian clock gene whose expression is strongly induced by the increased levels of blood-borne glucocorticoids that accompany acute and chronic stress. Here we show that dexamethasone rapidly increases the levels of H3K4me3 and H3K9/14ac in the promoters of both genes. Furthermore, the effect of dexamethasone on these genes, regarding both mRNA levels and the abundance of H3K4me3 and H3K9/14ac in their promoters, can be inhibited by the presence of nicotinamide, a metabolic molecule which has been shown to possess anxiolytic properties.

Nicotinamide treatment reduces the levels of histone H3K4 trimethylation in the promoter of the mper1 circadian clock gene and blocks the ability of dexamethasone to induce the acute response.

Circadian rhythms, which measure time on a scale of 24h, are generated by one of the most ubiquitous endogenous mechanisms, the circadian clock. SIRT1, a class III histone deacetylase, and PARP-1, a poly(ADP-ribose) polymerase, are two NAD(+)-dependent enzymes that have been shown to be involved in the regulation of the clock. Here we present evidence that the metabolite nicotinamide, an inhibitor of SIRT1, PARP-1 and mono(ADP-ribosyl) transferases, blocks the ability of dexamethasone to induce the acute response of the circadian clock gene, mper1, while it concomitantly reduces the levels of histone H3 trimethylation of lysine 4 (H3K4me3) in the mper1 promoter. Moreover, application of alternative inhibitors of SIRT1 and ADP-ribosylation did not lead to similar results. Therefore, inhibition of these enzymes does not seem to be the mode by which NAM exerts these effects. These results suggest the presence of a novel mechanism, not previously documented, by which NAM can alter gene expression levels via changes in the histone H3K4 trimethylation state.

mRNA levels of the linker histone variant, H1o, in mitotically active human diploid fibroblasts as a function of the phases of the cell cycle and cumulative population doublings.

Senescence and differentiation have many similarities with respect to certain aspects of gene expression and cell cycle related events. One linker histone variant tightly associated with differentiation is the H1 variant, H1o. The work of this investigation has focused on the expression of H1o during the phases of the cell cycle and as a function of increasing cumulative population doublings (CPD) in an in vitro model ageing cell system, namely, human diploid fibroblasts. Increased H1o mRNA levels were found during the S phase of the cell cycle contrary to H1o protein relative synthesis rates, which were found to be increased during the Go phase of the cell cycle. These results were obtained in actively proliferating cell populations. However when the proliferative rate of the overall population begins to drop (CPD 50), H1o mRNA levels tend to remain stable throughout the Go, G1 and S phases. On the other hand, no changes in the H1o relative synthesis rates were found as a function of increasing CPD. Uncoupling of H1o protein and mRNA levels has been observed in numerous differentiating systems. The analogous mode in which H1o gene expression is regulated in both these two systems reinforces the opinion that senescence and differentiation may have similarities at the level of chromatin remodelling.

Structure and expression of LeMA-1, a tomato protein belonging to the SEC18-PAS1-CDC48-TBP-1 protein family of putative Mg(2+)-dependent ATPases.

cDNA clones of a tomato protein, called Lycopersicum esculentum putative Mg(2+)-dependent ATPase (LeMA-1), were isolated from a cDNA library. Sequence comparison of the tomato protein with other genes in the database revealed that the protein is highly homologous to a human protein called TBP-1 and a yeast Tat-binding-analogue protein YTA1A. All three proteins belong to the recently discovered protein family of putative Mg(2+)-dependent ATPases and form within this family a subgroup of proteins involved in controlled protein degradation and possibly also in transcriptional regulation. Expression of the mRNA of LeMA-1 could be monitored in several plant tissues. LeMA-1 is the first member of this subgroup of proteins isolated from plants.

Chloroplast rps15 and the rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning non-chloroplast-encoded RNA polymerase.

Transcription of plastid genes and transcript accumulation were investigated in white leaves of the albostrians mutant of barley (Hordeum vulgare) and in heat-bleached leaves of rye (Secale cereale) as well as in normal green leaves of both species. Cells of white leaves of the mutant and cells of heat-bleached leaves bear undifferentiated plastids lacking ribosomes and, consequently, plastid translation products, among them the subunits of a putative chloroplast RNA polymerase encoded by the plastid genes rpoA, B, C1 and C2. The following results were obtained. (i) Plastid genes are transcribed despite the lack of chloroplast gene-encoded RNA polymerase subunits. The plastid origin of these transcripts was proven. This finding provides evidence for the existence of a plastid RNA polymerase encoded entirely by nuclear genes. (ii) Transcripts of the rpo genes and of rps15, but not of genes involved in photosynthesis and related processes (psbA, rbcL, atpI-H), were abundantly accumulated in ribosome-deficient plastids. In contrast, chloroplasts accumulated transcripts of photosynthetic, but not of the rpo genes. (iii) Differences in transcript accumulation between chloroplasts and ribosome-deficient plastids are due to different relative transcription rates and different transcript stability. (iv) The observed differences in transcription are not caused by an altered pattern of methylation of plastid DNA. Thus, the prokaryotic plastid genome of higher plants is transcribed by two RNA polymerases. The observed differences in transcription between chloroplasts and undifferentiated plastids might reflect different functions of the two enzymes.

Construction of a chloroplast DNA library from rye (Secale cereale), a cereal plant of temperature-inducible chloroplast ribosome deficiency.

The chloroplast genomes of flowering plants are circular DNA molecules, 120 to 160 kilobase pairs long, encoding the rRNA, all tRNAs, and 21 r-proteins of the chloroplast translational apparatus as well as key protein components of the photosynthetic and carbon reduction cycle reactions. In this paper we describe some characteristics of the rye chloroplast (plastid) genome and the construction and characterization of a clone library of 93% of its DNA in a plasmid and a cosmid vector. The size of rye chloroplast DNA is estimated at 135 kbp, similar to that for wheat and rice but slightly smaller than the estimate for maize (139 kbp). Chloroplast ribosome deficiency is induced in rye seedlings by germination and growth at 32 degrees-34 degrees C; therefore these clones would be useful for analyzing the regulation of chloroplast ribosome synthesis in higher plants, a process that requires coordinate expression of genes located in the nucleus and the chloroplast.

A new rearrangement of angiosperm chloroplast DNA in rye (Secale cereale) involving translocation and duplication of the ribosomal rpS15 gene.

The mapping and nucleotide sequencing of the rpS15 gene in the rye chloroplast DNA has shown that it is located in the inverted repeat (IR) and thus has two copies/genome. This is in contrast to tobacco and liverwort chloroplasts where rpS15 occurs as single-copy gene localized in the small single copy region (SSC). The direction of transcription of both gene copies in rye is toward SSC; that in tobacco and liverwort is toward IR-II. Further sequence data have revealed that the 3' end of each rye rpS15 gene copy is only 352 base pairs away from the corresponding IR.SSC junction and that this rearrangement event in rye involves also a 3' downstream-encoded and highly conserved chloroplast gene designated ORF393 in tobacco and ORF392 in liverwort. The latter in rye starts in both the IRs, but continues to full length into the SSC only from the IR-II. The direction of transcription of the nontruncated gene is fixed toward IR-I, being thus the inverse of ORF393/392 in tobacco and liverwort. Northern blot analysis has shown that the rearranged rpS15 gene is actively transcribed in rye chloroplasts and etioplasts and that its transcription pattern is different from that recently reported for tobacco rpS15.