Transcriptional regulation of seed-specific gene expression - from PvALF/ ABI3 to phaseolin

The phaseolin (phas) promoter drives the copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues when a nucleosome is positioned over its three phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis leaves was accomplished by ectopic expression of the transcription factor PvALF (Phaseolus vulgaris ABI3-like factor), and application of abscisic acid (ABA). PvALF belongs to a family of seed-specific transcriptional activators that includes the maize viviparious1 (VP1) and the Arabidopsis abscisic acid-insensitive3 (ABI3) proteins. The major goal of the study is to gain insight to the regulation of seed-specific gene expression in three different aspects. First, since ABI3 (homolog of PvALF) is involved in ABA-mediated expression of several seed-specific protein genes in Arabidopsis, understanding its transcriptional regulation will provide insight to the mechanism by which PvALF expression is controlled. To achieve this, ABI3 promoter deletion analysis using either $-glucuronidase (gus) or green fluorescent protein (gfp) reporter gene fusions have identified various regulatory regions within the ABI3 promoter including two upstream activating sequences and a minimal seed specific expression region. In addition, a 405 bp 5' UTR was shown to play a negative role in ABI3 expression, possibly through post-transcriptional mechanisms. Second, placement of PvALF expression under control of an estradiol-inducible promoter permitted chronological ChIP analysis of changes in histone modifications, notably increased acetylation of H3-K9, as phas chromatin is remodeled (potentiated). A different array of changes (trimethylation of H3-K4) is associated with ABA-mediated activation. In contrast, H3-K14 acetylation decreased upon phas potentiation and increased on activation. Whereas decreases in histone H3 and H4 levels were detected during PvALF-mediated remodeling, slight increases occurred following ABA-mediated activation, suggesting the restoration of histone-phas interactions or the redeposition of histones in the phas chromatin. The observed histone modifications thus provide insight to the factors involved in euchromatinization and activation of a plant gene. Finally, ectopically expressed ABI5 and PvALF renders the activation of phas ABA-independent, suggesting ABI5 acts downstream of ABA during phas activation

RNA synthesis and processing in isolated HeLa cell nuclei

Isolated HeLa cell nuclei have been characterised in terms of their ability to transcribe, process and transport RNA. In terms of transcription, it was found that all three RNA polymerases were active in the isolated nuclei. The size and nuclear location of the products of RNA polymerase I and III suggested that transcription by these polymerases was occurring normally in vitro. However, the RNA synthesised by RNA polymerase II was found to be much smaller than expected from the reported size of HeLa cell transcription units, when analysed in denaturing gradients. This was in contrast to the results of Sarma et al (1976) which showed that the size of RNA synthesised by RNA polymerase II in isolated HeLa cell nuclei is large when analysed under non-denaturing conditions. A number of possible reasons for this small size of RNA were examined. The results obtained indicate that this small size of RNA polymerase II product was probably not due to;- i. A slow elongation rate by RNA polymerase II resulting in a "nascent transcript profile." ii. Degradation of RNA in the isolated nuclei. iii. The absence of nuclear and cytoplasmic factors during incubation of the nuclei. iv. Degradation of the DM template during isolation and incubation of nuclei. It was found, however, that the state of the chromatin template was important in determining the size of RM transcribed. Thus, addition of acetyl CoA to isolated nuclei, which acetylated the histones, caused an increase in the size of RNA polymerase II product. On the other hand methylation of histones with Ado-Met vitro was correlated with a decrease in the size of RM. It was also found that the ionic content of the incubation medium affected the size of RNA transcript synthesised by RNA polymerase II. In particular, substituting 90 mM (NH4)2SO4 for 75 mM KCl in the incubation medium increased the size of RNA. This effect is discussed in terms of recent results which suggest that the small size of RNA polymerase II transcript commonly observed in vitro might be due to premature termination of transcription. The small RNA transcribed by RNA polymerase II in vitro appears to be stable. However, hnRNA prelabelled in vivo reduced in size during incubation of isolated nuclei. Some of this RNA is released from the nuclei during incubation. This release of RNA was examined to determine whether it represented the specific transport of mRNA. Although the size of released RNA particles, their ability, and the size of released RNA were consistent with mRNA transport, other features were more consistent with the leakage of hnRNP particles. The released RNA resembled hnRNA in terms of binding to poly(U) Sepharose, and the protein associated with the released RNA were similar to hnRHP particle proteins. Although the released RNA had messenger activitiy in a wheat germ cell free translation system, it is not possible to rule out mRNA contamination as the cause of this stimulation. It therefore appears that the isolated nuclei system of Sarma et al (1976) may not be ideal either for examining the transcription or the processing and transport of mRNA. On the other hand, the results obtained in the present study suggest ways in which this system might be modified in order to achieve full length transcription by RNA polymerase II in vitro, and study the processing and transport of these transcripts