Chromatin RNA Polymerase Research Articles

SPT5 stabilizes RNA polymerase II, orchestrates transcription cycles, and maintains the enhancer landscape

Transcription progression is governed by multitasking regulators including SPT5, an evolutionarily conserved factor implicated in virtually all transcriptional steps from enhancer activation to termination. Here we utilize a rapid degradation system and reveal crucial functions of SPT5 in maintaining cellular and chromatin RNA polymerase II (Pol II) levels. Rapid SPT5 depletion causes a pronounced reduction of paused Pol II at promoters and enhancers, distinct from negative elongation factor (NELF) degradation resulting in short-distance paused Pol II redistribution. Most genes exhibit downregulation, but not upregulation, accompanied by greatly impaired transcription activation, altered chromatin landscape at enhancers, and severe Pol II processivity defects at gene bodies. Phosphorylation of an SPT5 linker at serine 666 potentiates pause release and is antagonized by Integrator-PP2A (INTAC) targeting SPT5 and Pol II, while phosphorylation of the SPT5 C-terminal region links to 3' end termination. Our findings position SPT5 as an essential positive regulator of global transcription.

Two species of chromatin-RNA polymerase II complex are commonly present in nuclei of various tissues of rats.

When rat liver nuclei were digested with nuclease, we found that the chromatin-bound RNA polymerase II was liberated as two distinct complexes, peak 1 and peak 2, which seemed to reflect different functional states in cell nuclei. We further examined their occurrence in nuclear digests of various tissues of rats and the following results were obtained. Upon digestion with micrococcal nuclease of nuclei from brain, spleen, testis and kidney, chromatin-bound RNA polymerase II was liberated as two distinct forms which sedimented differently in a sucrose density gradient. The sedimentation rate of peak 1 varied depending on the tissue nuclei examined. After high salt or RNase treatment of the nuclear digests, peak 1 from liver, brain, spleen and testis nuclei showed the same sedimentation rate as did kidney peak 1, the rate for which remained unchanged by these treatments. The results suggested that peak 1 complexes from various tissue nuclei had basically the same structural organization, and we confirmed this by electrophoretic studies on RNase-treated liver and kidney nuclear digests. Peak 2 from various tissue nuclei exhibited identical sedimentation rates. Thus, the chromatin-bound RNA polymerase II seems to exist commonly in two distinct states in cell nuclei of rats.

Two species of chromatin-RNA polymerase II complex released from rat liver nuclei by nuclease digestion

To investigate the structural organization of transcribing chromatin, rat liver nuclei were washed thoroughly to remove the free RNA polymerase and then digested with micrococcal nuclease or DNAase I. After extensive digestion with the nuclease in the presence of 0.1 to 0.25 m m-divalent cation, two species of the chromatin-RNA polymerase II complexes were separated by subsequent chromatography on a column of Bio-Gel A-15m (peak 1 and peak 2). The peak 1 complexes, recovered at the oligonucleosomal region, were RNA polymerase II-nucleosome monomer or dimer complexes. These complexes were further characterized by the possession of factors that enhanced chromatin transcription, and seemed to associate with ribonucleoprotein particles. The peak 2 complexes, eluted at the position of purified RNA polymerase II molecules, contained endogenous template, but were apparently not organized in the nucleosomal structure. The RNA polymerase II molecules in both complexes were found to be associated with nascent RNA synthesized in vivo. When the digestion with micrococcal nuclease was performed in the presence of 3 m m-MgCl 2, peak 2 was recovered as a relatively large chromatin (S 2-chromatin), while peak 1 was not extracted and remained in the residual chromatin (P 2-chromatin). Analysis of RNA transcripts synthesized from these chromatins indicated that the two forms of the enzyme transcribed different DNA sequences, and both of the products were found in the polysomal poly(A) + messenger RNA. These lines of evidence suggest that there are separate RNA polymerase II transcriptional systems, which transcribe different DNA sequences of the genome.

Chromatin RNA polymerase activity from soybean hypocotyls treated with gibberellic acid and AMO-1618

Chromatin isolated from control, AMO-1618 [2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine carboxylate] and gibberellic acid (GA) treated soybean hypocotyl tissue incorporates labeled nucleoside triphosphates into acid-insoluble RNA. Gibberellic acid, sprayed on intact soybean hypocotyls, is shown to have enhanced the level of chromatin RNA polymerase activity while chromatin isolated from hypocotyls pretreated with AMO-1618 exhibits a lower polymerase activity relative to the control. Chromatin extracted from the treated or untreated seedlings are all sensitive to the inhibition (in varying degrees) by the presence of actinomycin D, pyrophosphate, or ribonuclease. Thus enhanced (or decreased) RNA-synthesizing capacity of chromatin in response to chemical treatments may be due to enhanced (or decreased) synthesis of RNA polymerase.

Growth and RNA synthesis in maize seedlings treated with ethyl methanesulfonate

Corn seeds were treated with the chemical mutagen ethyl methanesulfonate and the resulting seedlings were examined for growth and RNA synthesis. Ethyl methanesulfonate treatments reduced shoot elongation, seedling growth, and chromatin-directed RNA polymerase activity of shoot tissues. The chromatin RNA polymerase activity of control seedlings peaked at 24 h. This peak was delayed up to 12 h by the chemical mutagen treatment. Saturation of embryonic axis chromatin with E. coli RNA polymerase indicated that ethyl methanesulfonate increased the amount of DNA available as template. In both the treated and control axes, the available template decreased with tissue differentiation.

Enhancement by 2,4-dichlorophenoxyacetic acid of chromatin RNA polymerase in soybean hypocotyl tissue

Chromatin isolated from control and 2,4-dichlorophenoxyacetic acid-treated soybean hypocotyl tissue incorporates labeled nucleoside triphosphates into acid-insoluble RNA. Chromatin RNA polymerase activity of soybeans depends on the presence of all 4 nucleoside triphosphates, Mg 2+ and Mn 2+. RNA synthesis is inhibited by pyrophosphate and actinomycin D. The product of the reaction is completely sensitive to ribonuclease. Chromatin preparations from 2,4-dichlorophenoxyacetic acid-treated plants have a higher polymerase activity than control chromatin. RNA polymerase of chromatin preparations shows no response to 2,4-dichlorophenoxyacetic acid added during incubation. RNA polymerase from Escherichia coli greatly augments RNA synthesis of chromatin preparations from control and 2,4-dichlorophenoxyacetic acid-treated plants, but both chromatin preparations exhibit similar saturation curves with added E. coli polymerase. Comparison of the RNA products of chromatin preparations from control and 2,4-dichlorophenoxyacetic acid-treated plants on methylated albumin kieselguhr columns reveals a change in the RNA product.

Hormonal effect on nucleolar RNA polymerase in normal liver and hepatoma cells

DNA-dependent RNA polymerase was found not only in chromatin fraction but also in nucleolar fraction of normal rat liver cells as well as rat ascites hepatoma cells. Nucleolar RNA polymerase was more sensitive to the administration of steroid hormone than chromatin RNA polymerase. In hepatoma cells, chromatin RNA polymerase was rather insensitive to the steroid hormone. The analytical results of in vitro synthesized RNA by nucleolar RNA polymerase did not show ribosomal precursor but only 6S RNA.