Vladimir A. Bondarenko

Nucleosome Remodeling Induced by RNA Polymerase II: Loss of the H2A/H2B Dimer during Transcription

RNA polymerase II (Pol II) must transcribe genes in a chromatin environment in vivo. We examined transcription by Pol II through nucleosome cores in vitro. At physiological and lower ionic strengths, a mononucleosome imposes a strong block to elongation, which is relieved at increased ionic strength. Passage of Pol II causes a quantitative loss of one H2A/H2B dimer but does not alter the location of the nucleosome. In contrast, bacteriophage SP6 RNA polymerase (RNAP) efficiently transcribes through the same nucleosome under physiological conditions, and the histone octamer is transferred behind SP6 RNAP. Thus, the mechanisms for transcription through the nucleosome by Pol II and SP6 RNAP are clearly different. Moreover, Pol II leaves behind an imprint of disrupted chromatin structure.

Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes

Remodeling machines play an essential role in the control of gene expression, but how their activity is regulated is not known. Here we report that the nuclear protein nucleolin possesses a histone chaperone activity and that this factor greatly enhances the activity of the chromatin remodeling machineries SWI/SNF and ACF. Interestingly, nucleolin is able to induce the remodeling by SWI/SNF of macroH2A, but not of H2ABbd nucleosomes, which are otherwise resistant to remodeling. This new histone chaperone promotes the destabilization of the histone octamer, helping the dissociation of a H2A–H2B dimer, and stimulates the SWI/SNF‐mediated transfer of H2A–H2B dimers. Furthermore, nucleolin facilitates transcription through the nucleosome, which is reminiscent of the activity of the FACT complex. This work defines new functions for histone chaperones in chromatin remodeling and regulation of transcription and explains how nucleolin could act on transcription.

Mechanism of Polymerase II Transcription Repression by the Histone Variant macroH2A

ABSTRACT macroH2A (mH2A) is an unusual histone variant consisting of a histone H2A-like domain fused to a large nonhistone region. In this work, we show that histone mH2A represses p300- and Gal4-VP16-dependent polymerase II transcription, and we have dissected the mechanism by which this repression is realized. The repressive effect of mH2A is observed at the level of initiation but not at elongation of transcription, and mH2A interferes with p300-dependent histone acetylation. The nonhistone region of mH2A is responsible for both the repression of initiation of transcription and the inhibition of histone acetylation. In addition, the presence of this domain of mH2A within the nucleosome is able to block nucleosome remodeling and sliding of the histone octamer to neighboring DNA segments by the remodelers SWI/SNF and ACF. These data unambiguously identify mH2A as a strong transcriptional repressor and show that the repressive effect of mH2A is realized on at least two different transcription activation chromatin-dependent pathways: histone acetylation and nucleosome remodeling.

SWI/SNF remodeling and p300-dependent transcription of histone variant H2ABbd nucleosomal arrays

A histone variant H2ABbd was recently identified, but its function is totally unknown. Here we have studied the structural and functional properties of nucleosome and nucleosomal arrays reconstituted with this histone variant. We show that H2ABbd can replace the conventional H2A in the nucleosome, but this replacement results in alterations of the nucleosomal structure. The remodeling complexes SWI/SNF and ACF are unable to mobilize the variant H2ABbd nucleosome. However, SWI/SNF was able to increase restriction enzyme access to the variant nucleosome and assist the transfer of variant H2ABbd–H2B dimer to a tetrameric histone H3–H4 particle. In addition, the p300‐ and Gal4‐VP16‐activated transcription appeared to be more efficient for H2ABbd nucleosomal arrays than for conventional H2A arrays. The intriguing mechanisms by which H2ABbd affects both nucleosome remodeling and transcription are discussed.

DNA supercoiling allows enhancer action over a large distance

Enhancers are regulatory DNA elements that can activate their genomic targets over a large distance. The mechanism of enhancer action over large distance is unknown. Activation of the glnAp2 promoter by NtrC-dependent enhancer in Escherichia coli was analyzed by using a purified system supporting multiple-round transcription in vitro. The data suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2,500 bp) but not over a short distance (110 bp). DNA supercoiling facilitates functional enhancer–promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.

Rationally designed insulator‐like elements can block enhancer action in vitro

Insulators are DNA sequences that are likely to be involved in formation of chromatin domains, functional units of gene expression in eukaryotes. Insulators can form domain boundaries and block inappropriate action of regulatory elements (such as transcriptional enhancers) in eukaryotic nuclei. Using an in vitro system supporting enhancer action over a large distance, the enhancer‐blocking insulator activity has been recapitulated in a highly purified system. The insulator‐like element was constructed using a sequence‐specific DNA‐binding protein making stable DNA loops (lac repressor). The insulation was entirely dependent on formation of a DNA loop that topologically isolates the enhancer from the promoter. This rationally designed, inducible insulator‐like element recapitulates many key properties of eukaryotic insulators observed in vivo. The data suggest novel mechanisms of enhancer and insulator action.

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase I. IDENTIFICATION OF THE KINASE AND ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN VITRO

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pγ, the regulatory subunit of PDE, and GTP/Tα, the GTP-bound α subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016–15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024–15029), we showed that Pγ is phosphorylated by a previously unknown kinase (Pγ kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pγ loses its ability to interact with GTP/Tα, but gains a 10–15 times higher ability to inhibit GTP/Tα-activated PDE than that of nonphosphorylated Pγ. Thus, we propose that the Pγ phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Tα-activated PDE. Here we demonstrate that all known Pγs preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pγ kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pγ indicates that all known Pγs contain a P-X-T-P-R sequence and that this sequence is required for the Pγ phosphorylation by Pγ kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pγ kinase activity exactly coelutes with Cdk5 and p35. The Pγ kinase activity (∼85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pγ. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pγ in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pγ complexed with GTP/Tα in mature vertebrate retinal photoreceptors.

A Possible Role of RGS9 in Phototransduction A BRIDGE BETWEEN THE cGMP-PHOSPHODIESTERASE SYSTEM AND THE GUANYLYL CYCLASE SYSTEM

In the current concept of phototransduction, the concentration of cGMP in retinal rod outer segments is controlled by the balance of two enzyme activities: cGMP phosphodiesterase (PDE) and guanylyl cyclase (GC). However, no protein directly mediates these two enzyme systems. Here we show that RGS9, which is suggested to control PDE activity through regulation of transducin GTPase activity (He, W., Cowan, C. W., and Wensel, T. G. (1998) Neuron 20, 95–102), directly interacts with GC. When proteins in the Triton X-100-insoluble fraction of bovine rod outer segments were isolated by two-dimensional gel electrophoresis and binding of GC to these proteins was examined using a GC-specific antibody, proteins (55 and 32 kDa) were found to interact with GC. However, the activity of GC bound to the 55-kDa protein was not detected. This observation was elucidated by the finding that the 55-kDa protein inhibited GC activity in a dose-dependent manner. Amino acid sequence showed that five peptides derived from the 55-kDa protein were identical to corresponding peptides of RGS9. Together with other biochemical characterization of the 55-kDa protein, these observations indicate that the 55-kDa protein is RGS9 and that RGS9 inhibits GC. RGS9 may serve as a mediator between the PDE and GC systems.

Chromatin structure can strongly facilitate enhancer action over a distance

Numerous DNA transactions in eukaryotic nuclei are regulated by elements (enhancers) that can directly interact with their targets over large regions of DNA organized into chromatin. The mechanisms allowing communication over a distance in chromatin are unknown. We have established an experimental system allowing quantitative analysis of the impact of chromatin structure on distant transcriptional regulation. Assembly of relaxed or linear DNA templates into subsaturated chromatin results in a strong increase of the efficiency of distant enhancer–promoter E–P communication and activation of transcription. The effect is directly proportional to the efficiency of chromatin assembly and cannot be explained only by DNA compaction. Transcription activation on chromatin templates is enhancer- and activator-dependent, and must be accompanied by direct E–P interaction and formation of a chromatin loop. Previously we have shown that DNA supercoiling can strongly facilitate E–P communication on histone-free DNA. The effects of chromatin assembly and DNA supercoiling on the communication are quantitatively similar, but the efficiency of enhancer action in subsaturated chromatin does not depend on the level of unconstrained DNA supercoiling. Thus chromatin structure per se can support highly efficient communication over a distance and functionally mimic the supercoiled state characteristic for prokaryotic DNA.

Binding of cGMP to GAF Domains in Amphibian Rod Photoreceptor cGMP Phosphodiesterase (PDE) IDENTIFICATION OF GAF DOMAINS IN PDE αβ SUBUNITS AND DISTINCT DOMAINS IN THE PDE γ SUBUNIT INVOLVED IN STIMULATION OF cGMP BINDING TO GAF DOMAINS

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Pαβ) and two identical regulatory subunits (Pγ). Biochemical studies have shown that amphibian Pαβ has high affinity, cGMP-specific, non-catalytic binding sites and that Pγ stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pγ-depleted membrane-bound PDE (20–40% Pγ still present), a single type of cGMP-binding site with a relatively low affinity (K d ∼ 100 nm) was observed, and addition of Pγ increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pγ reduced its ability to stimulate cGMP binding. Among these, the site involved in Pγ phosphorylation by Cdk5 (positions 20–23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pγ inhibition of the cGMP hydrolytic activity of Pαβ. In addition, the Pγ concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Pαβ heterodimer contains two spatially and functionally distinct types of Pγ-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Pαβ-Pγ interaction in which Pγ, by binding to one of the two sites in Pαβ, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.