Dimitar Angelov

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.

Preferential interaction of the core histone tail domains with linker DNA

Within chromatin, the core histone tail domains play critical roles in regulating the structure and accessibility of nucleosomal DNA within the chromatin fiber. Thus, many nuclear processes are facilitated by concomitant posttranslational modification of these domains. However, elucidation of the mechanisms by which the tails mediate such processes awaits definition of tail interactions within chromatin. In this study we have investigated the primary DNA target of the majority of the tails in mononucleosomes. The results clearly show that the tails bind preferentially to “linker” DNA, outside of the DNA encompassed by the nucleosome core. These results have important implications for models of tail function within the chromatin fiber and for in vitro structural and functional studies using nucleosome core particles.

DETERMINATION OF PARAMETERS OF EXCITED STATES OF DNA and RNA BASES BY LASER UV PHOTOLYSIS

Abstract— The mechanism of photodecomposition of nucleic acid bases in a neutral aqueous solution upon two‐step excitation of high‐lying electronic states by a powerful laser UV radiation is discussed. Experimental dependences of photodecomposition efficiency versus UV radiation intensity are measured both under picosecond and nanosecond laser UV irradiations. By comparison of experimental dependences with a theoretical model, we obtain some characteristics of excited states, such as lifetime t1 of the first electronic excited state S1 intersystem crossing yield φ, photosensitivity from an intermediate excited state and others for all five nucleic acid bases.

The Histone Octamer Is Invisible When NF-κB Binds to the Nucleosome

The transcription factor NF-κB is involved in the transcriptional control of more than 150 genes, but the way it acts at the level of nucleosomal templates is not known. Here we report on a study examining the interaction of NF-κB p50 with its DNA recognition sequence in a positioned nucleosome. We demonstrate that NF-κB p50 was able to bind to the nucleosome with an apparent association constant close to that for free DNA. In agreement with this, the affinity of NF-κB p50 binding does not depend on the localization of its recognition sequence relative to the nucleosome dyad axis. In addition, the binding of NF-κB p50 does not induce eviction of histones and does not perturb the overall structure of the nucleosome. The NF-κB p50-nucleosome complex exhibits, however, local structural alterations within the NF-κB p50 recognition site. Importantly, these alterations were very similar to those found in the NF-κB p50-DNA complex. Our data suggest that NF-κB p50 can accommodate the distorted, bent DNA within the nucleosome. This peculiar property of NF-κB p50 might have evolved to meet the requirements for its function as a central switch for stress responses.

Involvement of retinoblastoma protein and HBP1 in histone H1(0) gene expression.

ABSTRACT The histone H10-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H10 promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H10 gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H10 H4 box were therefore expected to link differentiation-dependent expression of H10 to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H10H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H10 gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H10, HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells.

The NH2 tail of the novel histone variant H2BFWT exhibits properties distinct from conventional H2B with respect to the assembly of mitotic chromosomes.

ABSTRACT We have studied the functional and structural properties of nucleosomes reconstituted with H2BFWT, a recently identified putative histone variant of the H2B family with totally unknown function. We show that H2BFWT can replace the conventional histone H2B in the nucleosome. The presence of H2BFWT did not affect the overall structure of the nucleosome, and the H2BFWT nucleosomes exhibited the same stability as conventional nucleosomes. SWI/SNF was able to efficiently remodel and mobilize the H2BFWT nucleosomes. Importantly, H2BFWT, in contrast to conventional H2B, was unable to recruit chromosome condensation factors and to participate in the assembly of mitotic chromosomes. This was determined by the highly divergent (compared to conventional H2B) NH2 tail of H2BFWT. These data, in combination with the observations that H2BFWT was found by others in the sperm nuclei and appeared to be associated with the telomeric chromatin, suggest that H2BFWT could act as a specific epigenetic marker.

HYDRATED ELECTRON FORMATION IN NANOSECOND and PICOSECOND LASER FLASH PHOTOLYSIS OF HEMATOPORPHYRIN IN AQUEOUS SOLUTION

Nanosecond (λcxc= 266, 355 and 532 nm) and picosecond (λcxc. = 355 nm) laser flash photolysis of hematoporphyrin (Hp) was performed in neutral (pH 7.4) and alkaline (pH 12) aqueous solution, as well as in the presence of 0.1% Triton X‐100. The dependence of the yield of photoproduced hydrated electrons (e−aq) on laser pulse energy was studied over a wide range of energies (0.2 to >1000 mJ cm−2). The results show that e˜, are predominantly formed in a two‐photon process at λexc= 266 and 355 nm. One‐photon quantum yields are higher at λexc= 266 nm than at λexc= 355 nm. Both one‐photon and two‐photon pathways are less efficient at higher Hp concentration, reflecting the influence of Hp self‐aggregation. Two‐photon eaq formation is more efficient when 30 ps pulses are used for excitation, as compared to 10 ns pulses. No e−aq could be detected at λexc= 532 nm. Nanosecond pulse‐induced transient spectra obtained at pH 7.4 are also discussed.

Binding of NF-κB to nucleosomes: effect of translational positioning, nucleosome remodeling and linker histone H1.

NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A–H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes.

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA.

COMPARISON OF THE EFFECTS OF HIGH-POWER U.V.-LASER PULSES AND IONIZING RADIATION ON NUCLEIC ACIDS AND RELATED COMPOUNDS

Abstract The photoinduced effects on nucleic acids and some of their components by high-power laser are compared to those observed under the influence of high energy irradiation (electrons and γ-rays). Similarities of the action of both kinds of radiation and the resulting transients are discussed on the basis of some model compounds.