Biomedical subjects

C Thiriet

Publications and source records attributed to C Thiriet.

10 recordsLinked to original sources

PubMed2001

A novel labeling technique reveals a function for histone H2A/H2B dimer tail domains in chromatin assembly in vivo.

During S phase in eukaryotes, assembly of chromatin on daughter strands is thought to be coupled to DNA replication. However, conflicting evidence exists concerning the role of the highly conserved core histone tail domains in this process. Here we present a novel in vivo labeling technique that was used to examine the role of the amino-terminal tails of the H2A/H2B dimer in replication-coupled assembly in live cells. Our results show that these domains are dispensable for nuclear import but at least one tail is required for replication-dependent, active assembly of H2A/H2B dimers into chromatin in vivo.

Animals↗

PubMed2001

Assembly into chromatin and subtype-specific transcriptional effects of exogenous linker histones directly introduced into a living Physarum cell.

The apparent diversity of linker histone subtypes may be related to their specific roles in defining functional states of chromatin in vivo. We have developed a novel method to study constitutive peptides throughout the cell cycle and have demonstrated that an exogenous linker histone could be introduced into a living cell of the slime mold Physarum polycephalum. Here, we have used this method to assess the functional differences between three somatic linker histone subtypes in vivo, and to demonstrate the general applicability of this method. Exogenous linker histone proteins H1 degrees, H5 and H1 were directly absorbed into living cell segments of the naturally synchronous Physarum macroplasmodia at precise cell cycle stages. Fluorescence microscopy, native nucleoprotein gels and immunoblotting of nuclei and chromatin with subtype-specific antibodies revealed that exogenous linker histones were efficiently transported into nuclei and were integrated into chromatin. The immunoreactivity of a preparation of anti-H1 degrees antibodies that are blocked from binding to specific H1 degrees epitopes in native chromatin indicates that the exogenous linker histones were similarly associated into Physarum chromatin. Interestingly, linker histones were found to be less stably associated with Physarum chromatin during S-phase than during G(2)-phase. Furthermore, we show that exogenous linker histones incorporated in early G(2)-phase inhibited transcription and that the level of inhibition correlates with the apparent role of the linker histone subtype in regulating transcription in cells where it normally occurs.

Animals↗

PubMed2000

DNA sequence-dependent contributions of core histone tails to nucleosome stability: differential effects of acetylation and proteolytic tail removal.

Modulation of nucleosome stability in chromatin plays an important role in eukaryotic gene expression. The core histone N-terminal tail domains are believed to modulate the stability of wrapping nucleosomal DNA and the stability of the chromatin filament. We analyzed the contribution of the tail domains to the stability of nucleosomes containing selected DNA sequences that are intrinsically straight, curved, flexible, or inflexible. We find that the presence of the histone tail domains stabilizes nucleosomes containing DNA sequences that are intrinsically straight or curved. However, the tails do not significantly contribute to the free energy of nucleosome formation with flexible DNA. Interestingly, hyperacetylation of the core histone tail domains does not recapitulate the effect of tail removal by limited proteolysis with regard to nucleosome stability. We find that acetylation of the tails has the same minor effect on nucleosome stability for all the selected DNA sequences. A comparison of histone partitioning between long donor chromatin, acceptor DNA, and free histones in solution shows that the core histone tails mediate internucleosomal interactions within an H1-depleted chromatin fiber amounting to an average free energy of about 1 kcal/mol. Thus, such interactions would be significant with regard to the free energies of sequence-dependent nucleosome positioning. Last, we analyzed the contribution of the H2A/H2B dimers to nucleosome stability. We find that the intact nucleosome is stabilized by 900 cal/mol by the presence of the dimers regardless of sequence. The biological implications of these observations are discussed.

Acetylation↗

PubMed2000

The H3-H4 N-terminal tail domains are the primary mediators of transcription factor IIIA access to 5S DNA within a nucleosome.

Reconstitution of a DNA fragment containing a Xenopus borealis somatic type 5S rRNA gene into a nucleosome greatly restricts the binding of transcription factor IIIA (TFIIIA) to its cognate DNA sequence within the internal promoter of the gene. Removal of all core histone tail domains by limited trypsin proteolysis or acetylation of the core histone tails significantly relieves this inhibition and allows TFIIIA to exhibit high-affinity binding to nucleosomal DNA. Since only a single tail or a subset of tails may be primarily responsible for this effect, we determined whether removal of the individual tail domains of the H2A-H2B dimer or the H3-H4 tetramer affects TFIIIA binding to its cognate DNA site within the 5S nucleosome in vitro. The results show that the tail domains of H3 and H4, but not those of H2A and/or H2B, directly modulate the ability of TFIIIA to bind nucleosomal DNA. In vitro transcription assays carried out with nucleosomal templates lacking individual tail domains show that transcription efficiency parallels the binding of TFIIIA. In addition, we show that the stoichiometry of core histones within the 5S DNA-core histone-TFIIIA triple complex is not changed upon TFIIIA association. Thus, TFIIIA binding occurs by displacement of H2A-H2B-DNA contacts but without complete loss of the dimer from the nucleoprotein complex. These data, coupled with previous reports (M. Vettese-Dadey, P. A. Grant, T. R. Hebbes, C. Crane-Robinson, C. D. Allis, and J. L. Workman, EMBO J. 15:2508-2518, 1996; L. Howe, T. A. Ranalli, C. D. Allis, and J. Ausio, J. Biol. Chem. 273:20693-20696, 1998), suggest that the H3/H4 tails are the primary arbiters of transcription factor access to intranucleosomal DNA.

Animals↗

PubMed1999

Histone proteins in vivo: cell-cycle-dependent physiological effects of exogenous linker histones incorporated into Physarum polycephalum.

We detail a method which allows biochemical quantities of histone proteins to be introduced into a living eukaryotic cell. This method involves absorption of purified proteins into macroplasmodia of Physarum polycephalum. Further, since Physarum macroplasmodia exist as syncitial culture with completely synchronous nuclei with respect to cell cycle events, proteins may be introduced at specific points during the eukaryotic cell cycle. We show that a linker histone is absorbed whole into these cells and are properly transported to the nuclei of the cell. Furthermore, we also show incorporation of linker histone H5 inhibits the transcriptional activities occurring during the G2 phase in Physarum. This method will make it possible to introduce histones modified with structural probes into chromatin naturally assembled in vivo.

Animals↗

PubMed1999

Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum.

Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed.

Animals↗

PubMed1999

[Patient knowledge of asthma: results of a national survey in pneumology].

Knowledge of asthma and its treatment were evaluated in a survey of 1,000 asthmatics using a self-questionnaire with closed questions. As far as the disease was concerned, 87.4% of patients knew that the bronchi were the pathological organ and 70.6% that the disease persisted between attacks. Overall knowledge of the disease (pathological organ, persistence of the disease between attacks, chronic inflammation associated with acute bronchoconstriction) was adequate in only 25.6% of patients. It was associated with the educational level [higher > primary; p = 0.001], the grade of asthma [severe > mild; p = 0.007] and the number of medicines inhaled [(n > 3) > (N < 2); p = 0.001)]. As far as medicines were concerned, knowledge of their mechanisms of action varied according to therapeutic groups: antiallergics [78.6%], antibiotics [77.7%], antihistamines [74.5%], LA beta2-mimetics [45.9%], theophylline [31.9%], corticosteroids [31.7%], beta2-mimetics [24.8%]. No factor was statistically correlated with greater familiarity. However, only 7.2% of patients treated with metered-dose aerosols expressed handling problems and 78.8% of asthmatics questioned felt that they were sufficiently informed about their disease and its treatment. Information and education thus remain a priority in the management of asthma.

Adolescent↗

PubMed1998

Functionally relevant histone-DNA interactions extend beyond the classically defined nucleosome core region.

We demonstrate that core histones can affect the accessibility of a DNA element positioned outside of the classically defined nucleosome core region. The distance between a well positioned nucleosome and the binding site for the 5 S-specific transcription factor TFIIIA was systematically varied and the relative binding affinity for TFIIIA determined. We found that core histone-DNA interactions attenuate the affinity of TFIIIA for its cognate DNA element by a factor of 50-100-fold even when the critical binding region lies well outside of the classically defined nucleosome core region. These results have implications for the validity of parallels drawn between the accessibility of general nucleases to DNA sequences in chromatin and the activity of actual sequence-specific DNA binding factors.

Animals↗

PubMed1997

Antisera directed against anti-histone H4 antibodies recognize linker histones. Novel immunological probes to detect histone interactions.

We introduce a novel immunological approach to detect structural interactions between chromosomal proteins. Antigenically pure core histone H4 was prepared from chicken erythrocytes and used to produce anti-histone H4 antisera. IgG fractions were isolated from purified anti-H4 antibodies and used as antigens to produce "second generation" antisera. Epitopes cross-reacting with the second generation antisera were then identified within chromosomal proteins. These epitopes were presumed to mimic the complementary molecular surface of the original anti-H4 antibodies, and thus proteins containing these epitopes were putatively identified as specific ligands of H4 in chromatin. Surprisingly, we found this immunoreactivity was predominantly directed against H1 compared with H5 from chicken erythrocytes. Further, the immunoreactive epitopes were located within the C-terminal tail domain of the linker histones. These results suggest similar complementary interactions occur between H4 and the C-terminal tail domain of H1s in native chromatin. This could occur either within a single nucleosome as suggested by a previous report (Banères, J.-L., Essalouh, L., Jariel-Encontre, I., Mesnier, D., Garrod, S., and Parello, J. (1994) J. Mol. Biol., 243, 48-59) or between neighboring nucleosomes within the condensed chromatin fiber. The implications of these results with regard to the structure of the chromatin fiber and the future utility of this technique are discussed.

Animals↗

PubMed1995

Rapid and effective western blotting of histones from acid-urea-Triton and sodium dodecyl sulfate polyacrylamide gels: two different approaches depending on the subsequent qualitative or quantitative analysis.

An improved method for the electrophoretic transfer of histones from sodium dodecyl sulfate (SDS) and acetic acid-urea-Triton X-100 (AUT) polyacrylamide gels onto nitrocellulose membranes is described. In the case of SDS-gels, it was not essential to equilibrate them before transfer while for the AUT-gels, an equilibration step is essential to prevent the interference of Triton X-100 with the binding of histones to nitrocellulose. Transfer efficiency was different for different histone classes. Two procedures were developed: (1) one suitable for qualitative studies, and (ii) another for quantitative transfer.

Acetates↗