C Gorka

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasburg, Alsace, France

Are you C Gorka?

Claim your profile

Publications (33)260.83 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: General remodeling of chromatin is associated with events determining cell fate and the expression of specific genetic programs (1,2). In almost every case there is a tight link between these chromatin remodeling events and a drastic modification of the cell cycle parameters. One of the most striking examples of this phenomenon is early embryonic development. Indeed, transition periods have been defined during development characterized by the modification of both chromatin constituents and the proliferative capacities of cells (3–6). For instance, during Xenopus laevis early development, the midblastula transition (MBT) is characterized as a period after which the somatic replication-dependent type of H1 starts to accumulate and the embryonic H1 (B4) and HMG1 decrease dramatically (7). Moreover, in vertebrates, later during development, other subtypes of H1 accumulate:H1o in almost all vertebrates, H1t in mammalian spermatogenic cells and H5 in avian erythrocytes, again associated with a drastic modification of proliferative capacities of cells (7). To better understand the significance of these transitions, it is important to correlate chromatin remodeling events with cell cycle modification events (8–10). For instance, we showed previously that during early Xenopus development, the type of H1 expressed appeared to be more related to the frequency of cell division than any other cellular event (6,7).
    Methods in molecular biology (Clifton, N.J.) 02/1999; 119:443-54. · 1.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Individual anti-H1(0) monoclonal antibodies were screened in an immunolocalization assay to isolate clones able to recognize H1(0) in a differentiation-dependent manner using a murine erythroleukemia cell line. Two clones were selected, one recognizing H1(0) only in differentiating cells (clone 27 antibody), and the other recognizing the protein constitutively (clone 34 antibody). Both antibodies recognized a restricted region of the protein located at the N-terminal part of the globular domain. Amino acids 24-30, essential for the recognition of the protein by the clone 27 antibody, are extremely conserved in all known H1(0)-like proteins from sea urchin to human. Within these residues, proline 26, responsible for a bend in this region, plays a particularly important role in the epitope recognition. The region involved in the protein recognition by clone 34 antibody is larger and encompasses amino acids 20-30. However, proline 26 does not play an essential role in the structure of this epitope. Detailed analysis of the differential recognition of H1(0) in chromatin during cell differentiation and proliferation suggests that the modification of chromatin structure as well as that of H1(0) conformation can account for this effect. Indeed, in vitro study of H1(0)-four-way junction DNA interaction showed that the N-terminal tail domain of the protein can influence the recognition of H1(0) by these antibodies when the protein interacts with DNA. The two monoclonal antibodies described here therefore seem to be valuable tools for investigating fine modulations in chromatin structure and the concomitant changes occurring in the conformation of the protein.
    Journal of Biological Chemistry 02/1998; 273(2):1208-15. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Histone H1(0), a differentiation-specific member of the histone H1 family, accumulates in cells during the terminal phase of cell differentiation, in tissues composed of arrested cells or cells exhibiting little proliferation. Moreover, the induction of cell proliferation in vivo, i.e., after partial hepatectomy, is accompanied by a decrease in H1(0) content. These observations suggest that H1(0) may be involved in the arrest of cell proliferation in vivo. In order to investigate this possibility, we took advantage of the fact that after partial hepatectomy the initiation of cell division is not synchronous. The strategy was to know, at the level of a single cell, whether H1(0) decreases prior to the initiation of the S phase or whether a cell can initiate DNA replication having a significant amount of H1(0) in the nucleus. We defined new protocols to analyze H1(0) content and cell proliferation at the level of a single cell, both in situ and by flow cytometry. The simultaneous determination of the relative amount of H1(0) and the position of cells in the cell cycle showed that no significant difference in H1(0) content was detected in cells actively replicating their DNA compared to nondividing cells. These observations have been confirmed by the successive immunodetections of H1(0) and BrdU in situ on the same cells. Therefore, we show here that in vivo, cells can initiate DNA replication with significant amounts of H1(0) and that the decrease of H1(0) is not a prerequisite of cell division. We propose that the accumulation of H1(0) is not related to the arrest of cell proliferation, but is controlled in such a manner that the protein accumulates in slowly dividing cells and decreases in rapidly growing cells.
    Experimental Cell Research 05/1995; 217(2):528-33. · 3.56 Impact Factor
  • C Gorka, S Fakan, J J Lawrence
    [Show abstract] [Hide abstract]
    ABSTRACT: The localization of histone H1(0) in murine erythroleukemia cells which were induced to resume a differentiation program was studied in cells which have recovered their proliferative capacity after transient blockage in the G1 phase of the cell cycle. Previous studies have shown that histone H1(0) accumulation occurs at early times of induction and is probably related to the commitment itself. The distribution of the protein was determined by immunomicroscopy with monoclonal antibodies specific for histone H1(0). Our observations showed that the histone accumulates in nuclei. Immunoelectron microscopy further demonstrated the presence of histone H1(0) in condensed chromatin areas, including perinucleolar chromatin. Moreover, histone H1(0) also occurred in the perichromatin regions, previously described as preferential sites of pre-mRNA synthesis, suggesting that histone H1(0) is not fully excluded from active chromatin.
    Experimental Cell Research 04/1993; 205(1):152-8. · 3.56 Impact Factor
  • Biology of the Cell 01/1993; 79(3):282. · 3.49 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Histone H1(0) accumulation is associated with the terminal stage of differentiation. Unlike other H1 histones, it is able to accumulate in the absence of DNA synthesis, however the transcription of its gene is cell-cycle dependent. The regulation of H1(0)-gene expression has been studied during the induced differentiation of B16 cells and during reversion of the process, which may be achieved when induced cells are released into an inducing-agent-free medium. During the earlier period of induced differentiation, H1(0) mRNA showed over-expression when the cells were still proliferating. Then the amount of H1(0) mRNA decreased as the cells became arrested in G0-G1. H1(0) mRNA half-life measurements and run-on experiments demonstrated that such modulation of the amount of mRNA originated from a transcriptional control of H1(0)-gene expression. When induced cells reverted to a proliferative undifferentiated state, H1(0) mRNA decreased very rapidly, indicating that an active process was involved in this decay. This behavior differed from that observed in rat liver hepatocytes allowed to proliferate and de-differentiate after partial hepatectomy, or in murine erythroleukemia cells when the inducing agent was removed from the culture.
    European Journal of Biochemistry 10/1992; 208(3):775-9. · 3.58 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Histone H10 accumulation is associated with the terminal stage of differentiation. Unlike other H1 histones, it is able to accumulate in the absence of DNA synthesis, however the transcription of its gene is cell-cycle dependent. The regulation of H10-gene expression has been studied during the induced differentiation of B16 cells and during reversion of the process, which may be achieved when induced cells are released into an inducing-agent-free medium. During the earlier period of induced differentiation, H10 mRNA showed over-expression when the cells were still proliferating. Then the amount of H10 mRNA decreased as the cells became arrested in G0-G1. H10 mRNA half-life measurements and run-on experiments demonstrated that such modulation of the amount of mRNA originated from a transcriptional control of H10-gene expression. When induced cells reverted to a proliferative undifferentiated state, H10 mRNA decreased very rapidly, indicating that an active process was involved in this decay. This behavior differed from that observed in rat liver hepatocytes allowed to proliferate and de-differentiate after partial hepatectomy, or in murine erythroleukemia cells when the inducing agent was removed from the culture.
    European Journal of Biochemistry - EUR J BIOCHEM. 01/1992; 208(3):775-779.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We have studied the variation of histone H10 and of its coding mRNA during rat liver regeneration after partial hepatectomy. Our data showed that while H10 decreased when cell proliferation was initiated, H10 mRNA accumulated in a proliferation-dependent manner as did H3 mRNA. These results showed two interesting aspects of the regulation of H10 expression in vivo, confirming results we have obtained previously in vitro: first H10 mRNA accumulation is a proliferation-dependent event; second, H10 protein accumulation may be uncoupled from that of its coding mRNA.
    FEBS Letters 06/1991; 283(1):65-7. · 3.58 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Histone H1(0) is one of the potential candidates that may contribute to the onset and stabilization of a genetic program during induced differentiation of murine erythroleukemia cells. In an attempt to understand better the role of H1(0) in this process we have tried to determine at which level the regulation of its induced accumulation occurs. Protein H1(0) was found to increase by a factor of 3 while its mRNA increased by a factor of 14, due to activation of gene transcription. As shown by H1(0) half-life measurements, the difference between the actual amount of H1(0) and that expected from the amount of mRNA was not due to increased turnover of the protein. Fractionation of the translational apparatus at several times during induction, revealed that H1(0) mRNA was efficiently transferred to the high molecular weight polysomes. The rate of synthesis of H1(0) was also increased by a factor of 4. Taken together, these results suggest the existence of a strong control at the translational level, which regulates H1(0) accumulation.
    Journal of Molecular Biology 02/1991; 217(1):85-92. · 3.91 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Monoclonal antibodies (mAb) to ox liver histone H1 degree were produced and characterized. Two sets of mice were immunized either with pure H1(0) or with an H1(0)-yeast tRNA complex. Eleven hybridomas of various clonal origin were selected. Typing of the antibodies indicated that all but three IgM belonged to the IgG1 class and contained kappa light chains. Immunoblotting experiments using peptides derived from H1(0) or H5 treated by various proteolytic agents (trypsin, N-bromosuccinimide, cyanogen bromide, acetic acid), revealed that nine of the mAb reacted with the globular part of H1(0). More advanced characterization of the antigenic determinants allowed us to determine distinct regions within this globular part which are involved in the antigenic recognition. The peptopes could be subdivided into two groups. Three mAb bound to residues 24-27 and were specific for H1(0). Six mAb bound to residues 27-30 and were specific for H1(0) except one of them which strongly cross-reacted with H5 and GH5. Two mAb reacted with the entire histone H1(0) but failed to react with any of the peptides, suggesting that the corresponding epitope is a conformational antigenic determinant. In order to confirm the localization of the two distinct regions which are involved in the antigenic recognition, a synthetic decapeptide corresponding to the beginning of human H1(0) globular part (from residue 19 to residue 28) was synthesized. Inhibition experiments of the reaction between H1(0) and the various IgG1 mAb by increasing amounts of peptide-bovine serum albumin conjugates were then performed.
    European Journal of Immunology 07/1989; 19(6):1123-9. · 4.97 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Interactions of mammalian histones, H1-1 and H1(0), phi 0 from holothuria sperm and H5 with poly(dA-dT), poly(dG-dC) and poly(dG-me5dC) were measured by a nitrocellulose filter binding assay and circular dichroism. All of the proteins bound to every one of the polymers, but differed in the extent of binding, which depended on the polynucleotide/protein ratios and ionic strength. The order of retention of all polymers was phi 0 greater than H1-1 greater than H1(0). The binding of H1(0) to poly(dG-me5dC) was remarkably sensitive to ionic strength. The proteins caused changes in the spectral features of the polynucleotides, but differed in the type and extent of the change. Complexes prepared with H1-1 and H1(0) with all polymers showed a strongly negative psi spectrum. Complexes of poly(dA-dT) and phi 0, at a protein/polynucleotide ratio of 0.4, displayed a distinctive spectrum, giving the appearance of a Z-like DNA spectrum, at low ionic strength. At higher ionic strength the complexes showed a psi spectrum. Complexes of poly(dG-me5dC) in the Z or B conformation with phi 0 showed spectral features characteristic of a mixture of a Z-like and a psi spectrum. In contrast, H5 reduced the Z-DNA spectral features in the presence of Mg, and produced an inversion of the B spectrum up to a polynucleotide/protein ratio of 0.24. These findings demonstrate the ability of different proteins to produce changes in the conformation of DNA. This may reflect the ability of chromatin to undergo differential condensation, depending on both the base composition of DNA and the type of H1 histone bound to it.
    Biophysical Chemistry 10/1988; 31(3):275-86. · 2.28 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Interactions of mammalian histones, H1-1 and H1°, Φo from holothuria sperm and H5 with poly(dA-dT), poly(dG-dC) and poly(dG-me5dC) were measured by a nitrocellulose filter binding assay and circular dichroism. All of the proteins bound to every one of the polymers, but differed in the extent of binding, which depended on the polynucleotide/protein ratios and ionic strength. The order of retention of all polymers was Φo > H1-1 > H1°. The binding of H1° to poly(dG-me5dC) was remarkably sensitive to ionic strength. The proteins caused changes in the spectral features of the polynucleotides, but differed in the type and extent of the change. Complexes prepared with H1-1 and H1° with all polymers showed a strongly negative ψ, spectrum. Complexes of poly(dA-dT) and Φo, at a protein/polynucleotide ratio of 0.4, displayed a distinctive spectrum, giving the appearance of a Z-Iike DNA spectrum, at low ionic strength. At higher ionic strength the complexes showed a ψ spectrum. Complexes of poly(dG-me5dC) in the Z or B conformation with φo showed spectral features characteristic of a mixture of a Z-Iike and a ψ spectrum. In contrast, H5 reduced the Z-DNA spectral features in the presence of Mg, and produced an inversion of the B spectrum up to a polynucleotide/ protein ratio of 0.24. These findings demonstrate the ability of different proteins to produce changes in the conformation of DNA. This may reflect the ability of chromatin to undergo differential condensation, depending on both the base composition of DNA and the type of H1 histone bound to it.
    Biophysical Chemistry 09/1988; 31(3):275-286. · 2.28 Impact Factor
  • C Marion, J Roche, B Roux, C Gorka
    [Show abstract] [Hide abstract]
    ABSTRACT: The effectiveness of histone H1 subfractions H1-1 and H1(0) in inducing the ordered condensation of chromatin was examined by thermal denaturation, circular dichroism, electric birefringence, orientation mechanism, and orientational relaxation time measurements. Soluble rat liver chromatin was stripped of H1 by dissociation in 500 mM NaCl and long fragments of chromatin were subsequently reassociated with purified individual H1 subfractions for ratios of 1 and 2 mol of H1 per nucleosome. H1 subfractions behave differently with respect to their interactions with DNA in chromatin: although the orientation mechanisms of reconstituted chromatins are identical, H1(0) induces a less efficient protection of DNA than H1-1, as shown by nuclease digestion and by the length of free extended linker DNA determined by electric birefringence. This corresponds to a more extended structure of H1(0)-reconstituted chromatin as judged by the value of relaxation time. One can imagine that the replacement of H1 by H1(0) leads to a different structure or stability of the chromatin, confering a certain degree of flexibility of this region. This may be related to the functional role of H1(0) in DNA replication or transcription and may explain metabolic and evolutionary differences among H1 subfractions as recently suggested by Lennox [Lennox, R. W. (1984) J. Biol. Chem. 259, 669-672]. The extent of condensation when H1-depleted chromatin is overloaded with histones is probably a function of the electrostatic interactions between the basic C-terminal tails of histones and chromatin. Electric birefringence also reveals differences between native and reconstituted chromatins that are overlooked by several other criteria.
    Biochemistry 12/1985; 24(23):6328-35. · 3.38 Impact Factor
  • Biochemistry 11/1985; 24(23):6328-6335. · 3.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Micrococcal nuclease digestion and light scattering are used to compare native chromatins with various histone H1[0] contents. The experimental data show that the higher the H1[0] content, the greater the ability to form compact structures with increasing ionic strength, and the lower the DNA accessibility to micrococcal nuclease. On the contrary, reconstituted samples from H1-depleted chromatin and pure individual H1 fractions behave in such a way that samples reconstituted with pure H1 degree give rise to a looser structure, more accessible to nuclease than samples reconstituted with H1-1. This contradiction suggests that the effect of H1o on chromatin structure must originate from the interaction of this histone with other components in native chromatin among which other histone H1 subfractions are good candidates.
    Nucleic Acids Research 05/1985; 13(8):2843-53. · 8.81 Impact Factor
  • Roche J, Gorka C, Lawrence JJ
    Nature 01/1985; · 38.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Histone H1(0) has a number of unusual properties that set it apart from other H1 subtypes (for review see ref. 1). For example, H1(0) synthesis is not strictly coupled to DNA synthesis, it is absent from the embryonic liver of mice (but present shortly after birth) and its synthesis is hormone-dependent in some of the glands of adult rodents. All the H1 subtypes differ in their DNA binding properties, and H1(0) has been shown to be preferentially associated with nuclease-resistant chromatin. These features suggest that the H1(0) may have a role in developmental gene control. To investigate this further, we have fractionated the H1(0)-containing nucleosomes of chromatin from adult mouse liver. We report here that the gene for alpha-fetoprotein, which is expressed in embryonic tissue but repressed soon after birth, is preferentially associated with the H1(0)-containing nucleosomes. The related gene for albumin, which is expressed in both embryonic and adult tissues, is absent from the H1(0)-containing nucleosome fraction. These results support a role for histone H1(0) in the control of gene expression.
    Nature 01/1985; 314(6007):197-8. · 38.60 Impact Factor
  • Roche J, Gorka C, Lawrence JJ
    Nature. 01/1985;
  • [Show abstract] [Hide abstract]
    ABSTRACT: Histone H5 contains three tyrosines in the central, apolar region of the molecule. All three tyrosines can be spin labeled at low ionic strength. When the central globular domain is folded at high ionic strength, only one tyrosine becomes accessible to the imidazole spin label. Spin labeling the buried tyrosines prevents the folding of the globular structure, which, in turn, affects the proper binding of the H5 molecule to stripped chromatin. Chromatin complexes reconstituted from such an extensively modified H5 molecule show a weaker protection of the 168 base pair chromatosome during nuclease digestion. However, when only the surface tyrosine of the H5 molecule is labeled, such a molecule can still bind correctly to stripped chromatin, yielding a complex very similar to that of native chromatin. Our data supports the idea that not just the presence of the linker histone H5, but the presence of an intact H5 molecule with a folded, globular central domain in essential in the recognition of its specific binding sites on the nucleosomes. Our data also show that during the chromatin condensation process, the tumbling environment of the spin label attached to the surface tyrosine in the H5 molecule is not greatly hindered but remains partially mobile. This suggests that either the labeled domain of the H5 molecule is not directly involved in the condensation process or the formation of the higher-order chromatin structure does not result is a more viscous or tighter environment around the spin label. The folded globular domain of H5 molecule serves in stabilizing the nucleosome structure, as well as the higher-order chromatin structure.
    Journal of biomolecular Structure & Dynamics 11/1984; 2(2):319-32. · 2.98 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Rat liver chromatin is stripped of H1 histone by exposure to 0.5 M NaCl and reassociated with individual purified subfractions of H1 by salt-step dialysis. The effectiveness of proteins H1-1 and H1 in the condensation of DNA is monitored by transient electric birefringence and circular dichroism. Steady-state birefringence and relaxation time measurements show that reconstitutions are not perfect although some features of native chromatin are restored when a ratio of 2 moles of H1 per nucleosome is used. The amplitude of the positive birefringence is better recovered with H1-1 than with H1 but the values of relaxation times and molar ellipticities indicate that reconstituted samples exhibit a more compact and rigid structure compared to that of native chromatin.
    Biochemical and Biophysical Research Communications 07/1984; 121(2):530-7. · 2.28 Impact Factor

Publication Stats

410 Citations
260.83 Total Impact Points

Institutions

  • 1984–1999
    • Institut de Génétique et de Biologie Moléculaire et Cellulaire
      Strasburg, Alsace, France
    • Claude Bernard University Lyon 1
      Villeurbanne, Rhône-Alpes, France
  • 1989–1992
    • Unité Inserm U1077
      Caen, Lower Normandy, France