Nikolai D. Belyaev

Differential underacetylation of histones H2A, H3 and H4 on the inactive X chromosome in human female cells

It has previously been shown that the acetylated forms of histone H4 are depleted or absent in both constitutive, centric heterochromatin and in the facultative heterochromatin of the inactive X chromosome (Xi) in female cells. By immunostaining of metaphase chromosomes from human lymphocytes with antibodies to the acetylated isoforms of histones H2A and H3, we now show that these histones too are underacetylated in both Xi and centric heterochromatin. Xi shows two prominent regions of residual H3 acetylation, one encompassing the pseudoautosomal region at the end of the short arm and one at about Xg22. Both these regions have been shown previously to be sites of residual H4 acetylation. H2A acetylation on Xi is higher overall than that of H3 or H4 and is particularly high around the pseudoautosomal region, but not at Xg22. The results suggest that the acetylated isoforms of H3 and H4 have at least some effects on chromosomal structure and function that are not shared by acetylated H2A.

Stage-dependent redistributions of acetylated histones in nuclei of the early preimplantation mouse embryo

In the preimplantation mouse embryo, activation of the embryonic genome is accompanied by a transient enrichment of histone H4 acetylated at lysines 5, 8, and 12 at the nuclear periphery (Worrad et al., 1995: Development 121:2949–2959). In the present report, we use laser‐scanning confocal microscopy and a new panel of antibodies to define the distribution of specific acetylated isoforms of the other three core histones in mouse embryos at the 1‐ to 4‐cell stage. We find that histone H3 acetylated at lysine 9 and/or 18 (H3.Ac9/18) and the single acetylated form of H2A (H2A.Ac5) become transiently enriched at the nuclear periphery in the 2‐cell embryo. In contrast, H3.Ac14, H3.Ac23, and acetylated H2B, like H4.Ac16, remain distributed throughout the nucleoplasm. The staining intensity with antisera to H3.Ac9/18, even at the periphery was weak compared to that obtained with antisera to acetylated H4. A brief period of culture, however, in the presence of the inhibitor of histone deacetylases trichostatin A (TSA) or trapoxin increased labeling. Thus, the steady‐state level of H3.Ac9/18 at the nuclear periphery and H3.Ac14 and H3.Ac23 in the nuclear interior is relatively low, but turnover remains high. The localization of selected acetylated isoforms of H3 and H2A at the nuclear periphery was independent of ongoing transcription or of cytokinesis, but did require DNA replication. We propose a model in which the selective, replication‐dependent acetylation and deacetylation of zygotic chromatin at the nuclear periphery mediates the programming of zygotic transcription. Mol. Reprod. Dev. 47:421–429, 1997.

Histone H4 acetylation in plant heterochromatin is altered during the cell cycle

Abstract.Using polyclonal antibodies directed against acetylated isoforms of histone H4 (H4 acetylated at lysine positions 5, 8, 12, 16 and H4 tetraacetylated), indirect immunofluorescence revealed hyperacetylation for all H4 variants at the nucleolus organizer region (NOR) of metaphase chromosomes of the field bean Vicia faba. The transcriptionally inactive and late-replicating heterochromatin regions proved to be hypoacetylated at lysine positions 5, 8 and 12. The remaining chromatin showed average fluorescence. These patterns were altered when deacetylase was blocked by exposure of root tip meristems to trichostatin A for more than 2 h prior to fixation. Under these conditions, all lysine positions, except lysine 8, appeared to be hyperacetylated at the NOR and in addition at the prominent heterochromatin domains. This observation represents a hitherto unique switch of histone acetylation pattern during the cell cycle. This is apparently caused by deposition of acetylated H4.Ac5, 12 and 16 or by acetylation directly after replication, which later on becomes reduced (H4.Ac16) or even reversed (H4.Ac5 and 12) by deacetylase before cells enter mitosis.

Differential immunostaining of plant chromosomes by antibodies recognizing acetylated histone H4 variants

Metaphase chromosomes ofVicia faba were exposed to antibodies recognizing defined acetylated isoforms of histone H4. After indirect immunostaining with antibodies directed against H4 acetylated on lysines 5, 8 and 12 respectively, the entire chromosome complement was labelled. The brightest signal appeared at the nucleolus organizing region (NOR). The large genetically inert heterochromatic regions, which are composed of late replicating tandemly repetitive DNA sequences, remained unlabelled. Thus, the chromosomal distribution of histones H4 acetylated at positions of lysine 5, 8 and 12 is broadly correlated with the intensity of transcription and the sequence of replication of the field bean chromatin during interphase. Antibodies against H4 acetylated at lysine 16 also caused a strong signal at the NOR but otherwise a uniform fluorescence along the chromosome.

Differences of histone H4 acetylation and replication timing between A and B chromosomes of Brachycome dichromosomatica

Differences are demonstrated between A (transcriptionally active) and B (transcriptionally inactive) chromosomes that are characterized by a different level of histone H4 acetylation and a different timing of DNA replication. These differences between the chromatin of A and B chromosomes were found after immunolabelling of chromsomes of Brachycome dichromosomatica with antibodies specific for different acetylated forms (lysine 5, 8, 12 and 16) of histone H4. In contrast to the A chromosomes, which are labelled brightly in their entirety, the transcriptionally inactive B chromosomes are faintly labelled with antibodies against H4Ac5 and H4Ac8. No such difference between the chromosomes is found after immunostaining with the other antibodies H4Ac12 and H4Ac16. Analysis of DNA replication timing in root-tip meristems suggests that B chromosomes are labelled late in S-phase compared with A chromosomes. After C-banding the B chromosome appeared to have a similar amount of heterochromatin to the A chromosomes.

Cell cycle-dependent and lysine residue-specific dynamic changes of histone H4 acetylation in barley.

Histone acetylation affects chromatin conformation and regulates various cellular functions, such as transcription and cell cycle progression. Although mitosis dependent transcriptional silencing and large-scale chromatin structural changes are well established, acetylation of histone H4 during the mitosis is poorly understood in plants. Here, the dynamics of acetylation of histone H4 in defined genome regions has been examined in the fixed barley cells throughout the mitosis by three-dimensional microscopy. Patterns of strong acetylation of the two lysine residues K5 and K16 of histone H4 in the barley genomes were found to be different. In interphase nuclei, H4 acetylated at K16 was associated with the gene-rich, telomere-associated hemispheres, whereas K5 acetylation was detected in centromeric regions where the heterochromatin is distributed. Regions of strong K5 acetylation changed dynamically as the cell cycle proceeded. At prometaphase, centromeric acetylation at K5 decreased suddenly, with accompanying rapid increases of acetylation in the nucleolar organizing regions (NORs). Reverse changes occurred at telophase. On the other hand, the strongly acetylated regions of the K16 showed changes compatible with transcriptional activities and chromosome condensation throughout the cell cycle. Telomeric acetylation at K16 was detected throughout the cell cycle, although it was reduced at metaphase which corresponds to the most condensed stage of the chromosomes. It is concluded that dynamic changes in H4 acetylation occur in a lysine residue-, stage-, and region-specific manner and that they correlate with changes in the chromosome structure through the cell cycle.

The acetylation patterns of histones H3 and H4 along Vicia faba chromosomes are different

The acetylation pattern of H3 was studied on field bean chromosomes by means of indirect immunofluorescence using polyclonal antibodies recognizing H3 isoforms acetylated at lysine positions 9/18, 14 and 23. H3 was found to be hypoacetylated at lysine residues 9/18 and 14 within the heterochromatic regions composed of tandem repetitive Fok-I elements. Hyperacetylation of these residues was observed at the nucleolar organizing region (NOR) and in heterochromatic regions composed of repeats other than Fok-I elements. In contrast, H4 was underacetylated (H4.Ac5, 8, 12) or uniformly acetylated (H4.Ac16) at all heterochromatic regions, and acetylated above the average at all four lysines only within the NOR. Acetylation of lysine-23 of H3 was uniform, except for the NOR that showed no fluorescence. Inhibition of deacetylase during and after replication of heterochromatin by trichostatin A had no influence on the acetylation status of H3 but mediated an increase in acetylation of lysines 5, 12 and 16 of H4 above the average in the field bean heterochromatin. Thus, the chromosomal acetylation patterns of H4 and H3 of this species revealed common and divergent features. Whereas the acetylation level of H4 correlates well with the potential transcriptional activity and inversely with the time of replication of defined chromatin domains of Vicia faba, this is not generally true for H3.

Association between the cholesteryl ester transfer protein TaqI-detectable B polymorphism and low high-density lipoprotein cholesterol concentration in Saudis.

Plasma concentrations of HDL (high-density lipoprotein) cholesterol are low in the Saudi Arabian population. A B polymorphism at the CETP (cholesteryl ester protein transfer) locus that is detectable with the restriction enzyme Taq I is a genetic determinant of the plasma HDL cholesterol concentration. We assessed the relationship between the Taq I B CETP polymorphism and lipid and apolipoprotein concentrations in a study sample of 335 Saudi residents. The Taq I B1 and B2 allele frequencies were 0.54 and 0.46 respectively, similar to those in other populations. HDL cholesterol levels in B2B2 homozygotes were significantly higher than in B1B1 homozygotes [1.01 (0.3) compared with 0.92 (0.2) mmol/l; mean (S.D.); P=0.03]. There was also a significant difference between the B2B2 and B1B1 homozygotes with regard to apolipoprotein AI concentration [123.6 (16.4) compared with 113.7 (13.9) mg/dl; P=0.04]. This genetic variation was independent of metabolic risk factors known to influence HDL cholesterol levels. The allele frequency of the Taq I B CETP polymorphism and its relatively modest impact on HDL cholesterol concentrations argue against an important role for this allele, or for strongly linked loci, in determining the low levels of HDL cholesterol seen in the Saudi population.

Comparative analysis of topographic distribution of acetylated histone H4 by using confocal microscopy and a deconvolution system

Recent data demonstrate the close links between histone acetylation and basic cell processes. The spatial distribution of acetylated histone H4 was studied using two three-dimensional microscopical systems, confocal microscopy and a deconvolution system. Both systems provide satisfactory three-dimensional digital images. Acetylation of histone H4 at lysine 5 occurred throughout the whole nucleus except for the nucleolus, and highly acetylated regions clustered close to the surface of the nucleus. Internal details of these regions, invisible by confocal microscopy, were revealed by the deconvolution system. On the other hand, the deconvolution system failed to detect scattered nuclear signals which were clearly observed by the confocal microscopy. Deconvolution of the images obtained by confocal microscopy made the image a little clearer, but was not particularly useful.