Histone Assembly Research Articles

Histone Structures: Targets for Modifications by Molecular Assemblies

The core histone proteins contain modification sites that are key elements in the regulation of the cell cycle, DNA replication and repair with histone assembly, control of gene expression, and transcriptional elongation. Much work has been done on the various molecular assemblies that remodel nucleosomes, methylate, ubiquitinate, and cause ADP-ribosylation of histones, and acetylate and phosphorylate core histone tails. The core histones are the final targets of the enzymes in the molecular assemblies. What structural changes in the histones are correlated with these modifications? This paper considers the high-resolution structure of the histone octamer and stresses the importance of histone docking sequences in the binding of the two (H2A-H2B) dimers to the (H3-H4)(2) tetramer. There is an extensive acid-base area of interaction between histone octamers in crystals at high salt, which may have implications for nucleosome remodeling. We show that there are regions of high alpha-helix probability in all core histone N-terminal tails in regions where lysine acetylation occurs. There are also consensus sequences spanning up to eight amino acid residues between some histone tail regions. Circular dichroism studies using synchrotron radiation at wavelengths as low as 130 nm are promising for the accurate measurement of changes of histone secondary structure related to function.

Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex

Sir2 is an NAD-dependent histone deacetylase required for transcriptional silencing. To study the mechanism of Sir2 function, we examined the biochemical properties of purified recombinant Drosophila Sir2 (dSir2). First, we performed histone deacetylation assays and found that dSir2 deacetylates a broad range of acetylated lysine residues. We then carried out in vitro transcription experiments and observed that dSir2 does not repress transcription with either naked DNA templates or chromatin assembled from native (and mostly unacetylated) histones. It was possible, however, that repression by dSir2 requires an acetylated histone substrate. We therefore tested the transcriptional effects of dSir2 with native histones that were hyperacetylated by treatment with acetic anhydride. Assembly of the hyperacetylated histones onto DNA yields a soluble histone-DNA complex that differs from canonical nucleosomal chromatin. With this hyperacetylated histone-DNA complex, we observed potent (50- to 100-fold) NAD-dependent transcriptional repression by purified dSir2. In contrast, repression by dSir2 was not observed in parallel experiments in which histones were hyperpropionylated with propionic anhydride. We also found that dSir2 mediates the formation of a nuclease-resistant fast-sedimenting histone-DNA complex in an NAD-dependent manner. Unlike dSir2, the dHDAC1 deacetylase does not strongly repress transcription or generate a nuclease-resistant histone-DNA complex. Furthermore, with yeast Sir2, the transcriptional repression we observe correlates with deacetylation activity in vitro and silencing activity in vivo. These findings suggest that deacetylation by Sir2 causes a conformational change or rearrangement of histones into a transcriptionally repressive chromatin structure.

Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks.

The cytotoxic T lymphocyte protease granzyme A (GzmA) initiates a novel caspase-independent cell death pathway characterized by single-stranded DNA nicking. The previously identified GzmA substrate SET is in a multimeric 270-420-kDa endoplasmic reticulum-associated complex that also contains the tumor suppressor protein pp32. GzmA cleaved the nucleosome assembly protein SET after Lys(176) and disrupted its nucleosome assembly activity. The purified SET complex required only GzmA to reconstitute single-stranded DNA nicking in isolated nuclei. DNA nicking occurred independently of caspase activation. The SET complex contains a 25-kDa Mg(2+)-dependent nuclease that degrades calf thymus DNA and plasmid DNA. Thus, GzmA activates a DNase (GzmA-activated DNase) within the SET complex to produce a novel form of DNA damage during cytotoxic T lymphocyte-mediated death.

ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly.

The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI-containing factor, and NAP-1, a core histone chaperone, in an ATP-dependent process. We describe the isolation of Drosophila acf1 cDNA, which encodes the p170 and p185 forms of the Acf1 protein in ACF. Acf1 is a novel protein that contains two PHD fingers, one bromodomain, and two new conserved regions. Human WSTF, which is encoded by one of multiple genes that is deleted in Williams syndrome individuals, is the only currently known mammalian protein with each of the conserved motifs in Acf1. Purification of the native form of Acf1 led to the isolation of ACF comprising Acf1 (both p170 and p185 forms) and ISWI. Native Acf1 did not copurify with components of NURF or CHRAC, which are other ISWI-containing complexes in Drosophila. Purified recombinant ACF, consisting of Acf1 (either p185 alone or both p170 and p185) and ISWI, catalyzes the deposition of histones into extended periodic nucleosome arrays. Notably, the Acf1 and ISWI subunits function synergistically in the assembly of chromatin. ISWI alone exhibits a weak activity that is approximately 3% that of ACF. These results indicate that both Acf1 and ISWI participate in the chromatin assembly process and suggest further that the Acf1 subunit confers additional functionality to the general 'motor' activity of ISWI.

Cytoplasmic factors that control nuclear behavior in mammalian oocytes: A re‐evaluation of studies performed as a student of Yoshio Masui

Studies performed by the author in the laboratory of Dr Yoshio Masui are reviewed and interpreted in the light of subsequent findings. The first series of studies indicated that that chromosome condensation during meiotic maturation of mouse oocytes is controlled initially by a stable protein that decays during maturation and subsequently by an unstable protein synthesized after germinal vesicle breakdown. Cyclin B is present in immature oocytes, becomes partially degraded near metaphase I and then re-accumulates, suggesting that this may be protein whose activity was inferred from the original results. The second series of experiments indicated that factors which appear in the oocyte cytoplasm during maturation are able to remodel the sperm into metaphase-like chromosomes, and that the supply of these factors is limited. Recent work indicates that these factors are required for the assembly of histones onto the sperm DNA, and has identified two molecular species, mNAP-1 and NPM-3, known to promote replication-independent chromatin assembly in somatic cells, that are expressed in oocytes.

Cytoplasmic factors that control nuclear behavior in mammalian oocytes: A re-evaluation of studies performed as a student of Yoshio Masui

Studies performed by the author in the laboratory of Dr Yoshio Masui are reviewed and interpreted in the light of subsequent findings. The first series of studies indicated that that chromosome condensation during meiotic maturation of mouse oocytes is controlled initially by a stable protein that decays during maturation and subsequently by an unstable protein synthesized after germinal vesicle breakdown. Cyclin B is present in immature oocytes, becomes partially degraded near metaphase I and then re-accumulates, suggesting that this may be protein whose activity was inferred from the original results. The second series of experiments indicated that factors which appear in the oocyte cytoplasm during maturation are able to remodel the sperm into metaphase-like chromosomes, and that the supply of these factors is limited. Recent work indicates that these factors are required for the assembly of histones onto the sperm DNA, and has identified two molecular species, mNAP-1 and NPM-3, known to promote replication-independent chromatin assembly in somatic cells, that are expressed in oocytes.

Differential Silver-Staining Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis: A Nonisotopic Method for Characterizing Gel-Separated Histone–DNA Complexes

Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2and separated by SDS–PAGE. Differential detection was accomplished using two different silver staining protocols that preferentially stained either proteins or nucleic acids. A technique was developed which allows the relative proportion of linker histones and DNAs in each detected band to be determined, and is referred to as differential staining SDS–PAGE (DS-SDS–PAGE). DS-SDS–PAGE provides a novel, non-isotopic means for characterizing multiple nucleoprotein bands separated by polyacrylamide gel electrophoresis. In applying this method to a model linker histone–DNA study, we were able to detect both protein-DNA and protein-protein contacts that are important in linker histone assembly onto DNA.

Histone acetylation in chromatin structure and transcription.

'The amino termini of histones extend from the nucleosomal core and are modified by acetyltransferases and deacetylases during the cell cycle. These acetylation patterns may direct histone assembly and help regulate the unfolding and activity of genes.

The ability to organize sperm DNA into functional chromatin is acquired during meiotic maturation in murine oocytes

Following fertilization of meiotically mature eggs, the chromatin of the sperm becomes biochemically and structurally remodeled within the egg cytoplasm. Despite the essential role of the paternal genome during embryogenesis, little is known of when the activities that regulate this chromatin remodeling appear during oogenesis. To determine whether these activities were acquired during meiotic maturation, we inseminated maturing oocytes of mice shortly after germinal vesicle breakdown. As previously shown, insemination at this stage did not activate the maturing oocytes, which became arrested at metaphase II. Immunofluorescent analysis revealed that at 1 hr postinsemination the sperm chromatin was dispersed and contained protamines but was devoid of core histones H2B and H3. At 4 hr postinsemination, both protamine and core histones were detectable on the sperm chromatin. By 8 hr postinsemination protamines were absent, and histones stained maximally. The appearance of immunoreactive histones was correlated with a morphological transition of the sperm chromatin from the dispersed to a condensed state, which suggests that the assembly of the histones reflected modification of the chromatin to a somatic-like state in which it was competent to respond to the metaphase-promoting factor activity of the oocyte. Both the assembly of histones and chromatin condensation were reversibly blocked when protein synthesis was inhibited, indicating that the remodeling process required proteins synthesized during maturation. Injection of core histones into protein synthesis-inhibited oocytes failed to induce condensation of the sperm chromatin, which implies that correct remodeling requires synthesis during maturation of nonhistone proteins. To test the functional capacity of remodeled sperm chromatin, maturing oocytes were inseminated, allowed to continue maturation for 17 hr and then parthenogenetically activated. Following activation, the sperm-derived chromatin as well as that of the oocyte became decondensed within pronuclei and underwent DNA replication, indicating that sperm chromatin remodeled in maturing oocyte cytoplasm was functionally normal. When the postinsemination incubation time was reduced to 11 hr; however, neither the female nor the male pronuclei underwent DNA replication, implying that factors synthesized late during maturation are required for DNA replication after activation. Taken together, these results indicate that the ability to organize sperm DNA into functional somatic-like chromatin develops in oocytes during meiotic maturation, requires proteins synthesized during maturation, and can be expressed independently of activation.

The ability to organize sperm DNA into functional chromatin is acquired during meiotic maturation in murine oocytes

Following fertilization of meiotically mature eggs, the chromatin of the sperm becomes biochemically and structurally remodeled within the egg cytoplasm. Despite the essential role of the paternal genome during embryogenesis, little is known of when the activities that regulate this chromatin remodeling appear during oogenesis. To determine whether these activities were acquired during meiotic maturation, we inseminated maturing oocytes of mice shortly after germinal vesicle breakdown. As previously shown, insemination at this stage did not activate the maturing oocytes, which became arrested at metaphase II. Immunofluorescent analysis revealed that at 1 hr postinsemination the sperm chromatin was dispersed and contained protamines but was devoid of core histones H2B and H3. At 4 hr postinsemination, both protamine and core histones were detectable on the sperm chromatin. By 8 hr postinsemination protamines were absent, and histones stained maximally. The appearance of immunoreactive histones was correlated with a morphological transition of the sperm chromatin from the dispersed to a condensed state, which suggests that the assembly of the histones reflected modification of the chromatin to a somatic-like state in which it was competent to respond to the metaphase-promoting factor activity of the oocyte. Both the assembly of histones and chromatin condensation were reversibly blocked when protein synthesis was inhibited, indicating that the remodeling process required proteins synthesized during maturation. Injection of core histones into protein synthesis-inhibited oocytes failed to induce condensation of the sperm chromatin, which implies that correct remodeling requires synthesis during maturation of nonhistone proteins. To test the functional capacity of remodeled sperm chromatin, maturing oocytes were inseminated, allowed to continue maturation for 17 hr and then parthenogenetically activated. Following activation, the sperm-derived chromatin as well as that of the oocyte became decondensed within pronuclei and underwent DNA replication, indicating that sperm chromatin remodeled in maturing oocyte cytoplasm was functionally normal. When the postinsemination incubation time was reduced to 11 hr; however, neither the female nor the male pronuclei underwent DNA replication, implying that factors synthesized late during maturation are required for DNA replication after activation. Taken together, these results indicate that the ability to organize sperm DNA into functional somatic-like chromatin develops in oocytes during meiotic maturation, requires proteins synthesized during maturation, and can be expressed independently of activation.

Histone acetylation: influence on transcription, nucleosome mobility and positioning, and linker histone-dependent transcriptional repression.

We demonstrate using a dinucleosome template that acetylation of the core histones enhances transcription by RNA polymerase III. This effect is not dependent on an increased mobility of the core histone octamer with respect to DNA sequence. When linker histone is subsequently bound, we find both a reduction in nucleosome mobility and a repression of transcription. These effects of linker histone binding are independent of core histone acetylation, indicating that core histone acetylation does not prevent linker histone binding and the concomitant transcriptional repression. These studies are complemented by the use of a Xenopus egg extract competent both for chromatin assembly on replicating DNA and for RNA polymerase III transcription. Incorporation of acetylated histones and lack of linker histones together facilitate transcription by >10-fold in this system; however, they have little independent effect on transcription. Thus core histone acetylation significantly facilitates transcription, but this effect is inhibited by the assembly of linker histones into chromatin.

RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo.

In the yeast Saccharomyces cerevisiae, telomere repeat DNA is assembled into a specialized heterochromatin-like complex that silences the transcription of adjacent genes. The general DNA-binding protein Rap1p binds telomere DNA repeats, contributes to telomere length control and to telomeric silencing, and is a major component of telomeric chromatin. We identified Rap1p localization factor 2 (RLF2) in a screen for genes that alleviate antagonism between telomere and centromere sequences on plasmids. In rlf2 mutants, telomeric chromatin is perturbed: Telomeric silencing is reduced and Rap1p localization is altered. In wild-type cells, Rap1p and telomeres localize to bright perinuclear foci. In rlf2 strains, the number of Rap1p foci is increased, Rap1p staining is more diffuse throughout the nucleus, Rap1p foci are distributed in a much broader perinuclear domain, and nuclear volume is 50% larger. Despite the altered distribution of Rap1p in rlf2 mutant cells, fluorescence in situ hybridization to subtelomeric repeats shows that the distribution of telomeric DNA is similar in wild-type and mutant cells. Thus in rlf2 mutant cells, the distribution of Rap1p does not reflect the distribution of telomeric DNA. RLF2 encodes a highly charged coiled-coil protein that has significant similarity to the p150 subunit of human chromatin assembly factor-I(hCAF-I), a complex that is required for the DNA replication-dependent assembly of nucleosomes from newly synthesized histones in vitro. Furthermore, RLF2 is identical to CAC1, a subunit of yeast chromatin assembly factor-I (yCAF-I) which assembles nucleosomes in vitro. In wild-type cells, epitope-tagged Rlf2p expressed from the GAL10 promoter localizes to the nucleus with a pattern distinct from that of Rap1p, suggesting that Rlf2p is not a component of telomeric chromatin. This study provides evidence that yCAF-I is required for the function and organization of telomeric chromatin in vivo. We propose that Rlf2p facilitates the efficient and timely assembly of histones into telomeric chromatin.

Mapping of the Gene for the p60 Subunit of the Human Chromatin Assembly Factor (CAF1A) to the Down Syndrome Region of Chromosome 21

Exon trapping was used to clone portions of genes from the Down syndrome critical region (DSCR) of human chromosome 21. One trapped sequence showed complete homology with nucleotide sequence U20980 (GenBank), which corresponds to the gene for the p60 subunit of the human chromatin assembly factor-1 (CAF1A). We mapped this gene to human chromosome 21 by fluorescencein situhybridization, by the use of somatic cell hybrids, and by hybridization to chromosome 21-specific YACs and cosmids. The CAF1A gene localizes to YACs 745H11 and 230E8 of the Chumakovet al.(1992,Nature359: 380) YAC contig, within the DSCR on 21q22. This CAF1A, which belongs to the WD-motif family of genes and interacts with other polypeptide subunits to promote assembly of histones to replicating DNA, may contribute in a gene dosage-dependent manner to the phenotype of Down syndrome.

Purification of yeast histones competent for nucleosome assemblyin vitro

We have developed a procedure to purify nucleosomal assembly-competent histones as a mixture of H2A, H2B, H3 and H4 from isolated nuclei of the yeast Saccharomyces cerevisiae with a purity of 70-80%. The mixture contained each of the histone subunits approximately at the equi-molar ratio. Plasmid pBR322 DNA was assembled into nucleosomes with the purified yeast histones in the presence of nucleoplasmin from unfertilized eggs of the frog Xenopus laevis. The efficiency of assembly of yeast histones was comparable to that of core histones purified from HeLa cells. The length of DNA fragment wrapping around a core histone particle and the molar ratio of histone components in an assembled nucleosome particle were estimated to be 150 +/- 10 bp long and H2A:H2B:H3:H4 = 1.0:0.9:0:9:1.0, respectively.

Chromatin replication.

Just as the faithful replication of DNA is an essential process for the cell, chromatin structures of active and inactive genes have to be copied accurately. Under certain circumstances, however, the activity pattern has to be changed in specific ways. Although analysis of specific aspects of these complex processes, by means of model systems, has led to their further elucidation, the mechanisms of chromatin replication in vivo are still controversial and far from being understood completely. Progress has been achieved in understanding: 1. The initiation of chromatin replication, indicating that a nucleosome-free origin is necessary for the initiation of replication; 2. The segregation of the parental nucleosomes, where convincing data support the model of random distribution of the parental nucleosomes to the daughter strands; and 3. The assembly of histones on the newly synthesized strands, where growing evidence is emerging for a two-step mechanism of nucleosome assembly, starting with the deposition of H3/H4 tetramers onto the DNA, followed by H2A/H2B dimers.

Differences between some properties of acetylated and nonacetylated forms of HMG1 protein

There are data suggesting that HMG1 protein may be involved in DNA replication. Recently we have found that only the acetylated form of the protein generates tetramers, stimulates the activity of DNA polymerase α (EC 2.7.7.7) (with activated DNA as a template) and forms a specific complex with it. This paper compares some properties of the acetylated and nonacetylated forms of HMG1 protein and shows that it is only the acetylated form which serves as a histone assembly factor, increases the melting temperature of poly d[(A-T)] and stimulates the activity of DNA polymerase α when histone H1-depleted chromatin is used as a template.

Heterogeneous assembly of nascent core histones to form nucleosomal histone octamers in mouse FM3A cells.

We investigated the assembly of newly synthesized histones into nucleosomal histone octamers using an isopycnic centrifugation analysis of these proteins of mouse FM3A cells that had been labeled by culture with heavy amino acids. Cross-linked histone octamers or non-cross-linked core histones were separated electrophoretically from other proteins on sodium dodecyl sulfate/polyacrylamide gels before being banded to equilibrium in cesium formate/guanidinium chloride density gradients. The density of core histones rapidly came to equilibrium after culture of cells in the medium containing heavy amino acids for a time as short as 30 min, indicating that the density of histone octamers in the density-labeled cells reflects the content of these new (dense) core histones relative to old (light) ones in the octamer assembly. Cross-linked histone octamers from these cells were of heterogeneous densities as judged from a broad band in a density gradient. The position and width of the band was essentially unaffected, though the peak position of the density distribution within the band moved progressively from a less to a more dense area, when the time of culture was prolonged from 2 h to 16 h. It is concluded, therefore, that new and old histones are assembled into histone octamers in heterogeneous ratios in contradiction to a conservative or semi-conservative assembly model and it is also suggested that incorporation into chromatin of new histones is not necessarily restricted to newly replicated chromatin regions.

Assembly of new histones into nucleosomes and their distribution in replicating chromatin.

We studied the assembly of new histones into nucleosomes and their distribution in replicating chromatin in growing P815 mouse cells. New histones and new DNA were density-labeled with 13C, 15N, 2H-substituted amino acids together with [3H]arginine or with 5-iododeoxyuridine and [3H]thymidine, respectively, for 1 hr (approximately 20% of S phase). Mono- di-, tri-, tetra- and larger oligonucleosomes were isolated by sucrose gradient centrifugation of micrococcal nuclease-digested chromatin, and their density distribution was analyzed, without fixation, in metrizamide/triethanolamine density gradients [Russev, G. and Tsanev, R. (1976) Nucleic Acids Res. 3, 697-707] in which mono- and oligonucleosomes containing dense amino acids or 5-iododeoxyuridine separate from the corresponding normal nucleosomes. Under these conditions, approximately 74% of the new histones are found in nucleosomes on newly replicated DNA, and the remainder are on unreplicated DNA. The majority of new histones form entirely new nucleosomes; a minor fraction may form hybrid nucleosomes that also contain preexisting histones. New nucleosomes are distributed to both new daughter DNA molecules with approximately equal probability, and our evidence suggests, but does not prove, that they are distributed in a random manner along new DNA.

Identification of histone H2b as a heat-shock protein in Drosophila.

Total cell polypeptides synthesized, in cultured Drosophila cells under control (25 degrees C) and heat-shock (37 degrees C) conditions have been compared in two different two-dimensional polyacrylamide gel electrophoresis systems which, together, resolve polypeptides having a wide range of isoelectric points, including the most basic polypeptides of the cell. The electrophoresis of basic proteins showed that the most prominent basic polypeptide synthesized in heat shock comigrated with histone H2b. This heat-shock polypeptide was identified as histone H2b by two criteria: (a) it comigrated with authentic histone H2b in Triton-urea-acetic acid acrylamide gel electrophoresis after solubilization from nuclei with acid; and (b) partial proteolysis peptide maps of the basic heat-shock protein and histone H2b were identical. The synthesis of histone H2b was induced threefold in heat shock, whereas synthesis of the other histones was reduced from two- to tenfold. The noncoordinate synthesis of histones in Drosophila in heat shock provides an interesting system in which to investigate transcriptional and translational controls of histone synthesis as well as assembly of histones into chromatin.

Structure, Replication, and Transcription of the SV40 Genome

The chapter explains the structure, replication, and transcription of the SV40 genome. The purpose of the present essay is to examine the current status of ones knowledge on both the structure and the function of the SV40 genome, and to correlate these two aspects. Attempts have been made to understand replication and transcription mainly at the level of the DNA, but it now appears that the chromosome, rather than free DNA, is the actual template. The chapter discusses replication of the SV40 genome such as (a) replication of SV40 DNA, (b) gene product(s) and enzymes involved in DNA replication, and (c) assembly of histones on the SV40 genome. The chapter also discussed transcription of the SV40 genome such as (a) transcription of SV40 genome in vivo, (B) transcriptional control of gene expression, and (c) post-transcriptional control. It also discusses various models to study the structure, replication, and transcription of the SV40 genome.