Nonhistone Chromosomal Proteins Research Articles

HP1γ associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein, first identified in Drosophila, that plays a dose-dependent role in gene silencing. Three orthologs, HP1α, HP1β, and HP1γ, have been characterized in mammals. While HP1α and HP1β have been unambiguously localized in heterochromatin by immunocytochemical methods, HP1γ has been found either exclusively associated with euchromatin or present in both euchromatin and heterochromatin. Here, using an antibody directed against a peptide epitope at the carboxyl-terminal end of the molecule, we localize HP1γ in both euchromatin and heterochromatin compartments of interphase nuclei, as well as in the pericentromeric chromatin and arms of mitotic chromosomes of 3T3 cells. This dual location was also observed in nuclei expressing HP1γ as a fusion protein with green fluorescent protein. In contrast, when the distribution of HP1γ was analyzed with antibodies directed against an amino-terminal epitope, the protein was detectable in euchromatin and not in heterochromatin, except for transient heterochromatin staining during the late S phase, when the heterochromatin undergoes replication. These data suggest that the controversial immunolocalization of HP1γ in chromatin is due to the use of antibodies directed against topologically distinct epitopes, those present at the amino-terminal end of the molecule being selectively masked in nonreplicative heterochromatin.

A novel downstream positive regulatory element mediating transcription of the human high mobility group (HMG) I-C gene

The high mobility group (HMG) I proteins are small, non-histone chromosomal proteins that promote gene activation during development and within rapidly dividing cells. They do so by facilitating enhanceosome formation on inducible genes, via both protein/DNA and protein/protein interactions. The HMG I-C gene is tightly regulated, normally being expressed exclusively during embryonic development. However, HMG I-C expression is also observed frequently in a number of tumor types, and this expression has been shown to contribute to the malignant transformation process. With the aim of dissecting pathways that lead to aberrant expression of HMG I-C in tumor cells, we have analyzed HMG I-C gene regulation in the human hepatoma cell line PLC/PRF/5. One of the two HMG I-C transcripts detected in this cell line originates from a novel downstream initiation site at nucleotide −161 relative to the first methionine. Transcription from the downstream initiation site is mediated by a PRE located between nt −222 and −217. We show here that the Sp1 and Sp3 transcription factors interact with the PRE and transactivate the HMG I-C promoter in a cooperative fashion. This study provides the first characterization of this downstream HMG I-C promoter.

Organization of chromatin in cancer cells: role of signalling pathways

The role of mechanical and chemical signalling pathways in the organization and function of chromatin is the subject of this review. The mechanical signalling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. Intermediate filament proteins are associated with nuclear DNA, suggesting that intermediate filaments may have a role in the organization of chromatin. In human hormone-dependent breast cancer cells, the interaction between cytokeratins and chromatin is regulated by estrogens. Transcription factors, histone acetyltransferases, and histone deacetylases, which are associated with the nuclear matrix, are components of the mechanical signalling pathway. Recently, we reported that nuclear matrix-bound human and chicken histone deacetylase 1 is associated with nuclear DNA in situ, suggesting that histone deacetylase has a role in the organization of nuclear DNA. Chemical signalling pathways such as the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. The levels of phosphorylated histones are increased in mouse fibroblasts transformed with oncogenes, the products of which stimulate the Ras/MAPK pathway. Histone phosphorylation may lead to decondensation of chromatin, resulting in aberrant gene expression.Key words: histone acetylation, histone phosphorylation, nuclear matrix, cytoskeleton, histone deacetylase, cancer.

High mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 are significant target antigens of perinuclear anti-neutrophil cytoplasmic antibodies in autoimmune hepatitis

BACKGROUNDHigh mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 have been identified as novel antigens of perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCAs), and the existence of anti-HMG1 and anti-HMG2 antibodies...

The putative nuclear receptor mediator TIF1alpha is tightly associated with euchromatin.

Ligand-dependent transcriptional regulation by nuclear receptors is believed to be mediated by intermediary factors (TIFs) acting on remodelling of the chromatin structure and/or the activity of the transcriptional machinery. The putative transcriptional mediator TIF1alpha is a nuclear protein kinase that has been identified via its interaction with liganded nuclear receptors, including retinoic acid (RAR), retinoid X (RXR) and estrogen (ER) receptors. Here, we demonstrate that TIF1alpha is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome. Immunofluorescence confocal microscopy reveals that TIF1alpha exhibits a finely granular distribution in euchromatin of interphase nuclei, while it is mostly excluded from condensed chromatin and metaphase chromosomes. Immunoelectron microscopy shows that, in contrast to the heterochromatin protein HP1alpha, most of TIF1alpha is associated with euchromatin, where it is preferentially localised on regions known to be sites for RNA polymerase II (perichromatin fibrils and borders between euchromatin and heterochromatin). Early mouse embryos as well as embryonal carcinoma (EC) and embryonic stem (ES) cells express high levels of TIF1alpha. These levels dramatically decrease during organogenesis and upon differentiation of P19 EC cells, indicating that TIF1alpha is preferentially expressed in undifferentiated pluripotent cells in the course of development. Therefore, TIF1alpha could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation.

Phosphorylation of Heterochromatin Protein 1 by Casein Kinase II Is Required for Efficient Heterochromatin Binding inDrosophila

Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal protein with a dose-dependent effect on heterochromatin mediated position-effect silencing. It is multiply phosphorylated in vivo. Hyperphosphorylation of HP1 is correlated with heterochromatin assembly. We report here that HP1 is phosphorylated by casein kinase II in vivo at three serine residues located at the N and C termini of the protein. Alanine substitution mutations in the casein kinase II target phosphorylation sites dramatically reduce the heterochromatin binding activity of HP1, whereas glutamate substitution mutations, which mimic the charge contributions of phosphorylated serine, have apparently wild-type binding activity. We propose that phosphorylation of HP1 promotes protein-protein interaction between HP1 and target binding proteins in heterochromatin.

Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes.

Nonhistone chromosomal proteins HMG-14 and HMG-17 are closely related nucleosomal binding proteins that unfold the higher-order chromatin structure, thereby enhancing the transcription and replication potential of chromatin. Here we report that PCAF, a transcription coactivator with intrinsic histone acetyltransferase activity, specifically acetylates HMG-17 but not HMG-14. Using mass spectrum sequence analysis, we identified the lysine at position 2 as the predominant site acetylated by PCAF. Lysine 2 is a prominent acetylation site in vivo, suggesting that this PCAF-mediated acetylation is physiologically relevant. Experiments with HMG-17 deletion mutants and competition studies with various protein fragments indicate that the specific acetylation of HMG-17 is not determined solely by the primary sequence near the acetylation site. By equilibrium dialysis we demonstrated that acetylation reduces the affinity of HMG-17 to nucleosome cores. In addition, we found that the binding of HMG-14 and HMG-17 to nucleosome cores inhibits the PCAF-mediated acetylation of histone H3. Thus, the presence of HMG-14 and HMG-17 affects the ability of PCAF to acetylate chromatin, while the acetylation of HMG-17 reduces its binding affinity to chromatin. Conceivably, in HMG-17-containing chromatin, acetylation of HMG-17 precedes the acetylation of histones.

Structural and Kinetic Studies of a Cisplatin-modified DNA Icosamer Binding to HMG1 Domain B

The high mobility group (HMG) domain is a DNA-binding motif found in the non-histone chromosomal proteins, HMG1 and HMG2, and some transcription factors. Experimental evidence has demonstrated that HMG-domain proteins can play a role in sensitizing cells to the anticancer drug cisplatin. Fluorescence resonance energy transfer (FRET) experiments were performed in the present study to investigate structural changes that accompany complex formation between the HMG domain B of HMG1 and a cisplatin-modified, 20-base pair double-stranded DNA probe containing fluorescein and rhodamine tethered at its two ends. The binding affinity of HMG1 domain B for the cisplatin-modified DNA probe was investigated in fluorescence titration experiments, and a value of 60 +/- 30 nM was determined for the dissociation constant. Single photon counting methods were employed to measure the fluorescence lifetime of the fluorescein donor in the presence and absence of HMG1 domain B. These FRET experiments revealed a distance change that was used to estimate a bend angle of 80-95 degrees for the cisplatin-modified DNA upon protein binding. Stopped-flow fluorescence spectroscopic experiments afforded kinetic parameters for HMG1 domain B binding to the cisplatin-modified DNA probe, with kon = 1.1 +/- 0.1 x 10(9) M-1 s-1 and koff = 30 +/- 4 s-1.

Male Infertility and the Y Chromosome

Although it has been established since the 1970s thatdeletions of the long arm of the Y chromosome are as-sociated with spermatogenic failure, only in the last fewyears have these regions been described at the molecularlevel. In parallel, Y-linked genes and gene families thatmay explain the phenotypes of men carrying these de-letions have been identified. The first association be-tween spermatogenic failure and an underlying geneticcause was demonstrated by Tiepolo and Zuffardi (1976)in a report of six azoospermic patients carrying micro-scopically detectable deletions of the distal portion ofYq. In four patients, the deletion was de novo—that is,their fathers were tested and were found to carry intactY chromosomes. On this basis, Tiepolo and Zuffardi(1976) proposed the existence of a spermatogenesis fac-tor, called the “azoospermia factor” (AZF), encoded bya gene on distal Yq. However, the assumption that AZFrepresented a single Y-linked gene was overturned whenVogt et al. (1996) observed that Y chromosome micro-deletions follow a certain deletion pattern, with threerecurrently deleted nonoverlapping subregions in prox-imal, middle, and distal Yq11, designated “AZFa,”“AZFb,” and “AZFc,” respectively. In addition, it be-came clear that these deletions were not exclusively as-sociated with azoospermia (Reijo et al. 1996

Organization of chromatin in cancer cells: role of signalling pathways

The role of mechanical and chemical signalling pathways in the organization and function of chromatin is the subject of this review. The mechanical signalling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. Intermediate filament proteins are associated with nuclear DNA, suggesting that intermediate filaments may have a role in the organization of chromatin. In human hormone-dependent breast cancer cells, the interaction between cytokeratins and chromatin is regulated by estrogens. Transcription factors, histone acetyltransferases, and histone deacetylases, which are associated with the nuclear matrix, are components of the mechanical signalling pathway. Recently, we reported that nuclear matrix-bound human and chicken histone deacetylase 1 is associated with nuclear DNA in situ, suggesting that histone deacetylase has a role in the organization of nuclear DNA. Chemical signalling pathways such as the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. The levels of phosphorylated histones are increased in mouse fibroblasts transformed with oncogenes, the products of which stimulate the Ras/MAPK pathway. Histone phosphorylation may lead to decondensation of chromatin, resulting in aberrant gene expression.

Induction of High Mobility Group-I(Y) Protein by Endotoxin and Interleukin-1β in Vascular Smooth Muscle Cells: ROLE IN ACTIVATION OF INDUCIBLE NITRIC OXIDE SYNTHASE

Nonhistone chromosomal proteins of the high mobility group (HMG) affect the transcriptional regulation of certain mammalian genes. For example, HMG-I(Y) controls cytokine-mediated promoters that require transcription factors, such as nuclear factor-kappaB, for maximal expression. Even though a great deal is known about how HMG-I(Y) facilitates expression of other genes, less is known about the regulation of HMG-I(Y) itself, especially in cells in primary culture. Therefore we investigated the effect of endotoxin and the cytokine interleukin-1beta on HMG-I(Y) expression in vascular smooth muscle cells. Induction of HMG-I(Y) peaked after 48 h of interleukin-1beta stimulation (6.2-fold) in cells in primary culture, and this increase in mRNA corresponded to an increase in HMG-I(Y) protein. Moreover, immunohistochemical staining revealed a dramatic increase in HMG-I(Y) protein expression in vascular smooth muscle cells after endotoxin stimulation in vivo. This increase in HMG-I(Y) expression (both in vitro and in vivo) mirrored an up-regulation of inducible nitric oxide synthase, a cytokine-responsive gene. The functional significance of this coinduction is underscored by our finding that HMG-I(Y) potentiated the response of inducible nitric oxide synthase to nuclear factor-kappaB transactivation. Taken together, these studies suggest that induction of HMG-I(Y), and subsequent transactivation of iNOS, may contribute to a reduction in vascular tone during endotoxemia and other systemic inflammatory processes.

新規のＰ‐ＡＮＣＡ 抗ＨＭＧ１／ＨＭＧ２抗体 その炎症性疾患における意義

Besides proteinase 3 and myeloperoxidase, various target antigens have been identified that anti -neutrophil cytoplasmic antibodies (ANCA) can recognize. We have isolated a new member of perinuclear ANCA (P-ANCA) . Previously, we found that patients with ulcerative colitis (UC) had a novel P-ANCA against neutrophil 28-kD protein. Here, we purified the same antigens from HL-60 lysates by using reversed-phase HPLC. The N-terminus amino acids of these proteins were identical with those of high mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2. The 28-kD band detected by immunoblotting analysis using patient's serum completery disappeared after preincubation with HMG1/HMG2. A mono-clonal antibody to HMGI/HMG2 stained neutrophils selectivery in a P-ANCA pattern. These results indicate that HMG1/HMG2 can serve as the target antigens of P-ANCA. We then examined serum anti-HMG1/HMG2 antibodies by ELISA. The prevalence of the antibodies were 89% in autoimmune hepatitis, 70% in primary biliary cirrhosis, 48% in rheumatoid arthritis, 45% in systemic lupus erythematosus, and 35% in UC. The occurrence of the antibodies in these diseases seemed to be associated with the disease activity. Thus, anti-HMG1/HMG2 antibodies appear to be useful P-ANCA in some inflammatory diseases, especially in autoimmune liver diseases.

Analytical ultracentrifugation of chromatin.

The ability of analytical ultracentrifugation to elucidate chromatin structure/function relationships originates directly from its capacity to accurately measure key structural properties of complex macromolecular assemblies in solution. Figure 1 schematically illustrates the complex nature of chromatin. Newly replicated DNA is wrapped around core histone octamers spaced at approx 200 bp intervals to form nucleosomal arrays, which then interact with linker histones and numerous other nonhistone chromosomal proteins to form “chromatin.” Chromatin is conformationally dynamic, undergoing a number of short-range and long-range folding transitions to produce highly condensed interphase chromosomal fibers (Fig. 1). For short chromatin fragments studied in vitro, fiber condensation manifests both in the form of intramolecular conformational changes and reversible oligomerization (, , , ). In addition, the structure of chromatin fibers and functions such as transcription and replication are irrevocably linked; any given region of a chromosomal fiber can be either functionally active or inactive depending on both its specific complement of chromatin-associated proteins and its overall state of condensation (,). Consequently, to biochemically characterize chromatin structure/function relationships in vitro, one must be able to analyze both the intramolecular conformational dynamics and intermolecular interactions of an exceedingly complex macromolecular assembly (e.g., a 12-mer nucleosomal array containing one H1 molecule per nucleosome consists of >100 proteins and 2400 bp of DNA, has a molecular mass of approx 3.5×106 D, yet represents only roughly one millionth of an intact eukaryotic chromosome.) Open image in new window Fig. 1. Schematic illustration of the hierarchical relationships between DNA, chromatin, and interphase chromosomal fibers.

Mapping DNA interaction sites of chromosomal proteins. Crosslinking studies in yeast.

Eucaryotes use a common theme for packaging genomic DNA: 146 bp of DNA are wrapped around an octameric protein core consisting of two molecules each of the histones H2A, H2B, H3, and H4. Although this nucleosomal arrangement is ubiquitous throughout the genome, different chromosomal portions are nonetheless functionally and structurally distinct. This is owing to regional interactions between regulatory chromosomal proteins (i.e., transcription factors, structural components, histone-modifying enzymes) and specific portions of the chromosome. Therefore it is important to determine the natural sites of action of these chromatin (re-)modeling factors and to gain insight into the parameters governing their association with chromatin in vivo. To address this problem, Orlando and Paro () have used formaldehyde crosslinking to prevent redistribution of chromatin components during chromatin preparation, immunoprecipitation to isolate the chromosomal factor(s) to be investigated and hybridization to enable the detection of genomic sequences contacted by the factor(s) (see Chapter 33). A similar approach has been used by Braunstein et al. (), employing antibodies against sites of histone acetylation, to determine the acetylation state of histones in chromatin sites. We have streamlined and further developed this protocol and included a polymerase chain reaction (PCR) step to more sensitively and rapidly map the chromosomal distribution of regulatory proteins in Saccharomyces cerevisiae (Fig. 1). Open image in new window Fig. 1. Strategy for the mapping of chromosomal interaction sites of nonhistone chromatin associated factors. Yeast cells are crosslinked with formaldehyde in vivo (A) and subsequently disrupted mechanically. Chromatin fragments released from the cells are sheared by sonication (B) to an average size suitable for the desired accuracy and resolution of the mapping experiment. Antibodies recognizing a chromosomal non-histone protein are added and protein-DNA complexes are recovered by adsorption to proteinA sepharose beads (C). Samples from the INPUT and PRECIPITATE are heat-treated to reverse the crosslinks and DNA is isolated (D). The purified DNA is then used as template in multiplex PCR reactions with primer pairs derived from different chromosomal regions. Control PCR reactions with decreasing amounts of template are analyzed in parallel to assure that the PCR was still in the exponential phase to allow for a quantitative interpretation of the results (E). (F) Polyacrylamide gel electrophores’s is used to separate and visualize different sized PCR products. Bound as well as unbound sequences are equally represented in the genome and will be amplified accordingly from the INPUT sample. Only sequences specifically bound by the regulatory protein in vivo coimmunoprecipitate with this factor (the shaded area in the P1/P2 region symbolizes an interaction site) and will give rise to PCR products from the PRECIPITATE (F).

Biochemical Analysis of Nonhistone Protein Scaffold in Metaphase Chromosome of Common Wheat Triticum aestivum.

Removing the histone and most of the nonhistone from mitotic chromosomes, we studied the protein composition of the chromosomal nonhistone protein scaffold extracted and separated from isolated chromosomes by using one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). We found that the scaffold in the metaphase chromosome of Triticum aestivum consists of 16 nonhistone species. The molecular weights of these nonhistone protein range from 18KD to 90KD; the major nonhistone protein with the molecular weight of 30KD, 32KD, 37KD, 43KD, 44KD, 50KD, 53KD, 63KD, 78KD and 88KD which can be clearly recognized in the gel are abundant in the scaffold. Our findings demonstrated the existence of nonhistone proteins scaffold in metaphase chromosome of common wheat. This scaffold seems to be similar to the scaffold found in the metaphase chromosomes of humans and animals, in terms of SDS-PAGE profiles.

Linker histones versus HMG1/2: a struggle for dominance?

The linker histones (H1, H1 zero, H5, etc.) and a group of abundant non-histone chromosomal proteins (HMG1/2) bind to linker DNA in chromatin and exhibit both generalized and specific effects on gene transcription. The two classes of proteins share many features of DNA binding behaviour, although they are structurally unrelated. While the linker histones and HMG1/2 exhibit direct competition in binding to such structures as four-way junction DNA, whether they compete for binding to the nucleosome has not been investigated. The possibility for either opposite or synergistic effects on gene regulation must be considered at this point.

Isolation and separation of HMG proteins and histones H1 and H5 and core histones by column chromatography on phosphocellulose.

A single-step method to dissociate histones as well as nonhistone chromosomal proteins from chicken erythrocyte chromatin and their separation into histones H1, H5, core histones, and high mobility group proteins by column chromatography on phosphocellulose is presented. NaCl at 2.0 M is effective in dissociating both histones and nonhistone proteins. The core histones elute as a complex. The pH is a critical factor in separating H5 from the core histones.

The Heterochromatin Protein 1 Prevents Telomere Fusions in Drosophila

HP1 (Heterochromatin protein 1) is a conserved, nonhistone chromosomal protein that is best known for its preferential binding to pericentric heterochromatin and its role in position effect variegation in Drosophila. Using immunolocalization, we show that HP1 is a constant feature of the telomeres of interphase polytene and mitotic chromosomes. This localization does not require the presence of telomeric retrotransposons, since HP1 is also detected at the ends of terminally deleted chromosomes that lack these elements. Importantly, larvae expressing reduced or mutant versions of HP1 exhibit aberrant chromosome associations and multiple telomeric fusions in neuroblast cells, imaginal disks, and male meiotic cells. Taken together, these results provide evidence that HP1 plays a functional role in mediating normal telomere behavior in Drosophila .

AT-hook motifs identified in a wide variety of DNA-binding proteins.

The AT-hook is a small DNA-binding protein motif which was first described in the high mobility group non-histone chromosomal protein HMG-I(Y). Since its discovery, this motif has been observed in other DNA-binding proteins from a wide range of organisms. Using pattern searches and position-dependent matrices, we have extracted the AT-hook motifs present in a non-redundant protein sequence database. We have classified these motifs into three types according to their sequence similarity and have found that they are prevalent in many eukaryotic nuclear proteins in single or multiple copies. Furthermore, AT-hook motifs are frequently associated with known functional domains seen in chromatin proteins and in DNA-binding proteins (e.g. histone folds, homeodomains and zinc fingers). In general, it appears that the AT-hook motif is an auxiliary protein motif cooperating with other DNA-binding activities and facilitating changes in the structure of the DNA either as a polypeptide on its own [e.g. HMG-I(Y)] or as part of a multidomain protein [e.g. Swi2p in Saccharomyces cerevisiae or HRX (ALL-1) in Homo sapiens]. It is most interesting that this motif seems to be quite specific to known or predicted chromosomal/DNA-binding proteins, suggesting that it may act as a versatile minor groove tether.

Many Tumors and Many Genes: Genetics of Uterine Leiomyomata

Many Tumors and Many Genes: Genetics of Uterine Leiomyomata