Epigenetic Regulation of Ameloblast Differentiation by HMGN Proteins.

Dental enamel formation is coordinated by ameloblast differentiation, production of enamel matrix proteins, and crystal growth. The factors regulating ameloblast differentiation are not fully understood. Here we show that the high mobility group N (HMGN) nucleosomal binding proteins modulate the rate of ameloblast differentiation and enamel formation. We found that HMGN1 and HMGN2 proteins are downregulated during mouse ameloblast differentiation. Genetically altered mice lacking HMGN1 and HMGN2 proteins show faster ameloblast differentiation and a higher rate of enamel deposition in mice molars and incisors. In vitro differentiation of induced pluripotent stem cells to dental epithelium cells showed that HMGN proteins modulate the expression and chromatin accessibility of ameloblast-specific genes and affect the binding of transcription factors epiprofin and PITX2 to ameloblast-specific genes. Our results suggest that HMGN proteins regulate ameloblast differentiation and enamel mineralization by modulating lineage-specific chromatin accessibility and transcription factor binding to ameloblast regulatory sites.

V(D)J recombination: modulation of RAG1 and RAG2 cleavage activity on 12/23 substrates by whole cell extract and DNA-bending proteins.

Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.

Functional histone antibody fragments traverse the nuclear envelope.

Factors important in the translocation process of proteins across the nuclear membrane were studied by microinjecting either fluoresceinated nonimmune IgG and F(ab)2 or the corresponding molecules, prepared from antisera to histones, into the nucleus and cytoplasm of human fibroblasts. Intact IgG from both preparations remained at the site of injection regardless of whether it was injected into the nucleus or the cytoplasm. In contrast, nonimmune F(ab)2 distributed uniformly throughout the cell. The F(ab)2 derived from affinity-pure antihistone moves into the nucleus after cytoplasmic injection and remains in the nucleus after nuclear microinjection. The migration of the antihistone F(ab)2 into the nucleus results in inhibition of uridine incorporation in the nuclei of the microinjected cells. We conclude that non-nuclear proteins, devoid of specific signal sequences, traverse the nuclear membrane and accumulate in the nucleus provided their radius of gyration is less than 55A and the nucleus contains binding sites for these molecules. These findings support the model of "quasibifunctional binding sites" as a driving force for nuclear accumulation of proteins. The results also indicate that active F(ab)2 fragments, microinjected into somatic cells, can bind to their antigenic sites suggesting that microinjection of active antibody fragments can be used to study the location and function of nuclear components in living cells.

Localization of chromosomal protein HMG-1 in polytene chromosomes of Chironomus thummi.

The distribution of accessible antigenic sites in the chromosomal protein high mobility group one (HMG-1) in Chironomus thummi polytene chromosomes is visualized by immunofluorescence. The results indicate that (a) HMG-1 is distributed in a distinct banding pattern along the entire length of the chromosomes; (b) the banding pattern obtained with fluorescent antibody does not strictly correspond to that observed by phase-contrast microscopy; and (c) the amount of HMG-1 increases, and the fluorescent banding pattern changes, during the development of the organism. Our findings suggest that the protein may be involved in the modulation of the structure of selected loci in the chromosome.

Chromosomal proteins HMG-14 and HMG-17 are released from mitotic chromosomes and imported into the nucleus by active transport.

The high mobility group 14/17 (HMG-14/-17) proteins form specific complexes with nucleosome core particles and produce distinct footprints on nucleosomal DNA. Therefore, they could be an integral part of the chromatin fiber. Here we show that during the cell cycle these proteins are transiently dissociated from chromatin. They colocalize with the nuclear DNA in interphase and prophase but not in metaphase and anaphase. They relocate into the nucleus and colocalize again with the DNA in late telophase, concomitantly with the appearance of the nuclear envelope. Thus, these nucleosomal binding proteins are not always associated with chromatin. Using reconstituted nuclei and permeabilized cells, we demonstrate that these two small proteins, with a molecular mass <10 kD, are actively imported into the nucleus. We identify the major elements involved in the nuclear import of these chromosomal proteins: HMG-14/-17 proteins contain an intrinsic bipartite nuclear localization signal, and their entry into the nucleus through nuclear pores requires energy and the participation of importin alpha. These findings suggest that the cell cycle-related association of HMG-14/-17 with chromatin is dependent on, and perhaps regulated by, nuclear import processes.

Histone localization in polytene chromosomes by immunofluorescence.

Polytene chromosomes of Chironomus thummi were treated with antisera elicited by purified calf thymus histone fractions, and the location of each histone type was visualized by the indirect immunofluorescence technique. Each of the antisera produced specific and distinct patterns of fluorescence, suggesting that it is possible to use the indirect immunofluorescence technique to study the in situ organization of each histone in the various regions of the chromosomes. H1 and H2A antisera produced diffuse fluorescence patterns in acetic acid-fixed chromosomes which become more defined in formaldehyde-fixed preparations. Antisera to H2B, H3 and H4, when reacted with either formaldehyde- or acetic acid-fixed chromosomes, produce distinct banding patterns closely resembling the banding of acetoorcein-stained or phase-contrast-differentiated chromosomal preparations. These antisera produce corresponding patterns of fluorescence for each chromosome, suggesting that the overall organization of the histones is similar in the various bands. Because the dense band regions stain more brightly with antihistone sera than the less compacted interband areas, we believe that the number of antigenic sites of chromosome-bound histones is related to the amount of DNA present, and that the accessibility of histone determinants does not differ between the bands and interbands.

High mobility group proteins of amphibian oocytes: a large storage pool of a soluble high mobility group-1-like protein and involvement in transcriptional events.

Oocytes of several amphibian species (Xenopus laevis, Rana temporaria, and Pleurodeles waltlii) contained a relatively large pool of nonchromatin-bound, soluble high mobility group (HMG) protein with properties similar to those of calf thymus proteins HMG-1 and HMG-2 (protein HMG-A; A, amphibian). About half of this soluble HMG-A was located in the nuclear sap, the other half was recovered in enucleated ooplasms. This protein was identified by its mobility on one- and two-dimensional gel electrophoresis, by binding of antibodies to calf thymus HMG-1 to polypeptides electrophoretically separated and blotted on nitrocellulose paper, and by tryptic peptide mapping of radioiodinated polypeptides. Most, if not all, of the HMG-A in the soluble nuclear protein fraction, preparatively defined as supernatant obtained after centrifugation at 100,000 g for 1 h, was in free monomeric form, apparently not bound to other proteins. On gel filtration it eluted with a mean peak corresponding to an apparent molecular weight of approximately 25,000; on sucrose gradient centrifugation it appeared with a very low S value (2-3 S), and on isoelectric focusing it appeared in fractions ranging from pH approximately 7 to 9. This soluble HMG-A was retained on DEAE-Sephacel but could be eluted already at moderate salt concentrations (0.2 M KCl). In oocytes of various stages of oogenesis HMG-A was accumulated in the nucleus up to concentrations of approximately 14 ng per nucleus (in Xenopus), corresponding to approximately 0.2 mg/ml, similar to those of the nucleosomal core histones. This nuclear concentration is also demonstrated using immunofluorescence microscopy. When antibodies to bovine HMG-1 were microinjected into nuclei of living oocytes of Pleurodeles the lateral loops of the lampbrush chromosomes gradually retracted and the whole chromosomes condensed. As shown using electron microscopy of spread chromatin from such injected oocyte nuclei, this process of loop retraction was accompanied by the appearance of variously-sized and irregularly-spaced gaps within transcriptional units of chromosomal loops but not of nucleoli, indicating that the transcription of non-nucleolar genes was specifically inhibited by this treatment and hence involved an HMG-1-like protein. These data show that proteins of the HMG-1 and -2 category, which are usually chromatin-bound components, can exist, at least in amphibian oocytes, in a free soluble monomeric form, apparently not bound to other molecules. The possible role of this large oocyte pool of soluble HMG-A in early embryogenesis is discussed as well as the possible existence of soluble HMG proteins in other cells.

Autoantibodies to nucleosomal proteins: antibodies to HMG-17 in autoimmune diseases.

The relative amounts of autoantibodies against defined nucleosomal proteins present in serums from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and mixed connective tissue disease (MCTD) have been examined by an enzyme-linked immunoassay. Autoantibodies to nucleosomal proteins were detected in 45 percent of the patients with SLE, 18 percent of the MCTD patients, and none of the RA patients. The results suggest that, in SLE, antibodies are formed against a subset of nucleosomes which contain protein HMG-17.

Stimulation of RNA polymerase II elongation by chromosomal protein HMG-14.

The high-mobility group protein 14 (HMG-14) is a non-histone chromosomal protein that is preferentially associated with transcriptionally active chromatin. To assess the effect of HMG-14 on transcription by RNA polymerase II, in vivo-assembled chromatin with elevated amounts of HMG-14 was obtained. Here it is shown that HMG-14 enhanced transcription on chromatin templates but not on DNA templates. This protein stimulated the rate of elongation by RNA polymerase II but not the level of initiation of transcription. These findings suggest that the association of HMG-14 with nucleosomes is part of the cellular process involved in the generation of transcriptionally active chromatin.

Histone structure: asymmetric distribution of lysine residues in lysine-rich histone.

Structural studies on a very lysine-rich histone show that the carboxyl-terminal half of the molecule is enriched in lysine (and proline). which suggests that it is a site for binding to DNA. The amino-terminal half. containing most of the acidic residues. resembles small, nonhistone proteins and so might have specificity for factors other than DNA.

Chromatin unfolding and activation by HMGN(*) chromosomal proteins.

The high mobility group N (HMGN) proteins are a family of nuclear proteins that binds to nucleosomes, changes the architecture of chromatin, and enhances transcription and replication from chromatin templates. The intracellular organization of the HMGN (previously known as HMG-14/17) proteins is dynamic and is related to both cell-cycle and transcriptional events. These proteins roam the nucleus, perhaps as part of multiprotein complexes, and their target interactions are modulated by posttranslational modifications. Functional studies on HMGN proteins provide insights into the molecular mechanisms by which structural proteins affect DNA-dependent activities in the context of chromatin.

Assessment of the transcriptional activation potential of the HMG chromosomal proteins.

Chromosomal proteins HMG-14, HMG-17, and HMG-1 are among the most abundant, ubiquitous, and evolutionarily conserved nonhistone proteins. Analysis of their structure reveals features which are similar to those of certain transcription factors. The distribution of charged amino acid residues along the polypeptide chains is asymmetric: positive charges are clustered toward the N-terminal region, while negative charges are clustered toward the C-terminal region. The residues in the C-terminal region have the potential to form alpha helices with negatively charged surfaces. The abilities of HMG-14, -17, and -1 to function as transcriptional activators were studied in Saccharomyces cerevisiae cells expressing LexA-HMG fusion proteins (human HMG-14 and -17 and rat HMG-1) which bind to reporter molecules containing the beta-galactosidase gene downstream from a lexA operator. Fusion constructs expressing deletion mutants of HMG-14, -17, and -1 were also tested. Analysis of binding to the lexA operator with in vitro-synthesized fusion proteins shows that there are more sites for HMG-14, -17, and -1 binding than for LexA binding and that only the fusion constructs which contain the C-terminal, acidic domains of HMG-17 bind the lexA operator specifically. None of the LexA-HMG fusion protein constructs elevate the level of beta-galactosidase activity in transfected yeast cells. Thus, although HMG-14, -17, and -1 are structurally similar to acidic transcriptional activators, these chromosomal proteins do not function as activators in this test system.

Alleviation of histone H1-mediated transcriptional repression and chromatin compaction by the acidic activation region in chromosomal protein HMG-14.

Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes.

Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

Histone H1 is a specific repressor of core histone acetylation in chromatin.

Although a link between histone acetylation and transcription has been established, it is not clear how acetylases function in the nucleus of the cell and how they access their targets in a chromatin fiber containing H1 and folded into a highly condensed structure. Here we show that the histone acetyltransferase (HAT) p300/CBP-associated factor (PCAF), either alone or in a nuclear complex, can readily acetylate oligonucleosomal substrates. The linker histones, H1 and H5, specifically inhibit the acetylation of mono- and oligonucleosomes and not that of free histones or histone-DNA mixtures. We demonstrate that the inhibition is due mainly to steric hindrance of H3 by the tails of linker histones and not to condensation of the chromatin fiber. Cellular PCAF, which is complexed with accessory proteins in a multiprotein complex, can overcome the linker histone repression. We suggest that linker histones hinder access of PCAF, and perhaps other HATs, to their target acetylation sites and that perturbation of the linker histone organization in chromatin is a prerequisite for efficient acetylation of the histone tails in nucleosomes.

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.

Mitotic phosphorylation prevents the binding of HMGN proteins to chromatin.

Condensation of the chromatin fiber and transcriptional inhibition during mitosis is associated with the redistribution of many DNA- and chromatin-binding proteins, including members of the high-mobility-group N (HMGN) family. Here we study the mechanism governing the organization of HMGN proteins in mitosis. Using site-specific antibodies and quantitative gel analysis with proteins extracted from synchronized HeLa cells, we demonstrate that, during mitosis, the conserved serine residues in the nucleosomal binding domain (NBD) of this protein family are highly and specifically phosphorylated. Nucleosome mobility shift assays with both in vitro-phosphorylated proteins and with point mutants bearing negative charges in the NBD demonstrate that the negative charge abolishes the ability of the proteins to bind to nucleosomes. Fluorescence loss of photobleaching demonstrates that, in living cells, the negative charge in the NBD increases the intranuclear mobility of the protein and significantly decreases the relative time that it is bound to chromatin. Expression of wild-type and mutant proteins in HmgN1(-/-) cells indicates that the negatively charged protein is not bound to chromosomes. We conclude that during mitosis the NBD of HMGN proteins is highly phosphorylated and that this modification regulates the interaction of the proteins with chromatin.

Elevated expression of the c-fes proto-oncogene in adult human myeloid leukemia cells in the absence of gene amplification.

Expression of the 93-kd tyrosine kinase encoded by the human c-fes proto-oncogene (also known as FES) is restricted to mature hematopoietic cells of the granulocytic and monocytic lineages, suggestive of a function essential to normal myeloid differentiation. However, recent studies have shown that c-fes can transform fibroblasts if sufficient levels of gene expression are achieved. These findings indicate that strict regulation of the c-fes gene is critical to normal myeloid development, whereas elevated c-fes expression may contribute to malignant transformation. In the present study, we compared the c-fes messenger RNA (mRNA) levels in leukemia blasts from patients with myeloid or lymphoid leukemia with those of peripheral monocytes from a normal donor with the use of a quantitative ribonuclease protection assay. The presence of c-fes mRNA was readily detected in both acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) cells, but c-fes mRNA was present in low levels or was absent in lymphoid leukemia cells. The leukemia cells of two of five AML patients and four of four CML patients expressed more c-fes mRNA than monocytes from a normal donor, with more than a threefold elevation in the cells of one CML patient. No evidence of amplification or rearrangement of the c-fes gene was detectable by Southern blot analysis of myeloid leukemia DNA, suggesting that the variation in c-fes mRNA levels are related to differences in transcriptional activity and/or message stability. These results indicate that elevated c-fes expression is a common feature of myeloid leukemia cells that could potentially contribute to the leukemia phenotype.

Immunological detection of carcinogen-modified DNA fragments after in vivo modification of cellular and viral chromatin.

Antibodies specific for DNA modified by (+/-)-trans-7, 8-dihydrobenzo(a)pyrene-7,8-diol-9, 10-epoxide have been used to quantitate the relative modification level in fragments derived from pBR322 DNA from cellular DNA and in the coding and noncoding strands of simian virus 40 DNA. DNA fragments with a covalent molar modification level ranging from less than 1 to over 200 are resolved by agarose gel electrophoresis and transferred to diazobenzyloxymethyl cellulose paper. The paper is incubated with antibodies specific to carcinogen-modified DNA, and the location of the antibody is visualized by autoradiography after incubation with 125I-protein A. The binding of antibodies is directly proportional to the level of DNA modification. Using this technique, we find that linker DNA is about 2.5- to 3-fold more accessible to (+/-)-trans-7,8-dehydrobenzo(a)pyrene-7, 8-diol-9, 10-epoxide than nucleosomal core DNA and that under in vivo conditions the coding and noncoding strands of the simian virus 40 chromosome are equally accessible to trans-7,8-dihydrobenzo[a]pyrene-7,8-diol-9, 10-epoxide. The approach described allows assessment of the relative level of modification in any DNA sequence which can be subjected to gel electrophoresis.

Immunochemical visualization of binding of the chemical carcinogen benzo(a)pyrene diol-epoxide 1 to the genome.

antisera against DNA modified with r-7,t-8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-1) was elicited in rabbits. Such sera reached with either single- or double-stranded modified DNA but not with unmodified DNA, free benzo(a)pyrene, or proteins modified by BPDE-1. Indirect immunofluorescence studies indicated that the immunoglobulin G in the sera bound specifically to the nuclei of KD cells which were treated with BPDE-1. The intensity of fluorescence was proportional to the dose of BPDE-1 used to treat the cells. About 50% of the BPDE-1-DNA adducts remained bound to DNA 24 hr after the removal of the carcinogen. The location of BPDE-1-modified bases in Col E1 DNA was visualized by immunoelectron microscopy.