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.

Transcription factor FoxA (HNF3) on a nucleosome at an enhancer complex in liver chromatin.

Nucleosome-like particles and acetylated histones occur near active promoters and enhancers, and certain transcription factors can recognize their target sites on the surface of a nucleosome in vitro; yet it has been unclear whether transcription factors can occupy target sites on nucleosomes in native chromatin. We developed a method for sequential chromatin immunoprecipitation of distinct nuclear proteins that are simultaneously cross-linked to nucleosome-sized genomic DNA segments. We find that core histone H2A co-occupies, along with the FoxA (hepatocyte nuclear factor-3) transcription factor, DNA for the albumin transcriptional enhancer in native liver chromatin, where the enhancer is active. Because histone H2A on nuclear DNA is only known to exist in nucleosomes, we conclude that transcription factors can form a stable complex on nucleosomes at an active enhancer element in vivo.

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.

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.

HMGN4, a newly discovered nucleosome-binding protein encoded by an intronless gene.

We describe a newly discovered nuclear protein, HMGN4, that is closely related to the canonical HMGN2 nucleosome-binding protein. The protein is encoded by an intronless gene, which, in humans, is located in the hereditary hemochromatosis [correction of hemachromatosis] region at position 6p21.3. A single approximately 2-kb HMGN4 mRNA was found to be expressed, in variable amounts, in all human tissues tested; however, the HMGN4 transcript was significantly less abundant than that of HMGN2. The HMGN4 protein could be detected in HeLa cells by Western analysis with an antibody elicited against a unique region of the protein. Transfection of HeLa cells with a plasmid expressing HMGN4-GFP indicated that the protein localizes to the nucleus. Our results expand the multiplicity of the HMGN protein family and increase the known cellular repertoire of nucleosome-binding proteins.

HMGN3a and HMGN3b, two protein isoforms with a tissue-specific expression pattern, expand the cellular repertoire of nucleosome-binding proteins.

HMGN1 (HMG-14) and HMGN2 (HMG-17) are nuclear proteins that bind specifically to nucleosomes, reduce the compactness of the chromatin fiber, and enhance transcription from chromatin templates. Here we report that many vertebrates contain an additional type of HMGN protein named HMGN3 (Trip 7). The human HMGN3 gene is located on chromosome 6 and spans 32 kilobase pairs, which is nearly 10-fold longer than the closely related HMGN2 gene. However, the intron/exon boundaries of the HMGN3 gene are identical to those of HMGN1 and HMGN2. Unique within the HMGN family, the HMGN3 transcript undergoes alternative splicing and generates two different variants, HMGN3a and HMGN3b. The shorter variant, HMGN3b, arises from an additional splice site that truncates exon V and causes a frameshift. The resulting HMGN3b protein lacks the majority of the C-terminal chromatin-unfolding domain. Both splice variants are found in many vertebrates from frogs to man and are expressed in many tissues. The pattern of tissue-specific expression differs considerably from those of HMGN1 and HMGN2 at both the mRNA and the protein level. Our results expand the multiplicity of the HMGN protein family and raise the possibility that these nucleosome-binding proteins function as co-activators in tissue-specific gene expression.

High-mobility group proteins 14 and 17 maintain the timing of early embryonic development in the mouse.

The high-mobility group (HMG) proteins 14 and 17 are abundant chromosomal proteins that bind to nucleosomes and enhance transcription. We report that both mRNA species and both proteins are present throughout oogenesis and preimplantation development of the mouse. When antisense oligonucleotides targeting each mRNA species are injected into one-cell embryos, the proteins become depleted at the two- and four-cell stages and reaccumulate at the eight-cell stage. One-cell embryos injected with antisense oligonucleotides targeting both HMG-14 and HMG-17 cleave to the two-cell stage. Subsequent cleavages, however, are delayed compared with control-injected embryos. Nevertheless, these embryos ultimately reach the blastocyst stage. Similarly, injection into the nuclei of two-cell embryos of a peptide corresponding to the common nucleosome-binding domain of HMG-14 and HMG-17 delays progression to the four-cell stage. Furthermore, both RNA and protein synthesis is transiently reduced in antisense-injected embryos compared with injected controls. These results identify HMG-14 and HMG-17 as constitutive components of mouse oocyte and embryonic chromatin and establish a link between the structure of embryonic chromatin and the normal progression of embryonic development.

Dynamic binding of histone H1 to chromatin in living cells.

The linker histone H1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure. Histone H1 is generally viewed as a repressor of transcription as it prevents the access of transcription factors and chromatin remodelling complexes to DNA. Determining the binding properties of histone H1 to chromatin in vivo is central to understanding how it exerts these functions. We have used photobleaching techniques to measure the dynamic binding of histone H1-GFP to unperturbed chromatin in living cells. Here we show that almost the entire population of H1-GFP is bound to chromatin at any one time; however, H1-GFP is exchanged continuously between chromatin regions. The residence time of H1-GFP on chromatin between exchange events is several minutes in both euchromatin and heterochromatin. In addition to the mobile fraction, we detected a kinetically distinct, less mobile fraction. After hyperacetylation of core histones, the residence time of H1-GFP is reduced, suggesting a higher rate of exchange upon chromatin remodelling. These results support a model in which linker histones bind dynamically to chromatin in a stop-and-go mode.

Targeting of high mobility group-14/-17 proteins in chromatin is independent of DNA sequence.

Chromosomal proteins high mobility group (HMG)-14 and HMG-17 are nucleosomal-binding proteins that unfold the chromatin fiber and enhance transcription from chromatin templates. Their intracellular organization is dynamic and related to both cell cycle and transcription. Here we examine possible mechanisms for targeting HMG-14/-17 to specific regions in chromatin. Chromatin immunoprecipitation assays indicate that HMG-17 protein is not preferentially associated with chromatin regions containing transcriptionally active genes, or any type of specific DNA. We used a modification of the random amplified polymorphic DNA method to analyze DNA in various HMG-14/-17.nucleosome complexes. We found that although HMG-14 or HMG-17 proteins preferentially associate with core particles in which the DNA has a low frequency of CG dinucleotides, the genome does not contain consensus sequences that serve as specific targeting sites for the binding of either HMG-14 or HMG-17 proteins to nucleosomes. We used size exclusion and ion exchange chromatography to demonstrate that nuclei contain a large portion of HMG-17 associated with other proteins in a multiprotein complex. We suggest that these complexes regulate the dynamic organization of HMG-14/-17 in the nucleus and serve to target the proteins to specific sites in chromatin.

The accessibility of histone H3 tails in chromatin modulates their acetylation by P300/CBP-associated factor.

P300/CBP-associated factor (PCAF) is a transcriptional coactivator with intrinsic histone acetylase activity. Reversible acetylation of the core histone tails in chromatin has been linked to transcriptional regulation. Here we investigate the mechanism whereby PCAF acetylates its target in chromatin. We demonstrate that recombinant PACF preferentially acetylates the H3 tail in oligonucleosomes, as compared with nucleosome core particles. The rate of acetylation is directly related to the length of the oligonucleosomal substrate. Using a trypsin accessibility assay, we demonstrate that the rate of acetylation is also related to the accessibility of the H3 tail in uncondensed oligonucleosomes. We suggest that PCAF, and perhaps other histone acetyltransferases, acetylate chromatin templates more efficiently than core particle subunits and that this preference arises from an increased accessibility of the H3 tail in either condensed or uncondensed oligonucleosomes. Acetylation of the H3 tails by the native PCAF complex is not affected by the length of the oligonucleosomal substrate. Our results suggest that the accessibility of the H3 tail in chromatin is a major factor affecting their rate of acetylation and that component(s) in the native PCAF complex function to modify the organization of these tails in chromatin thereby enhancing their accessibility to PCAF.

NBP-45, a novel nucleosomal binding protein with a tissue-specific and developmentally regulated expression.

Here we characterize a novel murine nuclear protein, which we named NBP-45, that is related to the ubiquitous nuclear proteins HMG-14/-17, binds specifically to nucleosome core particles, and can function as a transcriptional activator. NBP-45 mRNA is expressed at low levels and in variable amounts in all mouse tissues tested but is especially abundant in RNA extracted from 7-day-old mouse embryos, suggesting that it functions in early embryonic development. NBP-45 is composed of 406 amino acids and is encoded by a single size transcript. The region spanning the N-terminal 85 amino acids contains three segments that are highly homologous to functionally important domains in the HMG-14/-17 protein family: the nuclear localization signal, the nucleosome binding domain, and the chromatin unfolding domain. The protein region spanning the C-terminal 321 amino acids has a 42% content of negatively charged residues. The first 23 amino acids contain a region necessary for nuclear entry of the protein, the region spanning residues 12-40 is the main nucleosomal binding domain of the protein, and the negatively charged, C-terminal domain is necessary for transcription activation. The functional domains of NBP-45 are indicative of a nuclear protein that binds to nucleosomes, thereby creating a chromatin region of high local negative charge. Our studies establish the nucleosomal binding domain as a protein motif that is present in other than just the ubiquitous HMG-14/-17 proteins. We suggest that the nucleosomal binding domain motif is a protein module that facilitates binding to nucleosomes in chromatin.

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.

Acetylation of novel sites in the nucleosomal binding domain of chromosomal protein HMG-14 by p300 alters its interaction with nucleosomes.

The reversible acetylation of histones is associated with structural alterations in the chromatin fiber that affect various DNA-related activities. Here we show that the histone acetyltransferase p300 specifically acetylates HMG-14, a nonhistone structural protein that binds to nucleosomes and reduces the compactness of the chromatin fiber. We identify 7 major acetylation sites, 6 of which are novel and have not been known to be acetylated in either HMG-14 or the closely related HMG-17 protein. All the acetylation sites involve evolutionarily conserved residues: 3 within the HMG-14/-17 nucleosomal binding domain and 4 in or near the bipartite nuclear localization domains of the proteins. In tissue culture cells the acetylation pattern is indicative of a selective process in which a subfraction of HMG-14 is preferentially acetylated. We find that the nucleosomal binding domain is a major target for acetylation in vivo and that the specific acetylation of HMG-14 by p300 weakens its interaction with nucleosome cores. Our results suggest that p300 modulates the interaction of HMG-14 with nucleosomes. Thus, p300 may affect chromatin-related activities not only by modifying histones or transcription factors but also by targeting structural nonhistone proteins.

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.

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.

Dynamic relocation of chromosomal protein HMG-17 in the nucleus is dependent on transcriptional activity.

Chromosomal proteins HMG-14/-17 are nucleosomal binding proteins, which alter the structure of the chromatin fiber and enhance transcription, but only from chromatin templates. Here we show that in tissue culture cells, HMG-17 protein colocalizes with sites of active transcription. Incubation of permeabilized cells with a peptide corresponding to the nucleosomal binding domains of HMG-14/-17 specifically arrested polymerase II-dependent transcription. In these cells the peptide displaces HMG-17 from chromatin and reduces the cellular content of the protein. These results suggest that the presence of HMG-14/-17 in chromatin is required for efficient polymerase II transcription. In non-permeabilized, actively transcribing cells, the protein is dispersed in a punctate pattern, throughout the nucleus. Upon transcriptional inhibition by alpha-amanitin or actinomycin D, the protein gradually redistributes until it localizes fully to interchromatin granule clusters, together with the splicing factor SC35. The results suggest that the association of HMG-17 with chromatin is dynamic rather than static, and that in the absence of transcription, HMG-17 is released from chromatin and accumulates in interchromatin granule clusters. Thus, the intranuclear distribution of chromosomal proteins which act as architectural elements of chromatin structure may be dynamic and functionally related to the transcriptional activity of the cell.