Bone formation beyond the skeletal envelope using calcium phosphate granules packed into a collagen pouch-a pilot study.

In this proof-of-concept, bone neoformation beyond the skeletal envelope is explored by using a collagen pouch (n= 6) packed with calcium phosphate (CaP) granules placed over the frontal bone in sheep (n= 3). At 13 weeks, macroscopic examination showed specimens covered by an adherent fibrinous envelope with slight vascularization. Histology revealed colonization of the implant by newly formed woven bone and fibrous connective tissue. Surface osteoblasts as well as material-filled macrophages, lymphocytes, polymorphonuclear cells and giant cells were also found in large quantities surrounding the newly formed bone tissue inside the collagen pouch. On the side facing the recipient bone, the collagen membrane had to a large extent been resorbed and bridging bone formation was clearly visible between the test article and recipient bone. On the other side facing soft tissue, the collagen pouch remained intact with a visible fibrous capsule. This study demonstrated that the use of a collagen sleeve as a container for CaP granules allows for good neoformation beyond the skeletal envelope with bridging bone formation clearly visible between the test article and recipient bone. Additionally, in this model, macrophages rather than osteoclasts appear to modulate CaP granule resorption and remodeling into new bone. This construct opens new perspectives for treatment methods that could be used for bone augmentation and restoration of cranio-maxillofacial defects and malformations.

Bioinformatical dissection of fission yeast DNA replication origins.

Replication origins in eukaryotes form a base for assembly of the pre-replication complex (pre-RC), thereby serving as an initiation site of DNA replication. Characteristics of replication origin vary among species. In fission yeast Schizosaccharomyces pombe, DNA of high AT content is a distinct feature of replication origins; however, it remains to be understood what the general molecular architecture of fission yeast origin is. Here, we performed ChIP-seq mapping of Orc4 and Mcm2, two representative components of the pre-RC, and described the characteristics of their binding sites. The analysis revealed that fission yeast efficient origins are associated with two similar but independent features: a ≥15 bp-long motif with stretches of As and an AT-rich region of a few hundred bp. The A-rich motif was correlated with chromosomal binding of Orc, a DNA-binding component in the pre-RC, whereas the AT-rich region was associated with efficient binding of the DNA replicative helicase Mcm. These two features, in combination with the third feature, a transcription-poor region of approximately 1 kb, enabled to distinguish efficient replication origins from the rest of chromosome arms with high accuracy. This study, hence, provides a model that describes how multiple functional elements specify DNA replication origins in fission yeast genome.

Accumulation of Molecular Aberrations Distinctive to Hepatocellular Carcinoma Progression.

Cancer develops through the accumulation of genetic and epigenetic aberrations. To identify sequential molecular alterations that occur during the development of hepatocellular carcinoma (HCC), we compared 52 early and 108 overt HCC samples by genome sequencing. Gene mutations in the p53/RB1 pathway, WNT pathway, MLL protein family, SWI/SNF complexes, and AKT/PI3K pathway were common in HCC. In the early phase of all entities, TERT was the most frequently upregulated gene owing to diverse mechanisms. Despite frequent somatic mutations in driver genes, including CTNNB1 and TP53, early HCC was a separate molecular entity from overt HCC, as each had a distinct expression profile. Notably, WNT target genes were not activated in early HCC regardless of CTNNB1 mutation status because ß-catenin did not translocate into the nucleus due to the E-cadherin/ß-catenin complex at the membrane. Conversely, WNT targets were definitively upregulated in overt HCC, with CTNNB1 mutation associated with downregulation of CDH1 and hypomethylation of CpG islands in target genes. Similarly, cell-cycle genes downstream of the p53/RB pathway were upregulated only in overt HCC, with TP53 or RB1 gene mutations associated with chromosomal deletion of 4q or 16q. HCC was epigenetically distinguished into four subclasses: normal-like methylation, global-hypomethylation (favorable prognosis), stem-like methylation (poor prognosis), and CpG island methylation. These methylation statuses were globally maintained through HCC progression. Collectively, these data show that as HCC progresses, additional molecular events exclusive of driver gene mutations cooperatively contribute to transcriptional activation of downstream targets according to methylation status. SIGNIFICANCE: In addition to driver gene mutations in the WNT and p53 pathways, further molecular events are required for aberrant transcriptional activation of these pathways as HCC progresses.

Comparative analysis demonstrates cell type-specific conservation of SOX9 targets between mouse and chicken.

SRY (sex-determining region Y)-box 9 (SOX9) is a transcription factor regulating both chondrogenesis and sex determination. Among vertebrates, SOX9's functions in chondrogenesis are well conserved, while they vary in sex determination. To investigate the conservation of SOX9's regulatory functions in chondrogenesis and gonad development among species, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using developing limb buds and male gonads from embryos of two vertebrates, mouse and chicken. In both mouse and chicken, SOX9 bound to intronic and distal regions of genes more frequently in limb buds than in male gonads, while SOX9 bound to the proximal upstream regions of genes more frequently in male gonads than in limb buds. In both species, SOX palindromic repeats were identified more frequently in SOX9 binding regions in limb bud genes compared with those in male gonad genes. The conservation of SOX9 binding regions was significantly higher in limb bud genes. In addition, we combined RNA expression analysis (RNA sequencing) with the ChIP-seq results at the same stage in developing chondrocytes and Sertoli cells and determined SOX9 target genes in these cells of the two species and disclosed that SOX9 targets showed high similarity of targets in chondrocytes, but not in Sertoli cells.

Microarray expression analysis of genes involved in innate immune memory in peritoneal macrophages.

Immunological memory has been believed to be a feature of the adaptive immune system for long period, but recent reports suggest that the innate immune system also exhibits memory-like reaction. Although evidence of innate immune memory is accumulating, no in vivo experimental data has clearly implicated a molecular mechanism, or even a cell-type, for this phenomenon. In this study of data deposited into Gene Expression Omnibus (GEO) under GSE71111, we analyzed the expression profile of peritoneal macrophages isolated from mice pre-administrated with toll-like receptor (TLR) ligands, mimicking pathogen infection. In these macrophages, increased expression of a group of innate immunity-related genes was sustained over a long period of time, and these genes overlapped with ATF7-regulated genes. We conclude that ATF7 plays an important role in innate immune memory in macrophages.

Rif1 binds to G quadruplexes and suppresses replication over long distances.

Rif1 regulates replication timing and repair of double-strand DNA breaks. Using a chromatin immunoprecipitation-sequencing method, we identified 35 high-affinity Rif1-binding sites in fission yeast chromosomes. Binding sites tended to be located near dormant origins and to contain at least two copies of a conserved motif, CNWWGTGGGGG. Base substitution within these motifs resulted in complete loss of Rif1 binding and in activation of late-firing or dormant origins located up to 50 kb away. We show that Rif1-binding sites adopt G quadruplex-like structures in vitro, in a manner dependent on the conserved sequence and on other G tracts, and that purified Rif1 preferentially binds to this structure. These results suggest that Rif1 recognizes and binds G quadruplex-like structures at selected intergenic regions, thus generating local chromatin structures that may exert long-range suppressive effects on origin firing.

The transcription factor ATF7 mediates lipopolysaccharide-induced epigenetic changes in macrophages involved in innate immunological memory.

Immunological memory is thought to be mediated exclusively by lymphocytes. However, enhanced innate immune responses caused by a previous infection increase protection against reinfection, which suggests the presence of innate immunological memory. Here we identified an important role for the stress-response transcription factor ATF7 in innate immunological memory. ATF7 suppressed a group of genes encoding factors involved in innate immunity in macrophages by recruiting the histone H3K9 dimethyltransferase G9a. Treatment with lipopolysaccharide, which mimics bacterial infection, induced phosphorylation of ATF7 via the kinase p38, which led to the release of ATF7 from chromatin and a decrease in repressive histone H3K9me2 marks. A partially disrupted chromatin structure and increased basal expression of target genes were maintained for long periods, which enhanced resistance to pathogens. ATF7 might therefore be important in controlling memory in cells of the innate immune system.

Temporal and spatial regulation of eukaryotic DNA replication: from regulated initiation to genome-scale timing program.

Replication origins are where pre-replication complexes are assembled during G1 phase. However, only a subset of the origins is actually "fired" to initiate DNA synthesis during S phase. Whereas factors involved in these steps are relatively well understood now, the mechanisms behind the origin specification, the choice of origins to be fired and determination of their timing are still under active investigation. Recent data show that the origin positions as well as the selection of those to be fired may be determined by multiple factors including sequences, chromatin context, epigenetic information, and some specific genomic features, but that the choice is surprisingly plastic and opportunistic. Timing regulation of firing, on the other hand, appears to be related to cell type-specific intrinsic chromatin architecture in nuclei. The conserved Rif1 protein appears to be a major global regulator of the genome-wide replication timing. Replication timing is regulated also by other factors including checkpoint signals, local chromatin structures, timing and quantity of pre-RC formation, and availability of limiting initiation factors.

Rif1 is a global regulator of timing of replication origin firing in fission yeast.

One of the long-standing questions in eukaryotic DNA replication is the mechanisms that determine where and when a particular segment of the genome is replicated. Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication and may affect the site selection and timing of origin firing. We identified rif1Δ, a null mutant of rif1(+), a conserved telomere-binding factor, as an efficient bypass mutant of fission yeast hsk1. Extensive deregulation of dormant origins over a wide range of the chromosomes occurs in rif1Δ in the presence or absence of hydroxyurea (HU). At the same time, many early-firing, efficient origins are suppressed or delayed in firing timing in rif1Δ. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that only partially overlap with the prereplicative complex assembly sites, although Rif1 tends to bind in the vicinity of the late/dormant origins activated in rif1Δ. The binding to the arm segments occurs through M to G1 phase in a manner independent of Taz1 and appears to be essential for the replication timing program during the normal cell cycle. Our data demonstrate that Rif1 is a critical determinant of the origin activation program on the fission yeast chromosomes.

The relationship between DNA replication and human genome organization.

Assessment of the impact of DNA replication on genome architecture in Eukaryotes has long been hampered by the scarcity of experimental data. Recent work, relying on computational predictions of origins of replication, suggested that replication might be a major determinant of gene organization in human (Huvet et al. 2007. Human gene organization driven by the coordination of replication and transcription. Genome Res. 17:1278-1285). Here, we address this question by analyzing the first large-scale data set of experimentally determined origins of replication in human: 283 origins identified in HeLa cells, in 1% of the genome covered by ENCODE regions (Cadoret et al. 2008. Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc Natl Acad Sci USA. 105:15837-15842). We show that origins of replication are not randomly distributed as they display significant overlap with promoter regions and CpG islands. The hypothesis of a selective pressure to avoid frontal collisions between replication and transcription polymerases is not supported by experimental data as we find no evidence for gene orientation bias in the proximity of origins of replication. The lack of a significant orientation bias remains manifest even when considering only genes expressed at a high rate, or in a wide number of tissues, and is not affected by the regional replication timing. Gene expression breadth does not appear to be correlated with the distance from the origins of replication. We conclude that the impact of DNA replication on human genome organization is considerably weaker than previously proposed.

Genome-wide studies highlight indirect links between human replication origins and gene regulation.

To get insights into the regulation of replication initiation, we systematically mapped replication origins along 1% of the human genome in HeLa cells. We identified 283 origins, 10 times more than previously known. Origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no origins in large GC-poor regions. Origin sequences are evolutionarily conserved, and half of them map within or near CpG islands. Most of the origins overlap transcriptional regulatory elements, providing further evidence of a connection with gene regulation. Moreover, we identify c-JUN and c-FOS as important regulators of origin selection. Half of the identified replication initiation sites do not have an open chromatin configuration, showing the absence of a direct link with gene regulation. Replication timing analyses coupled with our origin mapping suggest that a relatively strict origin-timing program regulates the replication of the human genome.