Distinct chromatin signature of histone H3 variant H3.3 in human cells.

Actively transcribed regions of the genome have been found enriched for the histone H3 variant H3.3. This variant is incorporated into nucleosomes throughout the cell cycle whereas the canonical isoforms are predominately deposited in association with replication. In order to obtain a global picture of the deposition pattern at the single cell level we expressed H3.3 in both normal and malignant human cells and analyzed nuclei using conventional and structured illumination imaging (SIM). We found that the distribution pattern of H3.3 in interphase differs from that of the canonical histone H3 variants and this difference is conveyed to mitotic chromosomes which display a distinct H3.3 banding pattern. Histone H3.3 localization positively correlated with markers for transcriptionally active chromatin and, notably, H3.3 was almost completely absent from the inactive X chromosome. Collectively, our data show that histone variant H3.3 occupies distinct intranuclear chromatin domains and that these genomic loci are associated with gene expression.

Establishment and characterization of a primary and a metastatic melanoma cell line from Grey horses.

The Grey horse phenotype, caused by a 4.6 kb duplication in Syntaxin 17, is strongly associated with high incidence of melanoma. In contrast to most human melanomas with an early onset of metastasis, the Grey horse melanomas have an extended period of benign growth, after which 50% or more eventually undergo progression and may metastasize. In efforts to define changes occurring during Grey horse melanoma progression, we established an in vitro model comprised of two cell lines, HoMel-L1 and HoMel-A1, representing a primary and a metastatic stage of the melanoma, respectively. The cell lines were examined for their growth and morphological characteristics, in vitro and in vivo oncogenic potential, chromosome numbers, and expression of melanocytic antigens and tumor suppressors. Both cell lines exhibited malignant characteristics; however, the metastatic HoMel-A1 showed a more aggressive phenotype characterized by higher proliferation rates, invasiveness, and a stronger tumorigenic potential both in vitro and in vivo. HoMel-A1 displayed a near-haploid karyotype, whereas HoMel-L1 was near-diploid. The cell lines expressed melanocytic lineage markers such as TYR, TRP1, MITF, PMEL, ASIP, MC1R, POMC, and KIT. The tumor suppressor p53 was strongly expressed in both cell lines, while the tumor suppressors p16 and PTEN were absent in HoMel-A1, potentially implicating significance of these pathways in the melanoma progression. This in vitro model system will not only aid in understanding of the Grey horse melanoma pathogenesis, but also in unraveling the steps during melanoma progression in general as well as being an invaluable tool for development of new therapeutic strategies.

Chromosomal dynamics of cell cycle regulator gene p21 during transcriptional activation.

The radial position of a gene within its chromosome territory (CT) in the interphase nucleus is thought to depend on the transcriptional activity of the gene and on transcriptional activity, gene density, and conformation of the chromosomal surrounding. In this study we analyzed the position of the cell cycle regulator gene p21 within the CT of human chromosome 6 (HSA6) upon transcriptional activation. Whereas the majority of active p21 genes is located in the interior of the CT of HSA6, induction of p21 transcription correlates with increased variation of gene localization within the CT and with a higher percentage of p21 genes located at the periphery of the CT. Additionally it demonstrates once more that transcription can take place throughout CTs. Comparison of the p21 locus with two non-coding regions on HSA6 showed that both non-coding sequences are located more frequently in the interior of the CT than p21 genes although they are situated in chromosomal neighborhoods with widely differing gene density and regional transcriptional activity. Thus our data support models describing an influence of the transcriptional activity of a gene on the localization within its CT. However, our data also indicate that additional factors such as chromatin remodeling are implicated in the positioning of genes within the respective chromosome territory.

Expression patterns of protein C inhibitor in mouse development.

Proteolysis of extracellular matrix is an important requirement for embryonic development and is instrumental in processes such as morphogenesis, angiogenesis, and cell migration. Efficient remodeling requires controlled spatio-temporal expression of both the proteases and their inhibitors. Protein C inhibitor (PCI) effectively blocks a range of serine proteases, and recently has been suggested to play a role in cell differentiation and angiogenesis. In this study, we mapped the expression pattern of PCI throughout mouse development using in situ hybridization and immunohistochemistry. We detected a wide-spread, yet distinct expression pattern with prominent PCI levels in skin including vibrissae, and in fore- and hindgut. Further sites of PCI expression were choroid plexus of brain ventricles, heart, skeletal muscles, urogenital tract, and cartilages. A strong and stage-dependent PCI expression was observed in the developing lung. In the pseudoglandular stage, PCI expression was present in distal branching tubules whereas proximal tubules did not express PCI. Later in development, in the saccular stage, PCI expression was restricted to distal bronchioli whereas sacculi did not express PCI. PCI expression declined in postnatal stages and was not detected in adult lungs. In general, embryonic PCI expression indicates multifunctional roles of PCI during mouse development. The expression pattern of PCI during lung development suggests its possible involvement in lung morphogenesis and angiogenesis.