Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable. A recently emerging paradigm for the epigenetic control and propagation of gene expression states involves the role of chromatin structure. Though historically viewed as a passive packaging structure primarily used to assemble the enormous amount of DNA into a eukaryotic nucleus, the nucleosome with its complement of core histones has emerged as a key target for regulating gene expression (Wolffe and Hansen 2001). The dynamic regulation of chromatin organization appears to be accomplished by macromolecular protein complexes that contain enzymatic activities that modify the tails of the core histones. The constellation of these histone modifications, including acetylation, phosphorylation, ubiquitination, and methylation, create both synergistic and antagonistic signals that correlate with the transcriptional activity of a gene (Wu and Grunstein 2000). This emerging "histone code" is hypothesized to create functionally distinct subdomains in chromatin that define active versus transcriptionally silent genes (Jenuwein and Allis 2001). Histone modifications and the chromatin-associated proteins that interpret these signals may represent an epigenetic marking system responsible for setting and maintaining heritable programs of gene expression during development.The role of histone acetylation/deacetylation [mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively] in modulating gene activity is now well established (Kuo and Allis 1998). The role of histone methylation in the regulation of chromatin structure and gene transcription has been...

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5CKOCKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.

We have previously identified 67 exons on a yeast artificial chromosome contig spanning 1.5 Mb around the multidrug resistance 1 gene region of human chromosome 7q21.1. In this study, we identified three novel cytoplasmic variants (MDC2-γ γ γ γ, MDC2-δ δ δ δ, and MDC2-ε ε ε ε) of the human metalloprotease-like disintegrin-like cysteine-rich protein 2 (MDC2) among these exons by screening a human brain cDNA library and also by using a reverse transcription polymerase chain reaction. Genomic sequence analysis strongly supported the idea that the variations in the cytoplasmic domain were generated by alternative splicing. The expression of MDC2 variant forms in human brain tissue and gliomas was examined by reverse transcription polymerase chain reaction and RNase protection assay. MDC2-ε ε ε ε was expressed only in the cortical and hippocampal regions in human brain, but not in gliomas. In contrast, MDC2-γ γ γ γ was a major form expressed in human gliomas. Specific expression of these cytoplasmic variants of MDC2 in human brain and its malignancies is discussed. Key words: MDC -ADAM -Alternative splicing -MDR regionThe mammalian ADAM (a disintegrin and metalloprotease) family is a recently identified gene family encoding membrane proteins, and has an extensive sequence similarity to snake venom disintegrin and metalloprotease. 1)Snake venom disintegrins are a family of anticoagulant peptides with a high cysteine content. 1) Typical ADAMs are cell-surface proteins that have multiple domains; pro, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domains. The ADAM family is expected to be involved in cell-cell interactions. For example, fertilin, the first ADAM described, has been implicated in integrin-mediated sperm-egg binding, 2, 3) and meltrin is required for myotube formation.4) In contrast, some members of this ADAM protein family have metalloprotease-like domains that are catalytically active and degrade specific substrates. An example of this type of ADAM is human tumor necrosis factor-α converting enzyme, which releases soluble tumor necrosis factor-α by proteolysis.5, 6) Because cellcell interaction and protease activity have important roles in metastasis and invasion of cancer cells, the ADAM family could be associated with malignant phenotype in cancer cells.We previously assembled a contig of 21 non-chimeric yeast artificial chromosomes (YAC) across 1.5 Mb of the multidrug resistance gene (PGY1 and PGY3) region on human chromosome 7q21.1. 7) Exon-trapping directly on the YACs resulted in an exon collection that includes 21 exons identical to the cDNA sequences of PGY1, PGY3, and sorcin, and 43 exons homologous or similar to human cDNA sequences. 8) In these 43 exons, nine exons were homologous to a cDNA coding a human metalloprotease/ disintegrin-like cysteine-rich protein (MDC), which is a member of the ADAM family.9, 10) These exons specifically detected a prominent 10 kb mRNA in the brain among various human tissues examined. 8) Sagan...