Abdominal-class homeodomain-containing (Hox) factors form multimeric complexes with TALE-class homeodomain proteins (Pbx, Meis) to regulate tissue morphogenesis and skeletal development. Here we have established that Pbx1 negatively regulates Hoxa10-mediated gene transcription in mesenchymal cells and identified components of a Pbx1 complex associated with genes in osteoblasts. Expression of Pbx1 impaired osteogenic commitment of C3H10T1/2 multipotent cells and differentiation of MC3T3-E1 preosteoblasts. Conversely, targeted depletion of Pbx1 by short hairpin RNA (shRNA) increased expression of osteoblastrelated genes. Studies using wild-type and mutated osteocalcin and Bsp promoters revealed that Pbx1 acts through a Pbx-binding site that is required to attenuate gene activation by Hoxa10. Chromatin-associated Pbx1 and Hoxa10 were present at osteoblast-related gene promoters preceding gene expression, but only Hoxa10 was associated with these promoters during transcription. Our results show that Pbx1 is associated with histone deacetylases normally linked with chromatin inactivation. Loss of Pbx1 from osteoblast promoters in differentiated osteoblasts was associated with increased histone acetylation and CBP/p300 recruitment, as well as decreased H3K9 methylation. We propose that Pbx1 plays a central role in attenuating the ability of Hoxa10 to activate osteoblast-related genes in order to establish temporal regulation of gene expression during osteogenesis.

Prostate cancer remains a life-threatening disease among men worldwide. The majority of PCa-related mortality results from metastatic disease that is characterized by metastasis of prostate tumor cells to various distant organs, such as lung, liver, and bone. Bone metastasis is most common in prostate cancer with osteoblastic and osteolytic lesions. The precise mechanisms underlying PCa metastasis are still being delineated. Intercellular communication is a key feature underlying prostate cancer progression and metastasis. There exists local signaling between prostate cancer cells and cells within the primary tumor microenvironment (TME), in addition to long range signaling wherein tumor cells communicate with sites of future metastases to promote the formation of pre-metastatic niches (PMN) to augment the growth of disseminated tumor cells upon metastasis. Over the last decade, exosomes/ extracellular vesicles have been demonstrated to be involved in such signaling. Exosomes are nanosized extracellular vesicles (EVs), between 30 and 150 nm in thickness, that originate and are released from cells after multivesicular bodies (MVB) fuse with the plasma membrane. These vesicles consist of lipid bilayer membrane enclosing a cargo of biomolecules, including proteins, lipids, RNA, and DNA. Exosomes mediate intercellular communication by transferring their cargo to recipient cells to modulate target cellular functions. In this review, we discuss the contribution of exosomes/extracellular vesicles in prostate cancer progression, in pre-metastatic niche establishment, and in organ-specific metastases. In addition, we briefly discuss the clinical significance of exosomes as biomarkers and therapeutic agents.

Although DNA replication has been thought to play an important role in the silencing of mating type loci in Saccharomyces cerevisiae, recent studies indicate that silencing can be decoupled from replication. In Schizosaccharomyces pombe, mating type silencing is brought about by the trans-acting proteins, namely Swi6, Clr1-Clr4, and Rhp6, in cooperation with the cis-acting silencers. The latter contain an autonomous replication sequence, suggesting that DNA replication may be critical for silencing in S. pombe. To investigate the connection between DNA replication and silencing in S. pombe, we analyzed several temperature-sensitive mutants of DNA polymerase ␣. We find that one such mutant, swi7H4, exhibits silencing defects at mat, centromere, and telomere loci. This effect is independent of the checkpoint and replication defects of the mutant. Interestingly, the extent of the silencing defect in the swi7H4 mutant at the silent mat2 locus is further enhanced in absence of the cis-acting, centromere-proximal silencer. The chromodomain protein Swi6, which is required for silencing and is localized to mat and other heterochromatin loci, interacts with DNA polymerase ␣ in vivo and in vitro in wild type cells. However, it does not interact with the mutant pol␣ and is delocalized away from the silent mat loci in the mutant. Our results demonstrate a role of DNA polymerase ␣ in the establishment of silencing. We propose a recruitment model for the coupling of DNA replication with the establishment of silencing by the chromodomain protein Swi6, which may be applicable to higher eukaryotes.The well studied system of mating type silencing in the budding yeast Saccharomyces cerevisiae has served as a paradigm for developmental regulation of gene regulation. Although the mating type phenotype of a homothallic strain is dictated by the MAT locus depending on whether it harbors the a-or ␣-specific alleles, two copies of the same genetic information are located at distant sites on the same chromosome, namely HML and HMR, which harbor ␣ and a alleles, respectively. However, these alleles are transcriptionally silent. The silencing is achieved by the cis-acting sequences E (essential) and I (important) that flank both HML and HMR loci (1, 2). In addition, several genes encode factors named mating type regulator/silent information regulator (MAR/SIR) that function in trans through the cis-acting sequences in keeping the HML and HMR loci silent. Extensive studies in S. cerevisiae have suggested that DNA replication is important for repression of the silent mating type loci HML and HMR (see Refs. 1 and 2 for reviews). These findings include a requirement of passage through S phase, a functional autonomous replication sequence (ARS) flanking the silent locus HMR, and a functional origin recognition complex for silencing (reviewed in Ref. 2). However, the requirement of DNA replication for silencing is obviated if the SIR1 silencing protein is recruited by alternative means, although passage through S phase is still essential (3, 4)....

Swi6/HP1, an evolutionarily conserved protein, is critical for heterochromatin assembly in fission yeast and higher eukaryotes. In fission yeast, histone deacetylation by histone deacetylases is thought to be followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/Suv39H1. H3-Lys-9-Me2 interacts with the chromodomain of Swi6/HP1. Swi6/HP1 is thought to act downstream of Clr4/Suv39, and further self-association of Swi6/HP1 is assumed to stabilize the heterochromatin structure. Here, we show that the self-association-defective mutant of Swi6 does not interact with Clr4. It not only fails to localize to heterochromatin loci but also interferes with heterochromatic localization of H3-Lys-9-Me2 (and thereby Clr4) and the endogenous Swi6 in a dominant negative manner. Thus, self-association of Swi6/HP1 helps in binding to and recruitment of Clr4 and thereby in establishment and maintenance of heterochromatin by a concerted rather than a sequential mechanism.Distinct sets of histone modifications organize chromatin into transcriptionally expressed or repressed structures. As in higher eukaryotes, fission yeast euchromatic regions are associated with hyperacetylated histones, particularly Lys-9 and Lys-14 acetylated and Lys-4 methylated histone H3. Conversely, heterochromatin assembly (silent mating type, centromere, rDNA, and telomere loci) involves deacetylation of histone H3 at Lys-9 and Lys-14 by Clr3, Clr6, and Sir2, followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/ Suv39H1. H3-Lys-9-Me2 is recognized by the chromodomain in Swi6/HP1 (1, 2). The property of Swi6 to form multimers is thought to cause folding of chromatin into a transcriptionally inactive structure (3). The RNAi pathway is also involved in heterochromatin assembly. The dcr1⌬, rdp1⌬, and ago1⌬ mutants are defective in H3-Lys-9 methylation and Swi6 recruitment at heterochromatin loci (4).Structural studies show that the chromoshadow domain (CSD) 3 in Swi6/HP1 contains a dimerization motif that creates a cleft for binding a pentapeptide (5, 6). Mutations in the conserved residues in the cleft inhibit interaction with the pentapeptide motif in proteins (6, 7). We found that mutation of one such residue (L315E) severely compromises the self-association property of Swi6. is not only defective in complementing the silencing defect in the swi6⌬ mutant but also exerts a dominant negative effect on silencing, accompanied by loss of heterochromatic localization not only of itself but also that of native Swi6 and H3-Lys-9-Me2 and, by implication, of Clr4. These results support a concerted rather than a sequential action of Clr4 and Swi6 in establishment and maintenance of heterochromatin. EXPERIMENTAL PROCEDURESStrains and Plasmids Used-All strains and plasmids used are listed in supplemental Tables 1 and 2, respectively.Media Compositions-All fission yeast media were prepared according to Moreno et al. (8). The serial dilution plate assay has been described earlier (9). Iodine staining assays for switching and silencing have ...

To our knowledge this is the first report that tempol and insulin can restore erectile function in diabetic rats by inhibiting apoptosis.