Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination.in ovo electroporation | basement membrane N eural crest cells (NCCs) belong to a transient population of embryonic multipotent stem cells that are induced at the interface between the neuroectoderm and the prospective epidermis. These cells delaminate from the epithelium and adopt a characteristic migratory pathway into the periphery, where they contribute to several tissues, notably cranial facial structures and peripheral nervous system (1). During delamination, NCCs undergo an epithelial-mesenchyme transition (EMT), characterized by loss of cell-cell contacts and polarity, as well as acquisition of migratory capabilities (2). Successful EMT and delamination are essential steps for the subsequent migration and differentiation of NCCs. Consequently, mutations in genes involved in NCCs EMT are often associated with congenital defects (3).Previous studies showed that Sry-related HMG box-9 (Sox9), a member of the SoxE family of transcription factors, plays several roles in NC formation, survival, EMT, and differentiation (4-6). In mice harboring a null mutation in Sox9, NCCs were still generated but died by apoptosis before or shortly after NC delamination, suggesting that Sox9 is required for NC survival (4). In chick, misexpression of Sox9 in the neural tube was sufficient to induce NC identity at the expense of neural fate but only inefficiently induced delamination and EMT (5). By contrast, coexpression of Sox9 with the zinc finger-type transcription factor, Snail2, promoted systematic ectopic EMT and NC delamination along the dorsal-ventral extent of the neural tube (4). In addition, in vitro studies showed that Sox9 physically interacts with Snail2 (6). Altogether, these data suggest that the partnership between Sox9 and Snail2 may underlie the ability of Sox9 to induce NC delamination and EMT. However, how the combination of Sox9 and Snail2 achieves this is unclear.The ability of Sox9 to initiate NC delamination appears to be regulated by protein kinase A (PKA)-mediated phosphorylation at two serine residues (S64 a...

No abstract

The ART (Activator of Replication and Transcription) protein of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8 (HHV-8), is encoded by the ORF50 gene. It is expressed as an immediate-early gene and plays a crucial role in the transition between latency and productive infection. HHV-8 ART is a transcriptional transactivator which can up-regulate viral gene expression. Transient expression assays showed that ART strongly activated ORF57 and K8 promoter-directed gene expression in both CV-1 and BJAB cells. The ART target site was mapped to a 40-bp region compassing nt 81904 to 81943 on the ORF57 promoter. When linked upstream to a heterologous SV40 promoter, this region by itself was able to confer ART responsiveness. This 40-bp segment contains a 16-bp consensus sequence which is also found in the K8 promoter region located between nt 74769 to 74784. Deletion of the fragment including this 16-bp consensus abrogated the ART responsiveness of the K8 promoter. The role of this 16-bp consensus in ART transactivation was further supported by site-directed mutagenesis. Mutations of the conserved nucleotides within the 16-bp consensus in the ORF57 promoter dramatically impaired its responsiveness to ART. Fusion protein analysis with chimeric proteins containing the DNA binding domain of yeast transactivator Gal4 (residues 1 to 147) and different ART segments defined an acidic C-terminal region (amino acids [aa] 527 to 634) as a potent activator. Deletions of this activation domain in the ART protein resulted in a decrease or loss of its ability to activate ORF57 and K8 promoters containing the ART responsive element in transfected cells. How the ART activation domain activates ORF57 and K8 gene expression through the 16-bp consensus sequence remains to be determined.

The seroprevalence of human herpesvirus 8 (HHV-8) was studied in 326 human immunodeficiency virus (HIV)-positive and -negative persons from Honduras; women constituted 77% (n = 251) of the subjects. Sera were tested for lytic HHV-8 antibodies by an IFA, and positive samples were confirmed by a radioimmunoprecipitation assay. Of the 326 persons tested, 58 (17.8%) had HHV-8 antibodies. Among the HIV-infected women, 22.7% were seropositive; 11.3% of the HIV-negative women were seropositive. HHV-8 seroprevalence was almost four times higher in HIV-positive female commercial sex workers (36%) than in HIV-negative female non-commercial sex workers (9.9%; odds ratio = 3.8, 95% confidence interval = 1.1-13; P = 0.01), suggesting that commercial sex work is a risk factor for HHV-8 infection. In the men studied, the overall HHV-8 seroprevalence was 22.6%, with a seropositivity rate of 28% for HIV-positive men compared with 12% for HIV-negative men.

SummaryRecent studies using defined transcription factors to convert skin fibroblasts into chondrocytes have raised the question of whether osteo-chondroprogenitors expressing SOX9 and RUNX2 could also be generated during the course of the reprogramming process. Here, we demonstrated that doxycycline-inducible expression of reprogramming factors (KLF4 [K] and c-MYC [M]) for 6 days were sufficient to convert murine fibroblasts into SOX9+/RUNX2+ cellular aggregates and together with SOX9 (S) promoted the conversion efficiency when cultured in a defined stem cell medium, mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in vitro, but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether, we provide a reprogramming strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues.