The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct,

The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineagespecific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J H rearrangement, whereas V H -DJ H and V j -J j rearrangements are severely impaired. D-J H coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination. The EMBO Journal (2012) 31, 945-958.

Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.

SUMMARY V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-strand breaks adjacent to signal sequences and creates hairpin coding ends. Here we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination using 13 highly purified polypeptides, thereby permitting variable domain exon assembly using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.

Freeze-dried Saos-2, human osteosarcoma cells, and extracts of Saos-2 cells contain all components necessary to induce ectopic new bone and marrow when implanted into athymic Nu/Nu nuce. On the other hand, human osteosarcoma cells of the U-2 OS strain failed to induce bone formation under the same experimental conditions. Our aim was to compare the relative expressions of known osteoinductive factors including bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGF-beta) in these two cell lines in an attempt to explain the unique bone-inducing ability of the Saos-2 cells. Saos-2 cells expressed mRNA for BMP-1, -2, -3, -4,-6, and TGF-beta 1. The non-osteoinductive U-2 OS cells expressed BMP-2, -4, -5, -6, and -7 as well as TGF-beta 1 mRNA, while levels of BMP-1 and BMP-3 mRNA were either not detectable or detectable at a very low level in U-2 OS cells. The presence of BMP-1 and -4 protein was confirmed in Saos-2 cells by immunofluorescence, and TGF beta protein was demonstrated by bioassay in both cell types. These findings suggest that Saos-2 cells are endowed qualitatively and quantitatively with sufficient amounts of many bone morphogenetic proteins-especially BMP-1, -3, and -4-to confer osteoinductivity upon these cells. However, the absence of osteoinductivity in U-2 OS cells, despite significant mRNA expression levels of several bone morphogenetic proteins, suggests that, even though expression of one or more bone morphogenetic proteins may be present, it may not necessarily be sufficient to confer osteoinductivity upon U-2 OS cells. U-2 OS cells may be non-osteoinductive because (1) they contain inhibitors to the BMPs or secrete inhibitory binding proteins, (2) they do not process BMPs correctly, or (3) the BMPs are inappropriately localized and sequestered within the U-2 OS cells. Saos 2 cells may be osteoinductive because (1) they uniquely express BMP-1, (2) they express an appropriate combination of interactive BMPs at appropriate levels, and/or (3) the Saos-2 cells elaborate as-yet-unidentified osteoinductive factor(s).

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Implants of defatted, freeze-dried Saos-2 human osteosarcoma cells grown to confluency induce de novo bone formation in athymic mice. These cells are also richly endowed with bone morphogenetic proteins and express mRNA for bone morphogenetic proteins 1, 2, 3, 4 and 6, as well as for transforming growth factor-beta 1. Our aim was to study whether the ability to induce bone formation is related to the level of expression of bone morphogenetic protein. We studied the osteoinductive abilities and levels of expression of bone morphogenetic protein of Saos-2 cells both during the growth phase and after confluency was reached. Subconfluent cells were at least 70% less effective in their osteoinductive ability than confluent cells. Comparison of bone morphogenetic protein mRNA expression in confluent and subconfluent cells revealed that the latter had lower expression of all the mRNAs studied. The expression of bone morphogenetic protein-1, bone morphogenetic protein-2, and bone morphogenetic protein-6 mRNAs was 2, 3, and 6 to 10-fold lower, respectively, in subconfluent cells. These results suggest that the ability of Saos-2 cells to induce de novo bone formation may be correlated with the relative expression of these proteins; the expression of bone morphogenetic proteins in Saos-2 cells also may be dependent on the cell cycle.