We have used the cloning of cancer-associated chromosomal aberrations as a means of discovering genes that play roles in growth and development. In the past, this strategy led to our discovery of six genes. The SIL gene was discovered because of its involvement in an interstitial deletion that is recurrent in T-cell acute lymphoblastic leukemias 1. SIL encodes a 150 kDa protein without clear homology to other known functional protein families or motifs 2. Sequence analysis suggests that Sil is a non-globular protein. This is a characteristic of proteins that seem to be able to diverge quickly during evolution and it is not seen, in general, in proteins that have catalytic activity. Following the structural characterization of this gene, genetic and biochemical studies were begun to investigate Sil function. The SIL knockout mouse dies during embryonic development between E8.5-10.53. Sil null embryos 7.5 to 8.5 days old are reduced in size with delayed development and regional apoptosis. In addition, they show failure of midline formation and randomization of left/right asymmetry. Detailed analysis of this phenotype has implicated Sil as a positive regulator of the sonic hedgehog pathway governing neural tube and notochord development 4. Several of these features were observed as well in zebrafish Sil null embryos 5.SIL is expressed in essentially all proliferating tissues: the expression being highest in fetal thymus, bone marrow, and fetal liver, but extremely low in adult muscle and brain. In tissue culture, SIL is an """"""""immediate early"""""""" gene whose mRNA expression is upregulated upon serum stimulation of quiescent fibroblasts. In contrast, contact inhibition, serum starvation, or proliferation arrest induced by terminal differentiation cause a decrease of SIL mRNA to undetectable levels 6. In continuously growing cells SIL displays an oscillatory protein accumulation during the cell cycle, despite having steady state levels of mRNA. These observations have suggested a possible role for Sil in cellular proliferation. We previously demonstrated an interaction of Sil with the mitotic regulatory protein, Pin1. We have now demonstrated post-translational phosphorylation of Sil within a critical region of the protein. Overexpression of Sil with a mutation in this region results in a premature exit of cells from the mitotic checkpoint. This same region is responsible for the interaction of Sil with Pin1. This escape from checkpoint arrest in the presence of the mutant Sil protein occurs in association with a progressive decline in Cdc2 activity apparently related to downregulation of the kinase activity of the Cyclin B1 complex caused by failure to maintain phosphorylation of threonine 161 on the Cdc2 subunit. We also provide data strongly suggesting that the degradation of Sil is mediated by the anaphase promoting complex (APC) following Sil ubiquitination. Our future plans include a refined dissection of the phosphorylation sites present in the critical region to determine which are the most critical for the phenotype that we have observed. We also hope to confirm the influence of the amino terminal portion of the protein on APC-mediated destruction by fusing it to another protein and demonstrating APC mediated degradation. We have also eliminated murine Sil from the cellular milieu by siRNA. Obviously, in contrast to the more focused regional phosphorylation site mutations, this approach eliminates the entire protein. The phenotype that we observe in this case also suggests a function for Sil in the mitotic checkpoint but expands the possible area(s) where the influence of Sil is felt. In the case of siRNA inhibition, G2 synchronized cells treated with an inducer of the mitotic checkpoint fail to accumulate in prometaphase and have increased apoptosis. An inducible human siRNA SIL construct has now been created and is being tested. So far it has yielded results similar to the constitutively activated murine Sil inhibitor. We hope to combine this construct with a set of targeted mutations of murine Sil so that we can dissect out the functions of different parts of the protein.Our work in the development of an in vivo conditional knockout model for Sil has gradually progressed. Using a BAC created by Dr. Aplan from this Branch we now have access to a readily rearranging and Sil-deleting construct which we have successfully crossed into an endogenous Sil+/- background. We expect, over the next few months, to have it in place in an endogenous Sil null background. There, assuming that the construct can """"""""rescue"""""""" the mouse from the Sil-null phenotype (which seems to be likely given the amount of Sil that is expressed from this construct) the influence of Sil on thymic development and lymphomagenesis will be studied.
| Erez, Ayelet; Perelman, Marina; Hewitt, Stephen M et al. (2004) Sil overexpression in lung cancer characterizes tumors with increased mitotic activity. Oncogene 23:5371-7 |
| Izraeli, S; Lowe, L A; Bertness, V L et al. (1999) The SIL gene is required for mouse embryonic axial development and left-right specification. Nature 399:691-4 |
