p21 as a transcriptional regulator of Bmp2 genestructural requirements and molecular partners

Abstract

Cell proliferation occurs through a series of stages that are collectively termed the cell cycle. In eukaryotes, the cell cycle is divided into four phases that are organized around the synthesis (S phase) and mitotic segregation (M phase) of the genome. The two other phases of the cycle, G1 and G2, represent gap periods during which cells check cell-intrinsic and extrinsic signals in order to ensure that the conditions are suitable for the S and M phase, respectively. There is also a quiescent state, known as G0, in which the cell exits the cell cycle and reaches a non-proliferative state. The cell cycle is regulated by a molecular network of proteins which operates in the cell nucleus and integrates signals from both outside and inside the cell. Core components of this machinery are a family of serine/threonine kinases named cyclin-dependent kinases (Cdk) which are activated by their associated cyclins. The kinase activity of cyclin-Cdk complexes is tightly regulated by a plethora of Cdk inhibitors (CKIs), which serve as brakes to halt cell cycle progression under unfavorable conditions. CKIs are divided into two families on the basis of their structure and specificities: The INK4 family (Inhibitors of Cdk4) containing p15INK4b, p16 INK4a, p18 INK4c and p19 INK4d only bind the monomeric forms of Cdk4 and Cdk6; and the Cip/Kip family (Cdk-interacting protein/Kinase-inhibitory protein) comprised by p21Cip1, p27Kip1 and p57Kip2 which control multiple cyclin-Cdk complexes by interacting with both the cyclin and Cdk subunits. However, recent data has shown that Cdk inhibitors of the Cip/Kip family can impact biological processes independently of their roles on cell cycle, and some of them seem to be Cdk-independent. Thus, it has been reported that Cip/Kip proteins can also modulate gene expression through direct binding to transcription factors. In particular, p21 can inhibit the activities of E2F1, c-Myc and STAT 3. However, the biological significance of the role of p21 in transcriptional regulation is still poorly understood. In collaboration with Isabel Fariñas’ group we have identified a biologically relevant role of p21 regulating bone morphogenetic (BMP) pathway in mouse neural stem cells (NSCs). Previous results have shown that Bmp2 expression is normally repressed by p21 at a transcriptional level and deleterious effects on self-renewal and multipotency, caused by the elimination of p21, are mediated by secreted BMPs. We have also identified a putative E2F binding site on Bmp2 promoter where p21 is detected by chromatin immunoprecipitation. Here we plan to further characterize genetically and biochemically the molecular mechanism by which p21 regulates the expression of BMPs by first, identifying the structural requirements for p21 effects on Bmp2 expression and second, identifying p21 partners that could be part of the repressor complex sitting on Bmp2 promoter. For our first goal we generated different mutants of p21. Using these mutants on Bmp2 promoter luciferase assays we have shown that Bmp2 gene is repressed by p21 at transcriptional level in an E2F-dependent, cell cycle-independent manner. The repressor activity of p21 was independent of its cyclin-binding ability and mapped to the N-terminus of the molecule. Using an in vitro pull down assay and proteomics, we have identified p54(nrb)/NonO as a new p21-interacting protein in the nucleus of NSCs. NonO is also a multifunctional protein involved in the binding and processing of nucleic acids which has also been described to function as a transcriptional regulator . However, a transcriptional effect of NonO on Bmp2 promoter has not been addressed and the biological significance of p21-NonO interaction in NSCs cultures should be investigated in future research.