Intracellular Notch1 May Induce a Conformational Change in CSL/DNA, without Forming ICN1/CSL/DNA Molecular Complex, in Vitro

Alexei A. Stortchevoi
2013 CellBio  
Intracellular Notch (ICN) initiates DNA transcription in cooperation with CSL that acts as repressor in the absence of ICN. The ICN mediates recruitment of MAML protein, leading to the formation of minimal transcriptional complex, MAML/ICN/CSL/DNA. Crystal structure reveals that different conformations exist between the free (CSL/DNA) and bound (ICN/MAML/CSL/DNA) forms. The significance of this modulation of the CSL/DNA molecular complex can be better understood by experimental approaches that
more » ... al approaches that aim to elucidate the cause and timing of these events. There are four orthologues of human ICN (ICN1-4). We studied interactions between human full-length ICN1 and CSL/DNA without involvement of MAML, in vitro, and found that 1) the EMSA profile of CSL/DNA is altered in the presence of ICN1 as a consequence of an intrinsic change(s) in CSL/DNA, and not due to the formation of an ICN/CSL/DNA molecular complex; 2) ICN1 destabilizes CSL/DNA. These findings indicate that human ICN1 functions to modulate the CSL/DNA molecular complex for subsequent recruitment of MAML, and that modulated CSL/DNA cannot accommodate ICN1 in the absence of MAML. The latter in turn, implies that the formation of the MAML/ICN1/CSL/DNA is likely to be a collective event, wherein preassembly of MAML and ICN1 as a binary complex co-localizes at the CSL/DNA promoter site, or the MAML/ICN1/CSL complex is pre-assembled prior to binding to the promoter, rather than ICN1 arriving at CSL/DNA ahead of MAML and/or other associated transcription factors. The novel finding that ICN1 destabilizes the CSL/DNA complex opens new possibilities of transcriptional regulation by Notch. A. A. STORTCHEVOI 74 cilitating transcription. This ICN function is supported by Mastermind-like, a human homologue of Drosophila protein (MAML) [20-22], Ski-interacting protein (SKIP) that binds both, CSL and ICN [23] and histone acetylase CBP/p300 [20]. MAML is also thought to control withdrawal of ICN1 from the transcription complex [20,24]. Other proteins involved in ICN-mediated transcription include negative regulator KyoT2 [25], transcriptional co-activator p300 [26], hypoxia-inducible fator 1 alfa (Hif-1a) [3,27], mediator protein Med220, cell-cycle depended kinase 7 (CDK7) and CDK8 [28], elongation factors FACT, Spt6, P-TEFb, reviewed [13] . While the number of transcription factors involved is extensive, and continues to grow, the sequence of the events leading to transcription and its turnover is far from been clearly understood. One of the lingering questions surrounding ICN mediated transcription events is whether ICN binds independently to CSL/DNA, or is dependent upon other factors to form the ICN/CSL/DNA complex. Some data point to a higher probability of an assisted rather than independent ICN1 binding to CSL/DNA in mammals: Timed ChIP/PCR study shows that the arrival and the departure of ICN1 at the CSL-occupied HES1 promoter occurs within the same 30 min. interval that is associated with MAML1 and SKIP binding to CSL/DNA [28] . Crystal structure of the CSL/HES1 promoter fragment with the ANK domain of murine ICN1 and N-terminal peptide of MAML1 shows that mutual dependence of these proteins is required to maintain stability of the association [29] . When crystal structures are compared between CSL/DNA [30] and MAML/ICN1/CSL/DNA (harboring the RAMANK domain of mouse ICN1) [31], it reveals conformational change occurs in CSL/DNA; this observation raises the important question of whether the change in CSL/DNA structure is induced by ICN1 only, or via a synergistic interaction between ICN1 and MAML. Human ICN1 is ~90% homologous to the mouse counterpart, with most of the discrepancies located within the C-terminal "Trans-Activation Domain" that is absent in the crystal structures, leaving the impact of interspecies differences open to speculation. We analyzed the impact of full-length human recombinant ICN1 on CSL/DNA binding. The study was designed to include only the initial trio: human HES1 promoter DNA, full-length human CSL and full-length human ICN1. We show that ICN1 does not form stable association with CSL/DNA in the absence of MAML (at the conditions used) but induces a change in the CSL/ DNA molecular complex that alters its electromobility in EMSA. Another effect of the ICN1is that it destabilizes the bond between the CSL protein and Hes1 promoter, and that the degree of CSL dissociation is dependent on the length of DNA fragment that is used for the EMSA. CSL and ICN1 Protein Expression in E. coli Recombinant ICN1 and CSL genes and cloning sites can be viewed in the supplement to this article. The genes were cloned in pET28a vector (Novagen, Madison, WI, USA) and expressed in BL21.DE3 strain
doi:10.4236/cellbio.2013.22010 fatcat:gcakolni4jgoxfubhvb6erlnj4