1. Mastermind Recruits CycC:CDK8 to Phosphorylate the Notch ICD and Coordinate Activation with Turnover 
Christy J. Fryer, J.Brandon White, Katherine A. Jones 
Molecular Cell 
Volume 16, Issue 4, Pages (November 2004)
DOI: /j.molcel

2. Figure 1 Kinetics of HES1 Transcriptional Activation by Notch
(A) HES1 RNA is induced by Notch signaling upon coculture of C33A cells with SN3T9 cells. HES1 transcription was analyzed by Northern blot of total RNA from control C33A cells, C33A cells cocultivated with control Ltk− cells for 2–24 hr, or C33A cells cocultivated with SN3T9 cells for times as indicated.
(B) Analysis of the levels of the Notch ICD, HES1, and CDK8 proteins in Notch signaling cells. HES1 and CDK8 proteins were analyzed by Western blot in the absence of coculture (lane 1) or at different points 30 min to 3 hr after coculture C33A cells (lanes 2–6). C33A cells were cocultured with SN3T9 cells for 1, 6, or 24 hr in the presence (lanes 11–13) or absence (lanes 8–10) of 1 mM lactacystin. Extracts were analyzed by SDS-PAGE and immunoblotted with an antibody to cleaved ICD.
(C) ChIP analysis of the binding of CBF, Notch ICD, and HES1 proteins to the human HES1 promoter. C33A cells were cocultured with either Ltk− cells or SN3T9 cells for the indicated times and immunoprecipitated with specific antisera.
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3. Figure 2
ChIP Analysis of the Recruitment of Transcription Factors to the HES1 Gene upon Notch Signaling
(A) A schematic diagram of the HES1 gene indicating the primer sets used for ChIP analysis of the HES1 promoter (−300 to +10) or third intron (+1158 to +1445). Rectangles indicate the three binding sites for HES1 (N-boxes), two binding sites for CBF1, and TATA box. For ChIP, C33A cells were cocultured with either Ltk− cells or SN3T9 cells and analyzed as in Figure 1C. Reactions shown in lanes 1–4 and those in lanes 5–8 were from separate experiments.
(B) CHIP analysis of transcription factor binding to the promoter (lanes 1–4) or intron (lanes 5–8) of the HES1 gene upon Notch signaling. PCR analysis of promoter and intron regions from the same experiment shown in (A), lanes 1–4.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
The Notch Cofactor, SKIP, Acts with MAM to Promote Notch Activation of the HES1 Promoter In Vivo
(A) SKIP facilitates MAM-dependent activation of the HES1 promoter. HeLa cells were transfected with ICD, MAM, and SKIP expression constructs using the indicated amounts of DNA (nanograms). Activation of HES1-LUC was monitored 48 hr after transfection and normalized to the control β-galactosidase gene. The data is represented as fold activation. Error bars, SD.
(B) Analysis of Notch ICD binding to the SKIP SNW domain in vitro. Purified recombinant FLAG-Notch1 ICD was incubated with the indicated GST-protein. The bound proteins were eluted and analyzed by immunoblotting with anti-FLAG antibody. The input FLAG-Notch1 protein is shown as a control (lane 1).
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
CDK8 Interacts Directly with MAM and Inhibits Notch Activation of the HES1 Gene In Vivo
(A) Western blot of CDK8, CDK9, or p300 interaction with full-length and mutant MAM proteins. HeLa nuclear extract was incubated with either GST (lanes 2, 5, and 9) or various GST-MAM proteins coupled to glutathione beads (lanes 3, 7, 8, and 10–12) as indicated. GST MAM-protein-coupled beads were incubated with binding buffer but without nuclear extract as controls (lanes 4 and 6). The input HeLa extract (IN) is shown in lanes 1 and 18. MAM interacts with CDK8 in vitro. Purified recombinant His-MAM was incubated with the indicated GST-protein (lanes 14–17) and analyzed as in Figure 3B but with an anti-His antibody. CDK9 does not interact with Notch enhancer proteins. HeLa nuclear extract was incubated with GST (lane 19), GST-CBF1 (lane 21), GST-ICD (lane 23), GST-MAM (lane 25), or GST-CycT1 (lane 26) and bound proteins analyzed by SDS-PAGE and immunoblotted with anti-CDK9 antisera. GST-protein-coupled beads were incubated without nuclear extract as controls (lanes 20, 22, and 24).
(B) HeLa cells were transfected with ICD, MAM, CDK8, mutant (kinase inactive) CDK8 (dnCDK8), or mutant CDK9 (dnCDK9) as indicated. The activation of HES1-LUC was analyzed as in Figure 3A. The activation by ICD+MAM was set to 100% and other data points are expressed as a percent change in activity. Error bars, SD.
(C) Endogenous CDK8, but not CDK9, accumulates in nuclear foci in cells that express MAM. HeLa cells were transfected with Myc-tagged MAM for 48 hr and the Myc-MAM protein (red), or endogenous CDK8 protein or CDK9 (green) protein, was visualized by indirect immunofluorescence and deconvolution microscopy. The cells were counterstained with DAPI (blue) to visualize cell nuclei.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
MAM and CDK8 Promote Modification and Turnover of the Notch ICD In Vivo
(A) MAM-directed destabilization of the ICD is blocked by treatment of cells with the proteasome inhibitor lactacystin. Myc-CBF1 and Myc-ICD were coexpressed in the presence or absence of MAM. Where indicated, lactacystin was added after 24 hr at 1 mM (lane 3), 10 mM (lane 4), or 50 mM (lane 5), and the expression of CBF and ICD in the total cellular extracts were monitored 48 hr posttransfection by SDS-PAGE and immunoblotting using an anti-Myc antibody. Expression of MAM alters the stability of ICD (lanes 6–13). Myc-ICD and Myc-CBF were transfected into HeLa cells and their stabilities determined by pulse-chase analysis. Chase times are indicated in minutes.
(B) Expression of kinase-active CDK8 destabilizes the ICD in vivo. Shown are Myc Western blots for expression of exogenous CBF and ICD proteins in HeLa cells after transfections in the presence or absence of wild-type or mutant CDK8 (kinase inactive) plus or minus cyclin C. Cells expressed CBF1 and either full-length ICD (lane 1–9) or an ICD lacking the PEST domain, ICD22 (lanes 10 and 11). The samples for lanes 1–6 were run on 8% Tris-glycine (TG) gels while the samples for lanes 7–11 were run on 3%–8% Tris-acetate (TA) gels to resolve the hyperphosphorylated ICD (P-ICD, lane 8).
(C) CDK8, but not CDK9, promotes turnover of the ICD in vivo. Cells were transfected with ICD, CBF1, CDK8, MCDK8, CDK9, or mutant CDK9 (kinase inactive), together with the appropriate cyclin partners, as indicated above each lane. Myc-ICD levels were assessed by Western blot with anti-Myc antisera.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 CycC:CDK8 Phosphorylates the Notch ICD In Vitro and In Vivo
(A) ICD is phosphorylated by CycC:CDK8 in vitro. CycC:CDK8 was immunoprecipitated from HeLa nuclear extract, incubated with GST-ICD proteins as indicated (lanes 1–3), and the in vitro kinase reactions were analyzed by SDS-PAGE. Recombinant His-CDK8/CycC was incubated with the GST-ICD proteins as indicated (lanes 4–9) and the in vitro kinase reactions were analyzed by SDS-PAGE. The upper panel shows the autoradiogram (32P) while the bottom panel is the Coomassie-stained gel. A schematic of the ICD proteins and the amino acid residues encompassed by each truncation are indicated. The M2-CT protein is the same as ICD-CT with the serine to alanine mutations indicated. The Ser-to-Ala point mutations in the ICD M1, M2, and M3 are also indicated.
(B) HeLa cells were transfected with ICD or M2 (see schematic). Activation of (CBF)8-LUC was monitored 48 hr after transfection and normalized to the control TK-renilla gene. The data is represented as fold activation. Error bars, SD.
(C) Mutation of putative CDK phosphorylation sites in the TAD or PEST motif alters the phosphorylation state and stability of the Notch ICD in vivo. HeLa cells were transfected with Myc-tagged CBF, ICD, ICD22, M1, M2, or M3 plus or minus CDK8. Total cellular extracts from independent experiments were analyzed on 8% TG gels (lanes 1–10) or 3%–8% TA gels (lanes 11–20) and immunoblotted with anti-myc antibody. The addition of CDK8 results in hyperphosphorylation of ICD (P-ICD) that can be resolved only on the 3%–8% TA gels.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7 Factors Controlling ICD Turnover and HES1 Activation
(A) Fbw7/Sel10 and CycC:CDK8 promote turnover of the Notch ICD in vivo. Expression of Myc-CBF and Myc-ICD proteins in HeLa cells were monitored 48 hr after transfection in the presence of either wild-type or mutant CycC:CDK8, or wild-type or dominant-negative Fbw7/Sel10, as indicated. All samples were run on 3%–8% TA gels to resolve the hyper-phosphorylated ICD (P-ICD). The data shown in lanes 1–6 and 7–11 are from two independent experiments. The Myc-CBF protein in the experiment in lanes 1–6 was run off the gel to better resolve the hyperphosphorylated ICD.
(B) Model for Notch-activation of the HES1 gene. The proteins bound to the promoter prior to signaling are indicated in light colors (CBF1, RNAPII, CBP/p300, CDK7, and Spt6), whereas proteins that are recruited together with the Notch ICD are shown in darker colors (MAM, SKIP, Med220, CDK8, CDK9/P-TEFb, FACT). Binding of MAM to p300 and CycC:CDK8 can promote phosphorylation of p300 (Fryer et al., 2002), hyperphosphorylation of the Notch ICD PEST domain (open circles), and facilitate Fbw7/Sel10-mediated ubiquitination of the ICD to disassemble the Notch enhancer complex.
Molecular Cell  , DOI: ( /j.molcel )