Figure 1.
ChIP-chip indicates strong N-Myc binding of the lif promoter in human neuroblastoma.
ChIP-chip data from previous study [29] was analyzed specifically for lif binding by N-myc. Several strong peaks were observed (brown). The lif promoter is greatly enriched in E-boxes including one canonical myc E-box CACGTG shown in purple and 6 non-canonical indicated by downward vertical arrows and listed in tabular form on the bottom left side. The 3 horizontal black bars labeled “a, b, and c” represent the locations of the 3 PCR products from ChIP PCR reactions in Fig. 4.The genomic locations are listed next to the E-boxes and refer to the last 4 digits of the location, with all being in the 28,970,000 base-pair range on chromosome 22.
Figure 2.
Expression of klf2, klf4, lif, and lin28b are linked to N-myc levels in human neuroblastoma.
qRT-PCR quantifies the changes in klf2, klf4, lif, and lin28b expression with similar 3–5 fold decreases in expression for each at each time point of Tet treatment (added daily). Error bars are standard deviation (S.D.) and where not evident it is because the S.D. is so low the bars are so small they do not show up. S.D. throughout this study were quite low and generally ranged from 0.5–5.5%. TET21N neuroblastoma were treated daily with Tet in this experiment. The data here were analyzed using the comparative Ct method, but then reanalyzed using the Pffafl method giving essentially identical results (Fig. S1).
Figure 3.
N-myc loss rapidly induces loss of expression of klf2, klf4, lif, and lin28b.
(A) RT-PCR for N-myc, klf2, and klf4 was conducted on Tet21N neuroblastoma cells treated for 8, 16, 24, and 72 hours were conducted along with a loading control (aasdh, a gene we have found does not vary with modulation of myc levels, for example in the TET21N array experiment). N-myc levels recovered from 8 hours onward due to a suboptimal dosing of active Tet. (B) qPCR indicates that klf2, klf4, lif, and lin28b levels rapidly decreased as after only 8 hours of Tet treatment and consequent short-term loss of N-myc. Error bars are S.D.
Figure 4.
Chromatin immunoprecipitation (ChIP) assays indicate that N-Myc directly binds lif and klf4 in neuroblastoma.
ChIP was conducted on Tet21N cells treated daily (3 days) or untreated with Tet. IgG was included as a control along with a 1∶50 and 1∶200 dilutions of input. The ChIP'd lif regions a, b, and c are represented by the black bars in Fig. 1. IgG and Input samples were run in parallel as controls.
Figure 5.
N-Myc regulates klf2, klf4, and lin28b as well as nanog, but not lif in neurospheres.
Control (N-myc flox/flox) and N-myc null (floxed, nestin-cre) neurospheres were used for qRT-PCR assays. Expression levels in controls were set to 100%. Error bars are S.D.
Figure 6.
A model of Myc stem-related function in neuroblastoma cells.
Two key functions are depicted: growth factor signaling through LIF and induction of stem-related gene expression through induced transcription of genes encoding KLF2, KLF4, and LIN28B. Together these programs are predicted to maintain a “blast”-like state in neuroblastoma tumors through transcriptional and miRNA functions.