E Proteins and Id2 converge on p57Kip2 to regulate cell cycle in neural cells

Abstract

A precise balance between proliferation and differentiation must be maintained during neural development to obtain the correct proportion of differentiated cell types in the adult nervous system. The basic helix-loop-helix (bHLH) transcription factors known as E proteins and their natural inhibitors, the Id proteins, control the timing of differentiation and terminal exit from the cell cycle. Here we show that progression into S phase of human neuroblastoma cells is prevented by E proteins and promoted by Id2. Cyclin-dependent kinase inhibitors (CKI) have been identified as key effectors of cell cycle arrest in differentiating cells. However, p57Kip2 is the only CKI that is absolutely required for normal development. Through the use of global gene expression analysis in neuroblastoma cells engineered to acutely express the E protein E47 and Id2, we find that p57Kip2 is a target of E47. Consistent with the role of Id proteins, Id2 prevents activation of p57Kip2 expression, and the retinoblastoma tumor suppressor protein, a known Id2 inhibitor, counters this activity. The strong E47-mediated inhibition of entry into S phase is entirely reversed in cells in which expression of p57Kip2 is silenced by RNA interference. During brain development, expression of p57Kip2 is opposite that of Id2. Our findings identify p57Kip2 as a functionally relevant target recruited by bHLH transcription factors to induce cell cycle arrest in developing neuroblasts and suggest that deregulated expression of Id proteins may be an epigenetic mechanism to silence expression of this CKI in neural tumors.

FIG. 1.

Effect of E proteins and Id2 on neuroblastoma cell proliferation. (A) Triplicate plates of SK-N-SH, IMR-32, and LAN-1 were transfected with expression vectors encoding E12, E47, E2-2, or the empty vector. Colonies were scored after 18 days of selection in G418. (B) SK-N-SH cells were transfected with an expression vector encoding Id2, a mutated Id2 lacking the HLH domain (Id2ΔHLH), or the empty vector, and the percentage of cells entering S phase was measured by incorporation of BrdU.

FIG. 2.

Activation of E47-ER in neuroblastoma cells induces E-box-mediated transcription and cell cycle arrest. (A) SK-N-SH cells were transfected with an E-box-luciferase construct and pBabe-E47-ER, pBabe-bHLH-ER, or pBabe vector. Luciferase activity was determined 36 h after transfection and addition of 4-OHT. Results are expressed as means ± standard deviations of triplicate assays normalized for transfection efficiency using β-galactosidase. (B) SK-N-SH cells stably expressing the fusion protein E47-ER or the empty vector were transfected with a plasmid encoding the E box of the immunoglobulin enhancer and treated with 4-OHT or vehicle control for 24 h. ChIP was performed using E47 antibody or normal rabbit IgG (NRIg), and precipitated DNA was amplified using primers specific for the E-box sequence or GAPDH. (C) SK-N-SH cells stably expressing either pBabe-E47-ER or pBabe vector alone were plated in triplicate and administered vehicle or 4-OHT for 18 days. The number of colonies is reported as the means ± standard deviations of the triplicate plating. (D) Activation of E47-ER induces inhibition of entry into S phase without apoptosis. SK-N-SH cells expressing either pBabe-E47-ER or pBabe vector were plated and treated with 4-OHT or vehicle for the indicated times. BrdU was added 90 min prior to the end of treatment with 4-OHT. Cells were processed for BrdU immunofluorescence (red), and nuclei were stained with DAPI (blue). (E) Quantitation of the experiment shown in panel D. The number of cycling cells is reported as the means ± standard deviations of three separate experiments. IP, immunoprecipitation.

FIG. 3.

Adenoviral expression of E47 and Id2 in human neuroblastoma cells. (A) Western blotting of E47 and Id2 in SK-N-SH cells harvested 8 h after infection with adeno-E47 (Ad-E47), adeno-Id2 (Ad-Id2), or adenovirus vector (Ad-vec). (B) Phase-contrast morphology of SK-N-SH cells infected with adeno-E47 or adenovirus vector demonstrates that E47 induces dendritic differentiation. (C) E47 induces expression of the neuronal somatodendritic differentiation marker MAP-2. SK-N-SH cells infected with adeno-E47 or adenovirus vector were harvested at the indicated times and assayed by Western blotting for MAP-2. (D) SK-N-SH cells were rendered quiescent by serum starvation, infected with adeno-Id2 or adenovirus vector, and labeled with BrdU. The percentage of BrdU-positive cells was determined at the indicated times after the infection.

FIG. 4.

Microarray analysis of the neuroblastoma cell line SK-N-SH after infection with adeno-E47 (Ad-E47) and adeno-Id2 (Ad-Id2). (A) Genes changed by adeno-E47 after 8 and 20 h. Genes located at the imprinted locus 11p15.5 are highlighted in boldface. (B) Genes changed by adeno-Id2 after 8 and 20 h. Genes reciprocally regulated by E47 and Id2 are indicated. Adenovirus vector, Ad-Vec.

FIG. 5.

Expression of chromosome 11p15.5 genes is reciprocally induced by E47 and repressed by Id2. Real-time PCR values of indicated transcript abundance in SK-N-SH cells infected with adeno-E47 (Ad-E47) or adenovirus vector (top panels) and adeno-Id2 (Ad-Id2) or adenovirus vector (bottom panels) for the indicated times. HPRT was used as an internal control.

FIG. 6.

Rapid induction of p57^Kip2 by E47 in multiple cell lines. (A) Northern blot analysis of p57^Kip2 following infection with adeno-E47 (Ad-E47) or adenovirus vector (Ad-vec) (upper panel) and adeno-Id2 (Ad-Id2) or adenovirus vector (lower panel). 28S rRNA is shown as a loading control. (B) Western blot analysis of E47, p57^Kip2, p27^Kip1, p21^Cip1, and α-tubulin from SK-N-SH cells infected with adenovirus vector and adeno-E47 for the indicated times. (C) Western blot from LAN-1 human neuroblastoma cells and TERT-immortalized human astrocytes transfected with an expression plasmid encoding E47 or the empty vector demonstrates specific induction of p57^Kip2. (D) SNB19, SF210, and U20S cells were transfected as described for panel C, whereas K562 and Raji cells were infected with adeno-E47 or adenovirus vector for 24 h before being analyzed by Western blotting.

FIG. 7.

p57^Kip2 is a direct target gene of E47. (A) Western blot analysis of SK-N-SH cells following transfection with pBabe-E47-ER, pBabe-bHLH-ER, or pBabe vector and treatment of the cells with 4-OHT for 24 h. (B) Northern blot analysis of SK-N-SH expressing pBabe-E47-ER and treated with CHX and 4-OHT. 28S rRNA is shown as a loading control. Western blotting for p57^Kip2 shows that induction of p57^Kip2 by E47-ER is fully inhibited by CHX. Asterisks indicate nonspecific bands. (C) Real-time quantitative RT-PCR for p57^Kip2 and HPRT mRNAs was done from SF210-E47-ER treated with CHX and 4-OHT for the indicated times. (D) Real-time quantitative RT-PCR for p57^Kip2 and HPRT mRNAs was done from SF210-E47-ER transfected with expression plasmids for CBP and pCAF and treated with 4-OHT. (E) Real-time quantitative RT-PCR for p57^Kip2 and HPRT mRNAs was done from U20S transfected with the indicated combinations of expression plasmids for E47, CBP, and pCAF.

FIG. 8.

Induction of the expression of p57^Kip2 is essential for E47-mediated cell cycle arrest of human neuroblastoma cells. SK-N-SH (A) and SF210 (B) cells expressing pBabe-E47-ER or pBabe vector were transfected with siRNA oligonucleotides expressing a scrambled sequence (CTR) or the p57^Kip2 sequence (p57^Kip2) before treatment with 4-OHT for 24 h. Expression of p57^Kip2, α-tubulin, and Cdk4 were analyzed by Western blotting. (C) Representative fields of SK-N-SH cells treated as described for panel A. BrdU was added for 1 h prior to fixation of the cells and staining with an antibody against BrdU (red). Nuclear DNA was stained with DAPI (blue). The fractions of SK-N-SH-E47-ER (D) and SF210-E47-ER (E) incorporating BrdU after the indicated treatments were quantitated by counting at least 2,000 DAPI-positive nuclei for each duplicate transfection. (F) Quantitation of luciferase expression in SK-N-SH cells transfected with an E-box-luciferase plasmid in the presence of plasmids encoding E47, Id2, or a constitutively active, unphosphorylatable Rb (PSM-Rb). (G) SK-N-SH cells were transfected with the indicated combinations of plasmids expressing E47, Id2, and PSM-Rb. Cellular lysates were analyzed for the expression of E47, p57^Kip2, Id2, PSM-Rb, and α-tubulin.

FIG. 9.

Reciprocal expression of Id2 and p57^Kip2 during differentiation of neuroblastoma cells and development of the mouse brain. (A) LAN-1 cells were treated with RA, and extracts were prepared on the indicated days and analyzed by Western blotting. (B) Adjacent sections from E15.5 mouse brain were immunostained for E47, Id2, and p57^Kip2. The alternative expression of Id2 and p57^Kip2 in the ventricular zone (VZ) and the mantle zone (MZ) of the inferior colliculus is depicted in the right lower panel. (C) Double immunofluorescence analysis of E47 (red) and p57^Kip2 (green, top panels) and Id2 (red) and p57^Kip2 (green, bottom panels) from the inferior colliculus of E15.5 mouse brain. E47 and p57^Kip2 colocalize in the MZ but not in the VZ, whereas cells expressing Id2 in the VZ are always p57^Kip2 negative.