Involvement of crosstalk between Oct4 and Meis1a in neural cell fate decision

Abstract

Oct4 plays a critical role both in maintaining pluripotency and the cell fate decision of embryonic stem (ES) cells. Nonetheless, in the determination of the neuroectoderm (NE) from ES cells, the detailed regulation mechanism of the Oct4 gene expression is poorly understood. Here, we report that crosstalk between Oct4 and Meis1a, a Pbx-related homeobox protein, is required for neural differentiation of mouse P19 embryonic carcinoma (EC) cells induced by retinoic acid (RA). During neural differentiation, Oct4 expression was transiently enhanced during 6-12 h of RA addition and subsequently disappeared within 48 h. Coinciding with up-regulation of Oct4 expression, the induction of Meis1a expression was initiated and reached a plateau at 48 h, suggesting that transiently induced Oct4 activates Meis1a expression and the up-regulated Meis1a then suppresses Oct4 expression. Chromatin immunoprecipitation (ChIP) and luciferase reporter analysis showed that Oct4 enhanced Meis1a expression via direct binding to the Meis1 promoter accompanying histone H3 acetylation and appearance of 5-hydoxymethylcytosine (5hmC), while Meis1a suppressed Oct4 expression via direct association with the Oct4 promoter together with histone deacetylase 1 (HDAC1). Furthermore, ectopic Meis1a expression promoted neural differentiation via formation of large neurospheres that expressed Nestin, GLAST, BLBP and Sox1 as neural stem cell (NSC)/neural progenitor markers, whereas its down-regulation generated small neurospheres and repressed neural differentiation. Thus, these results imply that crosstalk between Oct4 and Meis1a on mutual gene expressions is essential for the determination of NE from EC cells.

Figure 1. Induction of Meis1a/b expressions during neural differentiation of RA-primed P19 cells.

Aggregated P19 cells were treated with 5×10⁻⁷ M RA for various times and the expression levels of Meis1a/b mRNAs and proteins were analyzed by RT-PCR and WB with the anti-Meis1 antibody, respectively. (A) Expression patterns of Meis1a and Meis1b mRNAs during neural differentiation. (B) Quantification of the expression levels of Meis1a and Meis1b mRNAs indicated in A. (C) Expression patterns of Meis1a and Meis1b proteins during neural differentiation. (D) Quantification of the expression levels of Meis1a and Meis1b proteins indicated in C. (E) Expression levels of Meis1a/b mRNAs in mouse fetal brain. Total RNAs from the developing brain were analyzed by Northern blotting. (F) Detailed expression patterns of Meis1a and Oct4 mRNAs during neural differentiation. (G) Quantification of the expression levels of Meis1a and Oct4 mRNAs indicated in F. (H) Detailed expression patterns of Meis1a and Oct4 proteins during neural differentiation. (I) Quantification of the expression levels of Meis1a and Oct4 proteins indicated in H. Ribosomal large subunit protein P0 mRNA, β-actin and 28S ribosomal RNA were used as internal controls.

Figure 2. Involvement of Meis1a in neural differentiation of RA-primed P19 cells.

(A) Expression levels of Meis1a in S-Meis1a and AS-Meis1a cells with or without MIF. (B) Functional analysis of Meis1a in neuronal differentiation. Aggregated S-Meis1a and AS-Meis1a cells were treated with RA in the presence or absence of MIF for 4 days, additionally cultured in RA-free medium for 3 days, and stained with anti-β-tubulin (III) antibody, followed by Cy3-conjugated anti-mouse IgG antibody. Nuclei were stained with Hoechst 33258. Scale bar = 100 µm. (C) Quantification of the effect of Meis1a on neuronal differentiation indicated in B. (D) Effect of Meis1a on β-tubulin (III) expression. Differentiated S-Meis1a and AS-Meis1a cells with or without MIF were lysed and analyzed by WB with anti-β-tubulin (III) antibody. (E) Effect of Meis1a on GFAP-positive astrocyte differentiation. RA-treated S-Meis1a and AS-Meis1a cells were additionally cultured for 7 days and then stained with anti-GFAP antibody. (F) Quantification of the effects of Meis1a on astrocyte differentiation indicated in E. (G) Effect of Meis1a on GFAP and S100β expressions. Differentiated S-Meis1a and AS-Meis1a cells with or without MIF were analyzed by WB with the anti-GFAP and anti-S100β antibodies. *p<0.005 significantly different from MIF(−) control cells. n = 3.

Figure 3. Oct4 activates Meis1a expression.

Monolayer-cultured P19 cells were transfected with the pcDNA3-EF1-α-Oct4 expression vector and after 24 h Meis1a and Oct4 mRNAs and proteins were analyzed by RT-PCR (A) and WB (B), respectively. (C) Stimulatory effect of Oct4 on Meis1 promoter activity. P19 cells were transfected with Meis1(−926)-Luc and various amounts of pcDNA3-EF1-α-Oct4. After 24 h, luciferase activities and expression levels of Oct4 were analyzed. *p<0.001 significantly different from vacant vector introduced control cells. (D) Schematic presentation of Meis1 promoter-inserted luciferase reporter vectors. Meis1(−926)-Luc possesses three putative Oct4-BEs. (E) Functional analysis of Oct4-BEs. P19 cells were transfected with indicated Meis1-Luc vectors and pcDNA3-EF1-α-Oct4. After 24 h, luciferase activities were assayed. *p<0.001 significantly different from Meis1(−926)-Luc and Meis1(−335)-Luc. n = 3. (F) Association of Oct4, HDAC1, AcH3, 5mC and 5hmC with the Meis1 promoter. Genomic chromatin fragments from RA-treated aggregation form of P19 cells for 0 and 6 h were immunoprecipitated with the indicated antibodies and then DNAs were extracted. PCR was carried out using the primer set covering the −360 to −67 region of Meis1 promoter, in which 12 CpG sites exist. Aliquots of 10% antibody-untreated DNA samples were used for input DNA. (G) Quantification of the Meis1 promoter-bound Oct4, HDAC1, AcH3, 5mC and 5hmC indicated in F.

Figure 4. Meis1a suppresses Oct4 expression.

(A) Reduction of Oct4 by ectopic expression of Meis1a RNA. P19 cells were transfected with the vacant vector or pcDNA3-EF1-α-Oct4 and after 24 h, these aggregated cells were treated with RA for 0 and 6 h. Thereafter, expression levels of Oct4 and Meis1a proteins were analyzed by WB with anti-Oct4 and anti-Meis1 antibodies. (B) The suppressive effect of Meis1a on Oct4 promoter activity. P19 cells were co-transfected with Oct4(−1059)-Luc and various amounts of pcDNA3-EF1-α-Meis1a. After 24 h, luciferase activities and Meis1a expression levels were analyzed. *p<0.001 significantly different from vacant vector-introduced cells. n = 3. (C) Schematic presentation of Oct4 promoter-introduced luciferase reporters. Oct4(−1059)-Luc has four putative Meis1-BEs. (D) Functional analysis of Meis1-BEs. P19 cells were transfected with Oct4(−1059)-Luc, Oct4(−698)-Luc, Oct4(−506)-Luc, or Oct4(−254)-Luc together with 60 ng pcDNA3-EF1-α-Meis1a. After 24 h, luciferase activities were analyzed. *p<0.001 significantly different from Oct4(−698)-Luc, Oct4(−506)-Luc, and Oct4(−254) Luc. n = 3. (E) Occupation of the Oct4 promoter by Meis1a and HDAC1. Aggregated P19 cells were treated with RA for 0 and 6 h. Genomic chromatin fragments were immunoprecipitated with anti-Oct4 and anti-HDAC1 antibodies and DNAs were extracted. PCR was carried out using the primer set covering the −1062 to −778 region of the Oct4 promoter. Aliquots of 10% antibody-untreated DNA samples were used for input DNA. (F) Quantification of the Oct4 promoter-bound Meis1a and HDAC1 indicated in E. (G) Schematic presentation of the adjacent Meis1-BEs3/4 region. In this region, putative Pbx- and Hox-BEs also existed.

Figure 5. Stimulatory effect of Meis1a on the formation of neurospheres consisted of NSCs/neural progenitor cells.

(A) Effect of Meis1a on sphere formation during neural differentiation. Aggregated S-Meis1a and AS-Meis1a were treated with RA in the presence or absence of MIF. After 4 days, spheres were analyzed under a phase-contrast microscope. Scale bar = 100 µm. (B) Quantification of sphere sizes of S-Meis1a and AS-Meis1a indicated in A. More than 400 spheres of each sample were analyzed. (C) Effect of Meis1a on the cell growth during neural differentiation. (D and E) Aggregated S-Meis1a and AS-Meis1a cells were treated with RA together with or without MIF. Effects of Meis1a on the NSC/neural progenitor marker expressions. Cell lysates from RA-primed S-Meis1a (D) and AS-Meis1a (E) cells with or without MIF were analyzed by WB with anti-Nestin, anti-GLAST, anti-BLBP and anti-Sox1 antibodies. (F and G) Quantification of expression levels of NSC/neural progenitor markers in S-Meis1a and AS-Meis1a cells as shown in D and E, respectively.

Figure 6. Effect of Meis1a on Sox2 and Pax6 expressions.

(A) Expression patterns of Sox2 and Pax6 during neural differentiation. Aggregated P19 cells were treated with RA for various times and analyzed by WB with anti-Sox2 and Meis1 antibodies. (B) Quantification of expression levels of Sox2 and Pax6 as shown in A. (C and D) Stimulation of Sox2 and Pax6 mRNA and protein expressions by ectopic expression of Meis1a. Monolayer-cultured P19 cells were transfected with various amounts of pcDNA3-EF1-α-Meis1a and after 12 h, Sox2 and Pax6 mRNAs and proteins were analyzed by RT-PCR (C) and WB (D), respectively.