Histone demethylase Kdm4b functions as a co-factor of C/EBPβ to promote mitotic clonal expansion during differentiation of 3T3-L1 preadipocytes

Abstract

CCAAT/enhancer-binding protein (C/EBP) β is required for both mitotic clonal expansion (MCE) and terminal adipocyte differentiation of 3T3-L1 preadipocytes. Although the role of C/EBPβ in terminal adipocyte differentiation is well defined, its mechanism of action during MCE is not. In this report, histone demethylase Kdm4b, as well as cell cycle genes Cdc45l (cell division cycle 45 homolog), Mcm3 (mini-chromosome maintenance complex component 3), Gins1 (GINS complex subunit 1) and Cdc25c (cell division cycle 25 homolog c), were identified as potential C/EBPβ target genes during MCE by utilizing promoter-wide chromatin immunoprecipitation (ChIP)-on-chip analysis combined with gene expression microarrays. The expression of Kdm4b is induced during MCE and its induction is dependent on C/EBPβ. ChIP, Electrophoretic Mobility Shift Assay (EMSA) and luciferase assay confirmed that the promoter of Kdm4b is bound and activated by C/EBPβ. Knockdown of Kdm4b impaired MCE. Furthermore, Kdm4b interacted with C/EBPβ and was recruited to the promoters of C/EBPβ-regulated cell cycle genes, including Cdc45l, Mcm3, Gins1, and Cdc25c, demethylated H3K9me3 and activated their transcription. These findings suggest a novel feed forward mechanism involving a DNA binding transcription factor (C/EBPβ) and a chromatin regulator (Kdm4b) in the regulation of MCE by controlling cell cycle gene expression.

Figure 1

Bioinformatic analyses of the ChIP-on-chip data. Post-confluent growth-arrested 3T3-L1 preadipocytes were induced to differentiation as described. At 20 h ChIP-on-chip were performed and analyzed. (a) Distribution in 200-bp intervals of C/EBPβ binding regions relative to TSSs of the nearest RefSeq genes. (b) Mapping of C/EBPβ binding sites relative to the nearest RefSeq genes. >1 kb: more than 1 kb upstream from the TSS; Downstream: downstream from the 3′ end of the gene; UTR: untranslated region. (c) Average plot for conservation of the C/EBPβ binding regions among higher eukaryotes. (d) Sequence logo illustrating the top-scoring de novo motif for C/EBPβ-bound regions. (e) TRANSFAC matrices enriched around C/EBPβ-bound regions. (f) Overlap between the genes targeted by C/EBPβ (1039 genes) and the genes whose induction was dependent on C/EBPβ (481 genes)

Figure 2

Transactivation of Kdm4b by C/EBPβ during MCE of 3T3-L1 preadipocyte differentiation. (a) 3T3-L1 preadipocytes were transfected with control vector or C/EBPβ-expressing vector, together with reporter constructs containing the Kdm4b promoter (−1000 to −1 bp) or mutants. Luciferase activities were measured 36 h later and plotted. (b) 3T3-L1 cells were induced to differentiation and binding of C/EBPβ on the Kdm4b promoter was analyzed by ChIP-qPCR at times indicated. (c) 3T3-L1 cells were treated with control or C/EBPβ RNAi. After induction, the mRNA levels of C/EBPβ and Kdm4b at times indicated were analyzed by RT-qPCR. (d) Cell lysates were harvested at times indicated and subjected to western blotting with antibodies indicated. The C/EBPβ antibody detects two bands: liver-enriched activator protein (LAP, 34 kDa) and liver-enriched inhibitory protein (LIP, 21 kDa, an N-terminally truncated isoform of C/EBPβ). Hsp90 served as a loading control. (e) After induction, mRNA levels of the Kdm4 family members at times indicated were analyzed by RT-qPCR. Data normalized to 0 h time point of each gene. *P<0.05. (f–i) Overexpression of C/EBPβ promotes Kdm4b expression. 3T3-L1 preadipocytes were infected with retrovirus expressing flag-tagged C/EBPβ or with empty vector and induced to differentiation. (f) The overexpression of C/EBPβ (LAP) was confirmed by western blotting at 20 h. (g) At 16 h after induction, cells were labeled with EdU for 2 h and then subjected to FACS analysis. The percentages of cells in S phase were determined. (h) At 8 d after induction, cells were stained with Oil red O. (i) The effect of C/EBPβ overexpression on the expression of Kdm4b was detected by RT-qPCR

Figure 3

Kdm4b is required for MCE during 3T3-L1 preadipocyte differentiation. 3T3-L1 preadipocytes were infected with empty vector or infected with retrovirus expressing human Kdm4b (hKdm4b). Then cells were transfected with control RNAi or Kdm4b RNAi. After post-confluence, cells were induced to differentiation. (a) Cell lysates were harvested at 20 h and the expression of the indicated genes was detected by western blotting. (b) At 18 h after induction, cells were labeled with EdU for 2 h and then stained with Hoechst. The fluorescence of EdU (red) and Hoechst (blue) was detected with a fluorescence microscope. (c) DNA content was analyzed by PI staining and flow cytometry at 0 h and 20 h time points and analyzed. (d) Cell numbers were determined at times indicated after induction. Data normalized to 0 h time point. *P<0.05. (e) At 8 days after induction, cells were stained with Oil red O. (f) At 8 days after induction, the expression of adipocyte marker genes were detected by western blotting

Figure 4

Four cell cycle genes Cdc45l, Mcm3, Gins1 and Cdc25c were identified as C/EBPβ target genes. (a, b) 3T3-L1 cells were induced to differentiation. The expression profiles of C/EBPβ and the four cell cycle genes Cdc45l, Mcm3, Gins1 and Cdc25c were analyzed by RT-qPCR (a) and western blotting (b) respectively. (c, d) 3T3-L1 preadipocytes were transfected with control RNAi or C/EBPβ RNAi. After post-confluence, cells were induced to differentiation. The effect of knocking down C/EBPβ on the expression of the four cell cycle genes was detected by RT-qPCR (c) and western blotting (d) respectively

Figure 5

C/EBPβ binds to and transactivates the promoters of the four cell cycle genes. (a) Growth-arrested 3T3-L1 preadipocytes were induced to differentiation. At times indicated ChIP was performed to show the binding of C/EBPβ to the promoters of the four cell cycle gene. Data normalized to the IgG controls at each time point. One region of the insulin gene serves as a negative control. (b) EMSA was conducted with nuclear extracts from 3T3-L1 cells at 20 h after induction. Supershift experiments were performed with control IgG or C/EBPβ antibody. For competition analysis, unlabeled competitor probe of the C/EBP element in the C/EBPα gene promoter (Consensus) or unlabeled self probes mutated in the C/EBP element (Mut, shown in Supplementary Figure S4) were used. The positions of the C/EBPβ containing DNA–protein complex and the supershift band (SS) are indicated. (c) 3T3-L1 preadipocytes were transfected with reporter constructs with/out the C/EBP binding sites mutated and with/out the C/EBPβ expression vector. After 48 h, luciferase activity was analyzed and plotted. *P<0.05

Figure 6

Kdm4b acted as a co-factor of C/EBPβ to activate the promoters of the four cell cycle genes. (a) ChIP analysis indicated that Kdm4b was also associated with the promoters of the four cell cycle genes. (b) At 20 h post-induction, soluble chromatin was prepared for ChIPs and Re-ChIPs with the indicated antibodies. Recovery of the four cell cycle gene promoters was analyzed by PCR. (c, d) Interaction between C/EBPβ and Kdm4b. At 20 h post-induction, cell lysates were harvested and immunoprecipitated with antibody against C/EBPβ (c) or Kdm4b (d). Immunoprecipitate complexes were detected by western blotting with the indicated antibodies. The band detected by the C/EBPβ antibody is liver-enriched activator protein (LAP) (34 kDa). (e) 3T3-L1 preadipocytes were transfected with reporter constructs with/out the indicated expression vectors. After 48 h, luciferase activity was analyzed and plotted. *P<0.05

Figure 7

Kdm4b regulates the expression of C/EBPβ-targeted cell cycle genes through demethylation of H3K9me3. (a–d) Decrease of H3K9me3 modification level on the promoters of the four cell cycle genes during MCE. Upper panel: Schematic representation of the genomic locus. The regions from ‘a' to ‘d' were analyzed by ChIP-qPCR. The TSS is indicated by the arrow. Lower panel: ChIP-qPCR analysis of H3K9me3 occupancy on the sites in the gene loci indicated in the upper panel was performed at 0 h and 20 h after induction. Primers for ChIP-qPCR were available upon requested. (e) 3T3-L1 preadipocytes were treated with control RNAi or Kdm4b RNAi and then induced to differentiation. Cells were collected at times indicated and subjected to ChIP-qPCR analysis of H3K9me3 modification at position ‘c' of each gene promoter loci. Data were normalized to 0 h time point of control RNAi-treated cells. (f) 3T3-L1 preadipocytes were treated as in (e). The relative mRNA level of C/EBPβ-targeted cell cycle genes at times indicated was analyzed by RT-qPCR

Figure 8

Knockdown the expression of C/EBPβ and Kdm4b has no effect on pre-confluent 3T3-L1 cell proliferation. (a) 3T3-L1 preadipocytes were plated at 20% confluence, next day the cells were transfected with C/EBPβ or Kdm4b RNAi. Forty-eight hours later cell proliferation was determined by cell number counting at times indicated. (b) Cells were treated as in (a). At 48 h after RNAi transfection, cell cycle phase profiles were analyzed by PI staining followed by flow cytometry. (c, d) ChIP-qPCR analysis of C/EBPβ (c) and Kdm4b (d) binding on the promoters of mouse Cdc45l, Mcm3, Gins1 and Cdc25c was performed in pre-confluent 3T3-L1 cells. (e) Forty-eight hours after transfection of C/EBPβ or Kdm4b RNAi, cell lysates were harvested and the expression of the indicated genes was assessed by western blotting