Npac Is A Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells

Abstract

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells (ESCs). However, the related mechanisms remain obscure. Here, we show that Npac, a "reader" of histone H3 lysine 36 trimethylation (H3K36me3), is required to maintain mouse ESC (mESC) pluripotency since knockdown of Npac causes mESC differentiation. Depletion of Npac in mouse embryonic fibroblasts (MEFs) inhibits reprogramming efficiency. Furthermore, our chromatin immunoprecipitation followed by sequencing (ChIP-seq) results of Npac reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs. Interestingly, we find that Npac interacts with positive transcription elongation factor b (p-TEFb), Ser2-phosphorylated RNA Pol II (RNA Pol II Ser2P), and Ser5-phosphorylated RNA Pol II (RNA Pol II Ser5P). Furthermore, depletion of Npac disrupts transcriptional elongation of the pluripotency genes Nanog and Rif1. Taken together, we propose that Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA Pol II Ser2P and Ser5P.

Keywords: Histone H3K36me3; Npac; Pluripotency; Reprogramming; Transcriptional elongation.

Figure 1

Npac is required to maintain mESC pluripotency A. The Npac mRNA level decreased in mESCs cultured in LIF withdrawal ESC medium. The mRNA levels of Npac and Oct4/Pou5f1 were compared to those in the control cells cultured in normal ESC medium and normalized against Actb. B. The mRNA levels of the pluripotency genes Oct4/Pou5f1, Sox2, and Nanog were significantly decreased upon Npac KD. mESCs transfected with the empty pSUPER.puro vector were used as a control. C.Npac KD resulted in decreased protein levels of Oct4, Sox2, Nanog, and histone H3K36me3. β-actin served as a loading control. D.Npac KD caused up-regulation of the specific markers for endoderm and mesoderm. E. Representative bright field images (upper panel) of E14 cells transfected with control (empty vector), scrambled RNAi, or Npac RNAi-1 followed by 4 days of puromycin selection. AP staining was conducted on day 4 after transfection, and the results were shown at the bottom panel. Scale bar, 100 μm. F. Quantification of AP⁺ colonies for control (empty vector), scrambled RNAi, or Npac RNAi-1 transfected E14 cells. Data were shown as mean ± SE (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t-test). ESC, embryonic stem cell; mESC, mouse ESC; LIF, leukemia inhibitory factor; KD, knockdown; H3K36me3, histone H3 lysine 36 trimethylation; AP, alkaline phosphatase.

Figure 2

Depletion of Npac inhibits the efficiency of reprogramming A. Relative mRNA expression level of Npac determined by qRT-PCR in MEFs infected with OKSM alone or OKSM plus Npac-KD retrovirus (OKSM+Npac-KD). RNA was extracted from cells which were harvested 4 days after virus infection. mRNA expression levels were normalized against Actb. B. Depletion of Npac inhibited reprogramming efficiency. The numbers of GFP⁺ colonies which indicate putative iPSCs were counted from day 10 to day 14 after virus infection. C. The iPSCs generated from OKSM+Npac-KD presented weaker AP activity than those from OKSM alone. D. Graphical representation of the AP staining results shown in (C). E. The iPSCs generated from OKSM+Npac-KD expressed Oct4 and Nanog. Immunostaining was performed with anti-Oct4 and anti-Nanog antibodies in GFP⁺ iPSCs generated from OKSM+Npac-KD. F. EBs generated from GFP⁺ iPSCs which were induced by OKSM+Npac-KD were able to express lineage markers of ectoderm (Nestin), mesoderm (SMA), and endoderm (Gata4). MEF, mouse embryonic fibroblast; OKSM, the combination of transcription factors Oct4, Klf4, Sox2, and c-Myc; EB, embryoid body.

Figure 3

Changes of global gene expression upon Npac depletion in mESCs A. Microarray heatmap generated from the relative gene expression levels. Relative highly expressed genes were shown in red and lowly expressed genes in green. Npac was knocked down in E14 cells and selected for 96 h. Then whole-genome cDNA microarray hybridization was performed. Duplicates were chosen to ensure the reproducibility of results. GO analysis was performed relating to “biological process” for the up- or down-regulated genes, respectively. The enriched terms were classified into several function groups and listed in the figure. B. Heatmap of the down-regulated pluripotency genes upon Npac KD in mESCs. Genes were selected according to their known functions in pluripotency. Each selected gene was taken as individual tiles from the thumbnail-dendrogram duplicates. C. Heatmap of the up-regulated MAPK pathway-related genes upon Npac KD in mESCs. Genes were selected according to their known functions in the MAPK pathway. D. The protein levels of p-ERK1/2 and ERK1/2 were elevated in Npac-depleted cells as compared to the control (empty vector) cells. β-actin served as a loading control. E. ERK inhibitor (PD0325901, Sigma) triggered elevated expression of Nanog but did not rescue the down-regulated expression of Oct4 upon Npac KD. F. ERK inhibitor slightly brought down the up-regulated lineage markers in Npac-KD cells. GO, Gene Ontology; p-ERK1/2, phosphorylated ERK1/2.

Figure 4

Npac depletion may cause cellular apoptosis A. Heatmap of up-regulated cell death-related genes upon Npac KD in mESCs. Genes were selected according to their known functions in cell death. B. Cell cycle analysis by flow cytometry in the Npac RNAi-1 transfected cells and the control (empty vector) group. C. The representative flow cytometry pattern is shown. D. Apoptosis triggered by Npac KD was analyzed by Annexin V staining through flow cytometry. E. Graphical representation of the apoptosis cells detected by Annexin V staining. Data were shown as mean ± SE (n = 3). *, P < 0.05 (Student’s t-test).

Figure 5

Npac is mainly located to gene bodies and its genome-wide distribution resembles that of histone H3K36me3 A. Schematic diagram of the structure of the Nanog gene. Black boxes represent exons; solid lines represent introns; dashed line represents the promotor; gray boxes at the bottom represent the primers designed at specific areas of the Nanog gene. B. Npac is associated with the Nanog gene with high enrichment fold at its gene body. C. Npac is also associated with the gene bodies of other pluripotency genes including Tcf15, Prdm14, and Tcl1. D. Genome-wide distribution of Npac in mESCs. E. Genome-wide distribution of Npac resembles that of histone H3K36me3. H3K36me3_express and Npac_express represent the genome-wide distributions of H3K36me3 and Npac in expressed genes in E14 cells, respectively; H3K36me3_non-express and Npac non-express represent the genome-wide distributions of H3K36me3 and Npac in non-expressed genes in E14 cells, respectively. F. Genome-wide distributions of Npac and H3K36me3 in genes with different expression levels (high, middle, low, and no). H3K36me3_high/middle/low/no represent the genome-wide distributions of H3K36me3 in genes with high, middle, low, and no expression in mESCs, respectively. Npac_high/middle/low/no represent the genome-wide distributions of Npac in genes with high, middle, low, and no expression in mESCs, respectively. Each gene body is represented from 0% (TSS) to 100% (TTS). G. Npac and H3K36me3 ChIP-seq peaks at the gene bodies of the housekeeping gene (Actb) and pluripotency genes (Nucleolin, Nanog, and Tcl1) in mESCs. H. Npac and H3K36me3 ChIP-seq peaks at the gene bodies of the telomere maintenance-related genes (Rfc1, Terf1, and Rpa2) in mESCs. In (G) and (H), arrows denote TSS and transcription orientation. *, P < 0.05 (Student’s t-test). TSS, transcription start site; TTS, transcription termination site.

Figure 6

Npac could be involved in RNA Pol II-mediated transcriptional elongation A. Npac interacted with RNA Pol II. Cell lysate of wild-type ESCs was immunoprecipitated using either anti-RNA Pol II antibody (upper) or anti-Npac antibody (lower). Western blot was subsequently carried out with anti-Npac antibody (upper) and anti-RNA Pol II antibody (lower). B. Npac can be pulled down with RNA Pol II Ser5P and vice versa. C. Npac can be pulled down with RNA Pol II Ser2P and vice versa. D. Npac depletion led to down-regulation of RNA Pol II Ser2P and RNA Pol II Ser5P, while total RNA Pol II was not affected. β-actin served as a loading control. Arrows indicate the binds corresponding to RNA Pol II Ser5P, RNA Pol II Ser2P, and RNA Pol II, respectively. E. and F. The binding of RNA Pol II at the gene bodies of Nanog (E) and Rif1 (F) was significantly reduced in Npac-depleted cells. G. and H. The binding of RNA Pol II Ser2P at the gene bodies of Nanog (G) and Rif1 (H) was significantly reduced in Npac-depleted cells. I. and J. The distributions of H3K36me3 at the gene bodies of Nanog (I) and Rif1 (J) were significantly reduced in Npac-depleted cells. In (E), (G), and (I), “Nanog + 51 bp” indicates 51 bp downstream region from Nanog TSS. *, P < 0.05 (Student’s t-test). Co-IP, co-immunoprecipitation; RNA Pol II Ser5P, Ser5-phospharylated RNA Pol II; RNA Pol II Ser2P, Ser2-phospharylated RNA Pol II.

Figure 7

Npac interacts with p-TEFb and Npac depletion may lead to transcriptional elongation defect A. Npac interacted with Cdk9. B. Npac interacted with Cyclin T1. C. Workflow for the elongation recovery assay. D.Npac KD efficiency was not affected by the addition of DRB in Npac-depleted cells. E. Schematic showing the analyzed regions of Nanog. F. Changes in the transcription rate of exon 4 of Nanog upon Npac depletion. G. Changes in the transcription rate of exon 1 of Nanog upon Npac depletion. H. Schematic showing the analyzed regions of Rif1. I. Changes in the transcription rate of exon 2 of Rif1 upon Npac depletion. J. Changes in the transcription rate of exon 30 of Rif1 upon Npac depletion. In (F), (G), (I), and (J), each graph illustrates the RNA levels at different regions of Nanog/Rif1 at different recovery time after DRB block was released. Data were shown as mean ± SE (n = 3). p-TEFb, positive transcriptional elongation factor; Cdk9, Cyclin-dependent kinase 9; DRB, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole.

Figure 8

Model depicting the role of Npac in pluripotency A. In normal mESCs, Npac is expressed at a high level and histone H3K36me3 is enriched in the gene bodies of actively transcribed genes. Npac interacts with RNA Pol II Ser2P and Ser5P and recruits p-TEFb to promote productive elongation. B. In Npac-depleted cells, reduction of Npac leads to reduced enrichment of RNA Pol II Ser2P and histone H3K36me3 at the pluripotency genes, thus blocking productive transcriptional elongation. CDT, C-terminal domain.