Functional and molecular characterization of the role of CTCF in human embryonic stem cell biology

Abstract

The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting, X-chromosome inactivation and genomic architecture. In this study, we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes, including NANOG, SOX2, cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation, but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs, CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors, such as Cohesin, differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly, the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology.

Figure 1. Characterization of the hESC differentiation model.

A. 10X Phase contrast images of H9 pluripotent hESCs and H9 hESCs induced to differentiate with BMP4 for 5 days. 200 ng/ml BMP4 in TeSR was used in all panels. B. RT-PCR analysis showing decrease in mRNA levels of indicated genes upon BMP4 treatment of H9 cells. Expression levels are normalized to GAPDH and represented relative to pluripotent ESCs set to 1. C. Western analysis showing decrease in expression levels of indicated proteins upon BMP4 treatment of H9 cells. D. hCG ELISA from supernatant of BMP4-treated (yellow bars) and control-treated H9 (black bars hCG levels in control cells (hESCs) were barely detectable. E. RT-PCR analysis showing increase in mRNA levels of indicated genes upon BMP4 treatment of H9. Expression levels are normalized to GAPDH.

Figure 2. Effect of CTCF depletion on hESCs.

A. RT-PCR (left) and Western analysis (right) showing CTCF mRNA and protein levels, respectively, in H9 hESCs transfected with a scrambled siRNA (control si) or siCTCF. mRNA levels are normalized to GAPDH and represented relative to pluripotent hESC set to 1. B. BrDU proliferation assay in H9 showing that CTCF knockdown impairs proliferation. A₄₅₀ for siCTCF is normalized to that of control si (scrambled control) set to 1. Mean ± SEM represented. * represents p<0.05. C. Schematic of experimental design for Figures 2D, E and F. Each vertical line represents a 24 hr period. D. mRNA levels of indicated genes at 48 hrs and 96 hrs after CTCF knockdown in H9 and H1 hESCs. mRNA levels of indicated genes in control si and siCTCF were normalized to respective GAPDH levels. Subsequently, mRNA levels of siCTCF were normalized to control si set to 1. Mean ± SEM represented. E. RT-PCR analysis of indicated genes upon control si (black line) or siCTCF (red line) transfection followed by BMP4 treatment for 5 days in H9. mRNA levels were analyzed at the indicated days and normalized to GAPDH. Mean ± SEM represented. F. Immunofluorescence of NANOG and OCT4 proteins upon control si or siCTCF transfection followed by BMP4 treatment at indicated days in H9. Scale bar represents 360 µm.

Figure 3. CTCF associates with the NANOG-DPPA3-GDF3 locus in hESCs.

A. Schematic of the human NANOG locus that encompasses the co-regulated NANOG-DPPA3-GDF3 genes. Co-ordinates are represented relative to the NANOG transcription start site set to +1. Red amplicons represent PCR primers used for Chromatin Immunoprecipitation (ChIP) analyses. B. Chromatin Immunoprecipitation (ChIP)-qPCR analyses using α-IgG (control) and α-CTCF in H9 hESCs. Amplicons as in panel A. A PCR primer pair that amplifies a region within the DPPA3 gene (−74 kb upstream of NANOG transcription start site) was used as a negative control. Immunoprecipitated DNA is represented as a percentage of input amplified with the same PCR primers. Mean ± SEM represented.

Figure 4. CTCF sites within the NANOG locus can function as CTCF-dependent enhancer-blocking insulators.

A. Luciferase reporter assay measuring enhancer-blocking activity of CTCF sites from the NANOG locus. Enhancer-blocking plasmids contained a CMV enhancer, a CMV promoter-driven luciferase reporter, and 1.2 kb of DNA encompassing CTCF binding sites from relevant regions were cloned into either XhoI or Pst1 restriction enzyme sites. Luciferase activity was normalized to that of a co-transfected renilla-luciferase and normalized relative to pELuc (empty vector) set to 100%. 5′HS4 from the chicken β-globin locus was used as a positive control. Mean ± SEM represented. B. Same as above except cells were transfected with either a scrambled siRNA control or siCTCF before transfection of XhoI luciferase reporter constructs. **represents p<0.01. C. Same as panel B except cells were transfected with PstI luciferase reporter constructs. **represents p<0.01, *represents p<0.05.

Figure 5. Differential association of CTCF interacting partners and protein poly(ADP- ribosyl)ation within the NANOG locus in pluripotent and differentiated hESCs.

A. CTCF ChIP as in Figure 3 in H9 hESCs (black bars) and H9 hESCs differentiated with BMP4 for 5 days (Diff. hESC). The α-IgG (control) immunoprecipitates significantly lower levels of DNA relative to input (data not shown). Mean ± SEM represented. B. Western analyses of indicated proteins in H9 hESCs and 5 day BMP4-treated H9 hESCs (Diff. hESC). Mean ± SEM represented. C. Top panel: Immunoprecipitation of CTCF followed by Western blot analysis of indicated proteins. Bottom panel: Immunoprecipitation using an anti-PAR antibody followed by Western blot analysis with CTCF or Nucleolin. 1% Input was used in both top and bottom panels. D–F. As in Figure 3, except ChIP analysis of indicated proteins or modification. *represents p<0.05, **represents p<0.01. Mean ± SEM represented.