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. 2020 Mar 14;13(1):15.
doi: 10.1186/s13072-020-00336-w.

Alu Retrotransposons Modulate Nanog Expression Through Dynamic Changes in Regional Chromatin Conformation via Aryl Hydrocarbon Receptor

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Free PMC article

Alu Retrotransposons Modulate Nanog Expression Through Dynamic Changes in Regional Chromatin Conformation via Aryl Hydrocarbon Receptor

Francisco J González-Rico et al. Epigenetics Chromatin. .
Free PMC article

Abstract

Transcriptional repression of Nanog is an important hallmark of stem cell differentiation. Chromatin modifications have been linked to the epigenetic profile of the Nanog gene, but whether chromatin organization actually plays a causal role in Nanog regulation is still unclear. Here, we report that the formation of a chromatin loop in the Nanog locus is concomitant to its transcriptional downregulation during human NTERA-2 cell differentiation. We found that two Alu elements flanking the Nanog gene were bound by the aryl hydrocarbon receptor (AhR) and the insulator protein CTCF during cell differentiation. Such binding altered the profile of repressive histone modifications near Nanog likely leading to gene insulation through the formation of a chromatin loop between the two Alu elements. Using a dCAS9-guided proteomic screening, we found that interaction of the histone methyltransferase PRMT1 and the chromatin assembly factor CHAF1B with the Alu elements flanking Nanog was required for chromatin loop formation and Nanog repression. Therefore, our results uncover a chromatin-driven, retrotransposon-regulated mechanism for the control of Nanog expression during cell differentiation.

Keywords: Alu retrotransposons; Aryl hydrocarbon receptor; Chromatin conformation; Differentiation; Nanog.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of enhancer-blocking activity and histone methylation marks of the Alu elements located flanking the Nanog locus. a Scheme of the enhancer-blocking assay (EBA). b Insulator activity of NANOG x45s and x14s Alu elements using human HEK293 cell line. Constructs (blue bars) were transiently transfected and their activity analyzed by EBA. Data are showed as fold-enhancer blocking activity normalized to the reference pELuc vector. c Chromatin immunoprecipitation (ChIP) and re-ChIP for CTCF binding to the Nanog x45s Alu were done in NTERA2-wt cells left untreated (UT) or treated with 1 µM of RA for 48 h. For specificity, one primer for the qPCR reaction to amplify each Alu was located in a unique genomic sequence flanking the transposon (see Additional file 3: Table S2). Re-ChIP involved a first immunoprecipitation with CTCF antibody followed by a second immunoprecipitation with AhR antibody. Input DNAs, immunoprecipitation without specific antibodies and immunoprecipitation with GAPDH antibody were also preformed. d Analysis of the pattern of histone methylation marks in the regions of the Alu elements x45s and x14s flanking NANOG locus in NTERA2-wt UT, RA for 48 h and NTERA2-sh UT, RA for 48 h. Three biological replicates and three experimental replicates were done for panel B. Three biological replicates and two experimental replicates were done for panels C and D. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SD
Fig. 2
Fig. 2
Human Nanog locus forms a chromatin loop between x45s and x14s Alus upon cell differentiation in NTERA cell line. a Chromosome conformation capture (3C) assay using coordinates 3 and 6 as hooks. The relative crosslinking frequency was quantified in NTERA-wt cells untreated (UT, blue), treated with RA for 48 h (red) and in NTERA-sh cells UT (green), RA for 48 h (black). 3 + X (left) and 6 + X (right) primer combinations were addressed. b Chromosome conformation capture (3C) assay using coordinate 3 as hook. The relative crosslinking frequency was quantified in NTERA-wt cells untreated (UT, blue), treated with RA for 48 h (red) and in NTERA-CRISPRx45s cells UT (green), RA for 48 h (black). 3 + X primers combinations were addressed. c 3C assay using coordinate 6 as hook. The relative crosslinking frequency was quantified in NTERA-wt cells untreated (UT, blue), treated with RA for 48 h (red) and in NTERA-CRISPRx45s cells UT (green), RA for 48 h (black). 6 + X primers combinations were addressed. Three biological replicates and two experimental replicates were done for a. Two biological replicates and two experimental replicates were done for b and c. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SD
Fig. 3
Fig. 3
a 3C experiments with chaetocin (left) and deazaneplanocin-A (center) treatments, and CTCF siRNA transfection (right), in NTERA-wt UT (blue), treated with RA for 48 h (red), treated with chaetocin, deazaneplanocin-A or transfected with CTCF siRNA with or without RA (black and green, respectively). bNANOG mRNAs were quantified by RT-qPCR in NTERA2 cell line left untreated (UT) or treated with 1 µM RA for 48 h and/or chaetocin/deazaneplanocin-A for 48 h. GAPDH mRNA was used to normalize gene expression (A Ct) and 2−AACt to calculate variations with respect to control or untreated conditions. Three biological replicates and two experimental replicates were done for panels A. Four biological replicates and two experimental replicates were done for panel B. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SD
Fig. 4
Fig. 4
Dynamics of chromatin architecture-related proteins involved in the formation of Nanog chromatin loop during cell differentiation. a Scheme of the engineered chromatin immunoprecipitation (enChIP) 3× FLAG-dCas9 technique. b Chromatin immunoprecipitation (ChIP) for FLAG binding to the Nanog x45s and x14s Alus were done in NTERA2-wt cells left untreated (UT) or treated with 1 µM of RA for 48 h. ChIP was quantified by qPCR using specific oligonucleotides (see Additional file 3: Table S2). Input DNAs and immunoprecipitation without specifics antibodies were also preformed. c Table of main NANOG chromatin loop interacting proteins obtained with enChIP-dCas9 proteomic analysis (complete information enclosed in Additional file 3: Table S2). d Chromatin immunoprecipitation (ChIP) for CHAF1B, DDX5, KSRP, LAMIN A/C and PRMT1 binding to the Nanog x45s and x14s Alus were done in NTERA2-wt cells left untreated (UT) or treated with 1 µM of RA for 48 h. ChIP was quantified by qPCR using specific oligonucleotides (see Additional file 3: Table S2). Input DNAs and immunoprecipitation without specifics antibodies were also preformed for normalization and negative controls, respectively. Three biological replicates and three experimental replicates were done for panels B and D. *P < 0.05, **P < 0.01 and ***P < 0,001. Data are shown as mean ± SD
Fig. 5
Fig. 5
PRMT1 and CHAF1B drives the formation of NANOG locus Chromatin loop. a and b Chromosome Conformation capture (3C) assay using coordinate 3 as hook. The relative crosslinking frequency was quantified in NTERA-wt cells untreated (UT, blue), treated with RA for 48 h (red) and in NTERA-wt UT cells transfected with CHAF1B siRNA (a) or PRMT1 (b) (green), RA for 48 h (black). 3 + X primer combination was addressed. c Chromatin immunoprecipitation (ChIP) and re-ChIP for AhR and PRMT1 binding to the Nanog x45s Alu were done in NTERA2-wt cells treated with 1 µM of RA for 48 h. For specificity, one primer for the qPCR reaction to amplify each Alu was located in a unique genomic sequence flanking the transposon (see Additional file 3: Table S2). Re-ChIP involved a first immunoprecipitation with AhR antibody followed by a second immunoprecipitation with PRMT1 antibody. Input DNAs, immunoprecipitation without specifics antibodies and immunoprecipitation with GAPDH antibody were also preformed. d Analysis of the pattern of histone methylation marks in the regions of the Alu elements x45s and x14s flanking the Nanog locus in NTERA2-wt transfected with PRMT1 siRNA in conditions UT, RA for 48 h and NTERA2-sh UT, RA for 48 h. e Expression levels of NANOG mRNAs transfected with PRMT1 siRNA (left) or CHAF1B siRNA (right) were quantified by RT-qPCR in NTERA2 cell line left untreated (UT) or treated with 1 µM RA for 48 h. GAPDH mRNA was used to normalize gene expression (A Ct) and 2−AACt to calculate variations with respect to control or untreated conditions. Three biological replicates and two experimental replicates were done for a, b, c and d. Three biological replicates and three experimental replicates were done for e. *P < 0.05, **P < 0.01 and ***P < 0.001. Data are shown as mean ± SD
Fig. 6
Fig. 6
Scheme of proposed model of interaction between the protein complex and regulatory elements intervening in the regulation of NANOG’s expression in our model of carcinoma cell differentiation

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