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. 2022 Mar 1;36(5-6):313-330.
doi: 10.1101/gad.349039.121. Epub 2022 Feb 24.

Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states

Raymond Mario Barry  1   2 , Olivia Sacco  2 , Amel Mameri  3 , Martin Stojaspal  1   4 , William Kartsonis  1 , Pooja Shah  1 , Pablo De Ioannes  5 , Ctirad Hofr  4   6 , Jacques Côté  3 , Agnel Sfeir  2
Affiliations

Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states

Raymond Mario Barry et al. Genes Dev. .

Abstract

In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.

Keywords: 2C-like; EPC1; MERVL; RAP1; TIP60; ZSCAN4; telomere.

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Figures

Figure 1.
Figure 1.
Rap1 maintains gene transcription independent of telomeres. (A) Schematic representation of CRISPR/Cas9 gene-editing strategy to substitute Rap1 isoleucine 312 with arginine. Successful targeting creates an AciI restriction site to be used during genotyping. Single-stranded oligo donor (ssODN, black line) and cut site (red scissors) are indicated. (B) PCR genotyping of tail tip DNA from two Rap1I312R/+ heterozygous mice following gene targeting. (C) Example Sanger sequencing of a Rap1I312R/+ heterozygous mouse. (D) Representative image of IF-FISH in Rap1+/+, Rap1−/−, and Rap1I312R/I312R MEFs for Rap1 (green) and telomeres (red) using Rap1 antibody and a TTAGGG PNA probe, respectively. DAPI (blue) was used as a counterstain. (E) Rap1 Western blot from Rap1−/−, Rap1+/+, and Rap1I312R/I312R MEFs following subcellular fractionation into cytoplasmic (Cyt), nucleoplasmic (Nuc), and chromatin-bound (CBFs) fractions. α-Tubulin and histone H3 were loading controls for Cyt and CBFs, respectively. The blot is representative of n = 2 biological replicates. (F) Immunoblot for Rap1 from whole-cell lysates obtained from Rap1−/− (n = 2), Rap1+/+ (n = 3), and Rap1I312R/I312R (n = 3) MEFs. Rap1 relative abundance was determined by normalizing to γ-tubulin. (G) Hierarchically clustered heat map representing RNA-seq data for differentially expressed genes (DEGs) between Rap1+/+ and Rap1−/− MEFs (FC > 1.5; FDR < 0.1; n = 3 biological replicates per genotype). (Rap1IR/IR) Rap1I312R/I312R.
Figure 2.
Figure 2.
Rap1-I312R does not bind to genomic loci or DNA. (A) Dot blot for telomere repeats following ChIP using anti-HA antibody in cells expressing HA-tagged Rap1-WT, Rap1-I312R, or empty vector (EV) control (n = 2 biological replicates). Signal intensity was determined by normalizing to input. (B) High-throughput sequencing of ChIP samples as in A. Telomere binding was determined by calculating the proportion of reads with at least three telomere (TTAGGG)3/(CCCTAA)3 repeats. Data are the mean ± standard deviation of n = 4 biological replicates. (C) Summary of ChIP-seq peak calling analysis that identified 109 peaks in HA-Rap1-WT samples (MACS2; q < 0.05). Peaks were classified as subtelomeric if localized within 500 kb from the telomere. (D) Heat map representing ChIP-seq profiles of HA-Rap1-WT peaks at chromosome ends. (E) Heat map representing ChIP-seq profiles of HA-Rap1-WT interstitial peaks. (F) Electrophoretic mobility shift assay (EMSA) using His-tagged RAP (0–15 µM) and a 74-bp TTAGGG/AATCCC double-stranded DNA (100 nM). (G) EMSA using non-His-tagged RAP (0–15 µM) and a 74-bp TTAGGG/AATCCC double-stranded DNA (100 nM).
Figure 3.
Figure 3.
Proximity-dependent biotinylation reveals extratelomeric Rap1 binding partners. (A) Representative image of IF-FISH in Rap1−/− MEFs expressing BioID-Rap1-WT stained for BioID-Rap1 (magenta), biotin (green), and telomeres (red) using anti-Flag antibody, streptavidin, and TTAGGG PNA probe, respectively. DAPI (blue) was used as a counterstain. (B) Immunoblot for Rap1 and TRF2 following streptavidin pull-down in Rap1−/− MEFs expressing BioID-Rap1-WT (n = 3 biological replicates; clones C1, C2, and C3), BioID-Rap1-I312R (n = 4 biological replicates; clones C1, C2, C3, and C4), and BioID alone (EV). Where indicated, cells were treated with 50 µM biotin for 20 h prior to harvest. (C) Scatter plot representing log2 fold change in peptide spectrum match (PSM) of proteins identified by BioID-Rap1-WT versus BioID-Rap1-I312R. Fold change values were calculated relative to no biotin and BioID-alone controls. Each dot represents a unique protein. Known Tip60/p400 (green and purple) and shelterin (red) complex members are indicated. (D) Graphical representation of the top 10 biological processes overrepresented in BioID-Rap1-I312R streptavidin pull-down (FC > 2) using the PANTHER classification system statistical overrepresentation test. (E) Co-IP of Flag-tagged Tip60/p400 subunits (Tip60, Epc1, Epc2, Dmap1, Brd8, Ruvbl1, and Actl6a) and HA-Rap1 following cotransfection in HEK293T cells. (F) Reciprocal co-IP of HA-Rap1-I312R and Myc-tagged Epc1 or Epc2. Co-IP of HA-Rap1 or HA-Rap1-I312R with TRF2 was used as a control. (G) Reciprocal co-IP of HA-Rap1 and Myc-Tip60.
Figure 4.
Figure 4.
Rap1 forms a complex with and modulates the histone acetyltransferase (HAT) activity of Tip60/Epc1. (A) Schematic illustration of mouse Epc1 and Rap1 protein domains and deletion mutant constructs used for co-IP assays. (B) Co-IP of Flag-Epc1 deletion mutants and Myc-Rap1 in cotransfected HEK293T cells. Western blot analysis for input and IP sample was performed with the indicated antibody. (C) Co-IP of the Flag-Epc1 EPcA domain and Myc-Rap1. (D) Co-IP of Flag-Rap1 deletion mutants and Myc-Epc1. (E) Co-IP of Flag-Epc1, Myc-Tip60, and HA-Rap1. (F) Histone acetyltransferase (HAT) activity assays performed using co-IPs from E on core histones. Data are the counts per minute (CPM) mean ± standard deviation of n = 2 technical replicates; two-way ANOVA, and Tukey's multiple comparison test. (*) P-value < 0.0001. (G) HAT activity assay using affinity-purified human EPC1/TIP60 and RAP1 (increasing amounts) on short oligonucleosome chromatin. Bovine serum albumin (BSA) was a negative control. Data are CPM mean ± standard deviation of n = 3 technical replicates; two-way ANOVA, and Tukey's multiple comparison test. (*) P-value < 0.01.
Figure 5.
Figure 5.
Rap1 suppresses the 2C-like state by enhancing Tip60/p400 repression of 2C genes. (A) MA plot of log2 fold changes in gene expression in Rap−/− versus Rap1+/+ mESCs. Up-regulated (light red) and down-regulated (light blue) genes are indicated (FC > 1.5; FDR < 0.1; n = 5 biological replicates). 2C-stage genes are highlighted in dark red. (B) Hierarchically clustered heat map representing RNA-seq data of 2C genes up-regulated in Rap−/− versus Rap1+/+ mESCs (n = 5 biological replicates). (C) Volcano plot of differential repeat expression analysis in Rap1−/− versus Rap1+/+ mESCs. Up-regulated (red) and down-regulated (blue) repeats are indicated (FC > 2; FDR < 0.1; n = 5 biological replicates). (D) Kmeans (k = 5) clustered heat map representing RNA-seq data of 2C genes in Rap1+/+ (gray bar) and Rap1−/− (red bar) mESCs treated with siRNAs targeting Tip60/p400 subunits (Epc1, Tip60, p400, and Dmap1) or nontargeting (siNT) control (n = 3 biological replicates were used per condition). (E) ChIP-seq profiles of H4Ac and AcH2A.Z at the Zscan4d gene locus in Rap1+/+ (gray bar) and Rap1−/− (red bar) mESCs transfected with siRNA targeting Dmap1 or nontargeting (siNT) control. Profiles plotted are representative of n = 3 biological replicates. (F) Density plot centered at all MT2_Mm sites (n = 2667) for H4Ac and AcH2A.Z ChIP-seq data. Density plots are representative of n = 3 biological replicates. (G) ChIP-seq profiles of H4Ac and AcH2A.Z at a representative MERVL site in Rap1+/+ (gray bar) and Rap1−/− (red bar) mESCs transfected with siRNA targeting Dmap1 and nontargeting control (siNT). (H) Plot of the proportion of differentially expressed genes (DEGs) in Rap1+/+ versus Rap1−/− mESCs (FC > 1.5; FDR < 0.1) at fixed distances from the indicated MERVL (MT2_Mm, n = 2667; MT2B1, n = 7248; and MT2C_Mm, n = 1982) and LINE1 (Lx5, n = 16339; Lx3_Mus, n = 12651; L1Md_A, n = 16844; and L1Md_F n = 4016) repeat sequences. Proportions were calculated by dividing the number of DEGs by the total number of genes. (I) Plot of the proportion of up-regulated (blue) and down-regulated (red) DEGs in Rap1+/+ versus Rap1−/− mESCs (FC > 1.5; FDR < 0.1) at fixed distances from MT2_Mm repeat sequences.
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