Tet-mediated imprinting erasure in H19 locus following reprogramming of spermatogonial stem cells to induced pluripotent stem cells

Abstract

Selective methylation of CpG islands at imprinting control regions (ICR) determines the monoparental expression of a subset of genes. Currently, it is unclear whether artificial reprogramming induced by the expression of Yamanaka factors disrupts these marks and whether cell type of origin affects the dynamics of reprogramming. In this study, spermatogonial stem cells (SSC) that harbor paternalized imprinting marks, and fibroblasts were reprogrammed to iPSC (SSCiPSC and fiPSC). The SSCiPSC were able to form teratomas and generated chimeras with a higher skin chimerism than those derived from fiPSC. RNA-seq revealed extensive reprogramming at the transcriptional level with 8124 genes differentially expressed between SSC and SSCiPSC and only 490 between SSCiPSC and fiPSC. Likewise, reprogramming of SSC affected 26 of 41 imprinting gene clusters known in the mouse genome. A closer look at H19 ICR revealed complete erasure in SSCiPSC in contrast to fiPSC. Imprinting erasure in SSCiPSC was maintained even after in vivo differentiation into teratomas. Reprogramming of SSC from Tet1 and Tet2 double knockout mice however lacked demethylation of H19 ICR. These results suggest that imprinting erasure during reprogramming depends on the epigenetic landscape of the precursor cell and is mediated by TETs at the H19 locus.

Figure 1. Reprogramming and characterization of iPSC derived from SSC.

(A) Relative mRNA abundance determined by qPCR of three genes expressed in SSC (Pou5f1, Gfra1 and Id4) and a marker of differentiated spermatogonia (cKit) in the testicular population sorted for Gfra1 (Gfra1+), unsorted (Gfa1−), and somatic fibroblast controls. Different letters indicate significant differences between treatments based on ANOVA (p < 0.05). (B) H&E histological section of a subcutaneous teratoma generated by SSCiPSC injection into immunocompromised mouse. The central image shows a 4X overview of the teratoma [Scale bar: 500 μM]. Four areas are amplified (20X) in the side images showing derivatives from the three germ layers [Scale bar: 100 M]. (C) Representative 10X light microscope image of SSCiPSC colony morphology after alkaline phosphatase staining [Scale bar: 200 μM]. (D) Percentage of skin chimerism in pups derived from 3 different SSCiPSC lines (green bars) or fiPSC (red-brown bars).

Figure 2. Global transcriptome profile of imprinted and non-imprinted loci as revealed by RNAseq analysis.

(A) Hierarchical clustering of the nine biological samples analyzed; (B,C) Differentially expressed genes (FDR<0.05) between SSC and SSCiPSC (8124 genes, (B)) or fiPSC and SSCiPSC (490 genes, (C)); (D) Representation of the 41 known imprinting clusters along the mouse genome. Yellow squares depict clusters (15) with no differentially express genes, red squares denote clusters (26) with differentially expressed imprinted genes.

Figure 3. CpG methylation of H19 Imprinted Controlled region following reprogramming and differentiation.

Bisulfite sequencing of H19 ICR revealed that CpG methylation was almost completely erased following reprogramming of (A) SSCiPSC (68% in SSC vs 0–2% in SSCiPSC) in comparison to (B) fiPSC (48% in fibroblast vs 20–39.3% in fiPSC). (C) The loss of imprinting erasure in SSCiPSC is maintained even following differentiation.

Figure 4. Methylation at H19 following reprogramming of Tet1 and Tet2 KO (DKO).

(A) CRISPR/Cas mediated knockout of endogenous Tet1 and Tet2 loci by intra-cytoplasmic injection of in vitro transcribed Cas9 mRNA and sgRNA into mouse 2-cells embryos. CRISPR injection resulted in indels and two different genotypes. The CRISPR target sequence is indicated in green letters, whereas red letters indicates the indels generated. (B) Methylation at H19 ICR following reprogramming of Tet1 and Tet2 knockout SSC into three different lines of SSCiPSC.