Repetitive Sequence Stability in Embryonic Stem Cells

Abstract

Repetitive sequences play an indispensable role in gene expression, transcriptional regulation, and chromosome arrangements through trans and cis regulation. In this review, focusing on recent advances, we summarize the epigenetic regulatory mechanisms of repetitive sequences in embryonic stem cells. We aim to bridge the knowledge gap by discussing DNA damage repair pathway choices on repetitive sequences and summarizing the significance of chromatin organization on repetitive sequences in response to DNA damage. By consolidating these insights, we underscore the critical relationship between the stability of repetitive sequences and early embryonic development, seeking to provide a deeper understanding of repetitive sequence stability and setting the stage for further research and potential therapeutic strategies in developmental biology and regenerative medicine.

Keywords: DNA damage; embryonic stem cells; epigenetic regulation; repetitive sequences.

Figure 1

Classification and stability regulation mechanisms of repetitive sequences. (A) Classification of repetitive DNA sequences. Repetitive DNA sequences are divided into two classes: interspersed repetitive sequences and clustered tandem repetitive sequences. Tandem repetitive sequences are classified into satellites, minisatellites, and microsatellites. Interspersed repetitive sequences are mainly copies of transposable elements (TEs), including DNA transposons and retrotransposons. Among them, retrotransposons are mainly divided into two superfamilies: non-LTR retrotransposons and endogenous retroviruses. (B) Epigenetic mechanisms regulate repetitive DNA sequences (TEs and tandem repetitive sequences). (C) Rif1 is involved in DNA replication fork protection, DNA damage repair, telomere length homeostasis, ERV silencing, embryonic development, and TAD stabilization. (D) DNA topoisomerases and their special inhibitors mediate DNA cleavage and DNA damage on the telomeric DNA and purine–pyrimidine repeat sequences.

Figure 2

HR factors in replication fork protection and error-free DNA repair. (A) HR factors play a role in replication fork recognition and protection at S phase and error-free DNA repair at G2 phase in ESCs. The red and blue lines represent the sister chromatid. The curved red lines represent DNA replication. The incorrect symbol in the circle represents fork stalling. (B) BRCA1 deficiency indicates the inhibition of ALT events, the selection of DNA repair for repetitive sequences, satellite de-repression, and centromere instability. The red double line with a gap represents damaged DNA. The green lines represent repaired DNA. The curved blue lines represent centromeric RNA.

Figure 3

MMR inhibits recombination between diverged sequences. (A) MMR proteins play an important role in inhibiting spontaneous mutation, diverged recombination and repetitive sequence translocation. Different colors and shapes represent MMR proteins. (B) EXO1 or MSH2 inhibits recombination between diverged sequences, rather than recombination between identical, homologous sequences. The curved arrow represents the transfection of the reported plasmid into the cells. The black asterisks (*) represent GFP-mutant sites. The green box represents GFP expression. The red arrow represents increased GFP expression level.

Figure 4

LINE-1 retrotransposon drives DNA damage and transposition. (A) The structure of human LINE1 element. LINE-1 is constituted by a 5′ UTR with promoter, ORF1, ORF2, and a 3′ UTR with a poly(A) tail. ORF1 encodes RNA binding protein ORF1p (arrow shown), and ORF2 encodes a protein ORF2p (arrow shown), which has both endonuclease (EN) and reverse transcriptase (RT) activity. In the 3′ UTR of LINE-1 elements, G-rich sequences are the hallmark of young LINE-1 retrotransposon. ORF1p, ORF2p, and G-quadruplex structures contribute to its transposition. (B) LINE-1 RNA recruits Nucleolin/Trim28 to repress the 2-cell marker Dux and activate rRNA synthesis. LINE-1 knockdown in murine zygotes arrests preimplantation development at the two-cell and four-cell stages. (C) LINE1-encoded endonuclease cleaves genomic DNA and increases γH2AX levels. LINE-1 EN mutants use endogenous exposure 3′ OH GGGATT overhang as a primer to insert into dysfunctional telomeres. DSB sites induced by CRISPR/Cas9 promote de novo LINE-1 insertion, which is EN-independent and RT-dependent. The red arrow represents increased γH2AX level.

Figure 5

Dynamic chromatin structure in response to DNA damage. (A,B) CTCF depletion leads to spontaneous 2CLC reprogramming from mESCs. In addition, overexpression of Zscan4c in CTCF-deficient ESC increased 2CLC conversion, and Zscan4c may activate MERVL to promote 2CLC reprogramming in the context of weakened CTCF-induced TADs. (C) CTCF and cohesin establish γH2AX foci and nano-foci formation, and recruit DNA damage proteins to ensure proper DSB repair. Once CTCF depletion causes the loss of higher-order chromatin structures, it impairs bidirectional spread of γH2AX foci (double arrows shown) and DNA repair (one arrow shown) within TADs. (D) The distribution of SINE-B1/ALU and LINE-1 repeat sequences is correlated with the A/B compartment. LINE-1 is located in the B compartment, predominantly within the heterochromatin region, where heterochromatin protein HP1α and H3K9me3 modification bind around the nuclear membrane and nucleolus. SINE-B1/ALU aggregates in the A compartment, situated in the euchromatin region, where Pol II binds around the nucleoplasm.