AbstractThe recent discovery of N6-methyladenine (N6-mA) in mammalian genomes suggests that it may serve as an epigenetic regulatory mechanism1. However, the biological role of N6-mA and the molecular pathways that exert its function remain unclear. Here we show that N6-mA has a key role in changing the epigenetic landscape during cell fate transitions in early development. We found that N6-mA is upregulated during the development of mouse trophoblast stem cells, specifically at regions of stress-induced DNA double helix destabilization (SIDD)2,3,4. Regions of SIDD are conducive to topological stress-induced unpairing of the double helix and have critical roles in organizing large-scale chromatin structures3,5,6. We show that the presence of N6-mA reduces the in vitro interactions by more than 500-fold between SIDD and SATB1, a crucial chromatin organizer that interacts with SIDD regions. Deposition of N6-mA also antagonizes SATB1 function in vivo by preventing its binding to chromatin. Concordantly, N6-mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin. Repression of SIDD–SATB1 interactions mediated by N6-mA is essential for gene regulation during trophoblast development in cell culture models and in vivo. Overall, our findings demonstrate an unexpected molecular mechanism for N6-mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development.
MainSeveral recent studies have implicated N6-mA in epigenetic silencing, especially at long interspersed element 1 (LINE-1) endogenous transposons in mouse embryonic stem (mES) cells, in brain tissue under environmental stress, and in human lymphoblastoid cells and tumorigenesis1,7,8,9. Although these findings underscore the importance of N6-mA in mammalian biology and human disease, the underlying mechanisms of N6-mA-mediated gene silencing have thus far remained unclear.N6-mA is enriched at AT-rich regions in mammalian genomes1, especially in areas that interact with the nuclear architecture, such as matrix/scaffold attachment regions (M/SARs). M/SARs are liable to undergo DNA unpairing under torsional stress (known as SIDD or as base unpairing regions (BURs))2,3,4 during replication, transcription, and recombination3,5,6. A study that used genome-wide permanganate and S1 endonuclease mapping (single-stranded (ss)DNA-seq10) demonstrated a strong correlation between SIDD regions identified experimentally and those predicted by computational approaches11. SIDD regions in M/SARs are crucial for organizing large chromatin domains, such as establishing and maintaining heterochromatin–euchromatin boundaries12,13,14 and facilitating long-range interactions15.SATB1, a well-known SIDD-regulating protein, is mainly expressed in developing T cells16, epidermis17, and trophoblast stem cells (TSCs)18,19. SATB1 binds to SIDD and then stabilizes the DNA double helix2,20, thereby establishing and maintaining large-scale euchromatin and heterochromatin domains12,13,14. For example, SATB1 binds directly to key enhancers in pro- and pre-T cells, thereby activating gene expression in these cells while repressing the mature T cell fate21. During early embryogenesis, SATB1 and SATB2 form an intricate network that regulates ES cell differentiation22. In addition, there is emerging evidence that Satb1 is markedly upregulated during extraembryonic tissue development and promotes extraembryonic fates such as trophectoderm and primitive endoderm lineages18,19,23.In this work, we show that N6-mA contributes to TSC development and differentiation by antagonizing SATB1 function at SIDD. Moreover, ALKBH1, the DNA demethylase of N6-mA, highly prefers SIDD sequences as substrates24, which further strengthens the connection between N6-mA and DNA secondary structures during cell fate transitions.N
6-mA at SIDD during TSC developmentAs N6-mA is present at low levels (6–7 parts per million (ppm)) in mES cells1, we searched for conditions under which N6-mA levels were upregulated. Notably, N6-mA levels are positively correlated with the in vivo developmental potential (pluripotency) of these conditions as determined by tetraploid complementation (4N). N6-mA is greatly diminished under traditional 2i conditions (cultures with ERK and GSK3b inhibitors, which are 4N negative) but retained under some alternative 2i conditions (4N positive) (Extended Data Fig. 1a), which may explain discrepancies in the literature25.As previous studies showed that N6-mA demethylase Alkbh1-deficient mice developed trophectodermal defects26, we next investigated the role of N6-mA in the development and differentiation of TSCs. We leveraged a Cdx2-inducible expression system27 (iCdx2) to model TSC development in cell culture, which manifests a well-synchronized and efficient cell fate transition process (Fig. 1a). Consistent with previous work28, our RNA sequencing (RNA-seq) analysis showed that iCdx2 cells go through first a transition state, then a TSC-like cell (TSC-LC) state, before undergoing trophectoderm lineage differentiation (Extended Data Fig. 1b). The N6-mA level increased transiently at a time window that coincides with the emergence of TSC-LCs, and then tapered off during trophectoderm lineage differentiation (Fig. 1a). Accordingly, the expression of Alkbh1 inversely correlated with N6-mA levels during this cell fate transition (Extended Data Fig. 1b).Fig. 1: N6-mA is upregulated at SIDD regions during TSC development.a, Top, schematic of the iCdx2 ES cell-to-TSC fate transition system. Bottom, DNA dot blotting of N6-mA at different time points during differentiation. D, day. Experiments were repeated independently three times with similar results. For blot source data, see Supplementary Fig. 1. b, Average N6-mA reads per genomic content (RPGC) at different time points, centred at differentially increased N6-mA peaks (day 5 versus day 0). c, Mass spectrometry (MS) analysis of N6-mA. Left, schematic of matrix attachment region (MAR) DNA extraction. Middle, N6-mA levels from different chromatin fractions and mock control (buffer, nucleases, and proteinase K). One-way ANOVA (P