# The superpowers of imprinting control regions

Bertille Montibus, Franck Court, Philippe P. Arnaud

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Bertille Montibus, Franck Court, Philippe P. Arnaud. The superpowers of imprinting control regions. Genome Research, 2025, 35, pp.1-19. 10.1101/gr.281215.125 . hal-05424162

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# The superpowers of Imprinting Control Regions

Bertille Montibus¹, Franck Court¹ Philippe Arnaud¹*

¹Genetics, Reproduction and Development Institute (iGReD), CNRS, INSERM, Université Clermont Auvergne, Clermont-Ferrand, France.

*Correspondence: philippe.arnaud@uca.fr

**Running title:** Imprinting Control Region (ICR): A multifunctional cis-regulatory region

ORCID IDs: Bertille Montibus : 0000-0002-6895-3954; Franck Court : 0000-0003-0327-9708; Philippe Arnaud : 0000-0002-7937-8764
**Summary**

Genomic imprinting is a specialized mechanism of transcriptional regulation whereby approximately 200 mammalian genes are expressed monoallelically, according to their parental origin. This crucial developmental process is primarily controlled by discrete cis-regulatory elements known as imprinting control regions (ICRs), which play an essential role in directing allele-specific gene expression across large imprinted domains.

In this review, we highlight the features that define ICRs as a distinct class of cis-regulatory regions, from their ability to maintain germline-inherited DNA methylation to their multifunctional roles in transcriptional control. For each imprinted domain, we examine the diverse mechanisms by which individual ICRs integrate multiple regulatory functions to coordinate both proximal and distal imprinted gene expression. By uncovering the multifaceted roles of ICRs, this review provides a compelling framework for understanding, more broadly, the molecular basis of finely controlled gene expression.

**Keywords:** Epigenetic, cis-regulatory region, promotor, enhancer, insulator, transcriptional interference, lncRNA, 3d-chromatin structure.
The functional specialization of each cell type and tissue is a complex process in higher organisms. It depends on the cell's ability to establish its own transcriptional profile in response to developmental and environmental cues. This process involves the coordinated interaction of multiple factors that ensure the fine-tuned regulation of gene expression.

Among the regulatory mechanisms, cis-regulatory regions play a key role by precisely and timely recruiting transcription factors and other regulatory molecules to activate and maintain appropriate transcriptional activity. The most well-described types of cis-regulatory regions include proximal cis-acting promoters, as well as distal enhancers, silencers, and insulators.

This review focuses on a small family of cis-regulatory regions comprising just over twenty members: the Imprinting Control Regions (ICRs). These regions are essential for regulating approximately 200 imprinted genes in mammals, which are expressed in a parent-of-origin-specific manner. ICRs are emerging as "Swiss-army knife" regulatory elements that, by integrating epigenetic signals and influencing the three-dimensional chromatin structure, can function as promoters, enhancers, and/or insulators, thereby regulating both proximal and distal target genes in an allele-specific manner.

## Genomic Imprinting

In the mid-1980s, it was observed that fertilized mouse oocytes containing either two maternal or two paternal genomes failed to develop to term, despite their diploid composition (Barton et al. 1985). This work demonstrated the existence of functional differences between the two parental genomes and, in fact, highlighted the necessity of both a maternal and a paternal genome for proper mammalian development. Genetic studies in mouse embryos with uniparental disomy (UPD) of specific chromosomes or chromosomal regions (i.e., mice that inherit two maternal copies and no paternal copies, or vice versa, of a chromosome or part of a chromosome) have helped further define the genomic regions involved in this phenomenon. These studies identified several genomic areas where uniparental inheritance leads to lethality or abnormal growth and/or behavioral phenotypes, providing the foundation for the first imprinting map (Cattanach and Kirk 1985). Taken together, the studies provided strong evidence for the existence of genes whose expression status depends on whether they are inherited from the mother or the father, so-called imprinted genes.

The first to be identified in 1991, was the Insulin-like growth factor receptor (Igf2r), which is expressed from the maternal allele (Barlow et al. 1991). Since then, between 150 and 200 imprinted genes have been identified in humans and mice, with almost a third of them conserved between these two species (Babak et al. 2008; Perez et al. 2015; Andergassen et al. 2017; Tucci et al. 2019). Most of these imprinted genes are organised in evolutionarily conserved genomic clusters, which contain two or more maternally and paternally expressed genes in large regions of up to several megabases in size.

Although it is likely that the majority of genes subject to imprinting have been identified (DeVeale et al. 2012; Kelsey and Bartolomei 2012; Edwards et al. 2023), it cannot be ruled out that more refined analyses will lead to the identification of new imprinted genes (Henckel and Arnaud 2010). Technically and conceptually, it is indeed difficult to identify all the genes subject to imprinting. Beyond the canonical image of a gene that is always and exclusively expressed from a single parental allele, the genes subject to imprinting often have a more complex expression pattern. Often, mono-allelic expression is limited to certain isoform, certain tissues, with many of these genes being imprinting in placenta and/or brain only, at certain stages of development or even in certain individuals (polymorphic imprinting). This last case has been documented, for example, for the *IGF2R* and *WT1* genes in humans (Jinno et al. 1994; Monk et al. 2006). The development of higher resolution RNA-seq-based approaches to accurately detect and quantify mouse strain-specific polymorphisms on transcripts isolated from reciprocal hybrid biological material identified genes with more or less pronounced parental origin-specific allelic bias rather than a completely monoallelic expression pattern (Perez et al. 2015;
Andergassen et al. 2017). Single-cell RNA-seq performed in the mouse cortex further shows that monoallelic/biased expression is consistently found across all cells of major cortical cell types for most imprinted genes, highlighting that, at least in this tissue, bias is not an artefact due to a mix of cells with imprinted and non-imprinted expression for a given gene (Laukoter et al. 2020). These findings raise questions about the significance of this allelic bias and, more generally, about the definition of an imprinted gene. The observation that brain-specific deletion of the paternal, but not the maternal, allele of the paternally biased *Bcl2l1* gene results in the loss of specific neuron types provides proof of concept that this phenomenon has functional significance and supports that gene with allelic bias are imprinted genes (Perez et al. 2015).

In the animal kingdom, imprinting is restricted to placental mammals, suggesting a link between *in utero* fetal development and imprinting (Moore et al. 2015). This link is also found at a functional level, with many imprinted genes involved in the control of fetal and placental growth. Genomic imprinting also plays a key role in the postnatal regulation of brain function, behavioural development and metabolism. Consequently, its deregulation has been implicated in human pathologies. In addition to cancer or placenta-related pathologies (Monk 2015), alterations in imprinted gene dosage leads to congenital disorders, collectively termed imprinting disorders, of which 13 have been characterised to date (Eggermann et al. 2023). These disorders share clinical features including growth restriction, cognitive impairment, metabolic disorders, feeding difficulties and hypotonia (Eggermann et al. 2023).

Epigenetic modifications, particularly DNA methylation, play a central role in the regulation of genomic imprinting. These modifications primarily target discrete cis-regulatory elements known as imprinting control regions (ICRs), which are essential for orchestrating monoallelic gene expression across large imprinted domains. In this review, we examine, for each imprinted domain, the diverse mechanisms by which individual ICRs integrate multiple cis-regulatory activities to regulate both proximal and distal imprinted gene expression throughout the entire domain.

## Imprinting Control Regions

ICRs are genomic regions of a few kilobases, rich in the dinucleotide CpG (although not all of them have CpG island status), for which it has been functionally demonstrated that they control, in *cis*, the monoallelic expression of all imprinted genes within a cluster (Arnaud 2010; Kelsey and Feil 2013). Each imprinting domain is thus controlled by an ICR. One of the key features of these ICRs is that they are methylated only on one of the two parental alleles, and it is widely accepted that this parental allele-specific DNA methylation is essential for the imprinting mechanism (Bourc'his et al. 2001; Hata et al. 2002). Allelic DNA methylation originates from either the male or female gamete and is subsequently maintained throughout development in the embryo and then, in all adult tissues. These regions are therefore also referred to as gDMR for "germline differentially methylated region" (Arnaud 2010; Kelsey and Feil 2013). Following this definition, twenty "ubiquitous" gDMR/ICRs have been identified in mice (Proudhon et al. 2012; Xie et al. 2012), most of which are conserved in humans (Table 1). Only 3 of these ICRs, *H19*, *Rasgrf1* and *IG-DMRs*, all located in an intergenic region, are methylated on their paternal allele. The 17 ICRs with maternal methylation all coincide with a promoter region. Notably, two maternal gDMRs are present at the *Gnas* locus. The major ICR is located at the *Gnasxl/Nespas* promoter, and its deletion affect expression of all transcript of the locus, while a gDMR located at the 1A promoter is consider as subsidiary ICR and controls tissue-specific imprinted expression of the *Gnas* transcript only(Williamson et al. 2004, 2006) (Table 1). In addition, DNA methylation at ICRs interacts with histone modifications, leading to a distinct histone mark signature (Henckel et al. 2009). The methylated allele displays an heterochromatic signature, enriched with repressive marks such as H3K9me3 and H4K20me3. In contrast, the non-methylated allele is characterized by permissive marks, including H3K4me2/me3 (Fournier et al. 2002; McEwen and Ferguson-Smith 2010). Accordingly, at
the genome-wide level, ICRs are discrete regions with a four-mark signature comprising partial DNA methylation, H3K4me3, H3K9me3 and H4K20me3 in somatic cells.

With the exception of the *Impact* and *Slc38a4* loci, each ICR controls the imprinting of multiple genes within large domains ranging from 100kb to several Mb (Table 1, Figure 1). Of the two largest known imprinting clusters in mice, the *Snrpn* locus, is 3.25 Mb in size and contains 14 imprinted genes, while the *Kcnq1* locus is 845 kb in size but contains 12 imprinted genes (Table 1) (Barlow 2011). However, the size of a given cluster may vary depending on the tissue. Specifically, imprinted gene clusters expand and contract during development (Andergassen et al. 2017). In general, clusters expand from implantation, reaching a maximum in extra-embryonic tissues, and then retract in adulthood. This phenomenon is very well illustrated by the *Igf2r* locus, whose expansion to 10 Mb in the placenta brings six new genes, including *Arib1* and *Thbs2*, under the control of the ICR, allowing their maternal expression specifically in this tissue (Andergassen et al. 2017). This observation highlights the fact that an ICR can control genes over a very long distance and that its action can be tissue specific.

In addition to these canonical “ubiquitous” ICRs, ICRs exhibiting a parent-of-origin DNA methylation signature restricted to specific developmental stages have been identified. Twenty of these so-called transient ICRs have been identified in mice and several dozen in humans (Table 2) (Proudhon et al. 2012; Sanchez-Delgado et al. 2016; Yang et al. 2022). These transient gDMR/ICRs, described in more detail in a dedicated section below, extend the scope of allele-specific regulation during mammalian development, particularly during early embryogenesis.

<table><thead><tr><th>Coordinates<br>(mm39)</th><th>Locus/localisation<br>(*: identified in<br>gynogenetic/androgenetic embryos)</th><th>Methylation origin</th><th>Allelic DNA methylation<br>maintenance</th><th>Controlled genes<br>(Expression origin: red maternal, blue paternal; TE:transposable element)</th><th>Reference</th></tr></thead><tbody><tr><td>chr11:19,967,200-19,973,800</td><td>Spredb2 gene body *</td><td>Maternal</td><td>Pre-implantation</td><td>Actr2</td><td>Yang et al., 2022</td></tr><tr><td>chr1:63,237,859-63,240,159</td><td>80 kb upstream Zdbf2</td><td>Maternal</td><td>Pre-implantation and Placenta</td><td>Liz</td><td>Duffie' et al., 2014</td></tr><tr><td>chr1:168,821,520-168,822,120</td><td>1700017J07Rik *</td><td>Maternal</td><td>Pre-implantation</td><td>Zfp64</td><td>Yang et al., 2022</td></tr><tr><td>chr8:123587339-123593939</td><td>Cdh15 gene body</td><td>Maternal</td><td>Tissue-specific</td><td>Cdh15 short isoform</td><td>Proudhon et al., 2012</td></tr><tr><td>chr7:12704327-12705727</td><td>Zfp324 gene body *</td><td>Maternal</td><td>Pre-implantation</td><td>TE G36178</td><td>Yang et al., 2022</td></tr><tr><td>chr5:135377454-135381054</td><td>Fkbp6 promoter</td><td>Maternal</td><td>Pre-implantation and Placenta</td><td>Fkbp6 (paternal expression is observed in all embryonic stage tissues and in placenta)</td><td>Strogantsev et al., 2015</td></tr><tr><td>chr17:34617574-34618374</td><td>BC051142 promoter*</td><td>Maternal</td><td>Pre-implantation</td><td>Brd2</td><td>Yang et al., 2022</td></tr><tr><td>chr5:143743755-143744355</td><td>D130017N08Rik promoter*</td><td>Maternal</td><td>Pre-implantation</td><td>Ocm</td><td>Yang et al., 2022</td></tr><tr><td>chr1:37913981-37914881</td><td>Lipt1 gene body *</td><td>Maternal</td><td>Pre-implantation</td><td>Mitd1</td><td>Yang et al., 2022</td></tr><tr><td>chr7:127465372-127467572</td><td>Zfp668 gene body *</td><td>Maternal</td><td>Pre-implantation</td><td>Prss36</td><td>Yang et al., 2022</td></tr><tr><td>chr7:6679999-6681199</td><td>Zim1 last exon *</td><td>Maternal</td><td>Pre-implantation</td><td>TE G36138</td><td>Yang et al., 2022</td></tr><tr><td>chr13:47316673-47317624</td><td>AK008011 (Coro1C retrogene) promoter</td><td>Maternal</td><td>Tissue-specific</td><td>Pseudogene</td><td>Proudhon et al., 2012</td></tr><tr><td>chr6:47978140-47979732</td><td>Zfp777 last exon</td><td>Maternal</td><td>Pre-implantation</td><td>Zfp777 unaffected (biallelic expression in blastocyst)</td><td>Proudhon et al., 2012</td></tr><tr><td>chr7:6134799-6136399</td><td>Zfp787 last exon</td><td>Maternal</td><td>Pre-implantation</td><td>Zfp787 unaffected (biallelic expression in blastocyst)</td><td>Proudhon et al., 2012</td></tr><tr><td>chr9:44032497-44033097</td><td>Intergenic*</td><td>Paternal</td><td>Pre-implantation</td><td>none</td><td>Yang et al., 2022</td></tr><tr><td>chr14:119116412-119118312</td><td>Dzip1 gene body*</td><td>Maternal</td><td>Pre-implantation</td><td>TE G13933</td><td>Yang et al., 2022</td></tr><tr><td>chr5:144491810-144493410</td><td>Nptx2 gene body*</td><td>Maternal</td><td>Pre-implantation</td><td>TE G33380</td><td>Yang et al., 2022</td></tr><tr><td>chr10:13002744-13003744</td><td>Plagl1 last exon*</td><td>Maternal</td><td>Pre-implantation</td><td></td><td>Yang et al., 2022</td></tr><tr><td>chr17:87441028-87442628</td><td>Socs5 gene body*</td><td>Maternal</td><td>Pre-implantation</td><td></td><td>Yang et al., 2022</td></tr><tr><td>chr18:24838657-24839257</td><td>Intergenic*</td><td>Paternal</td><td>Pre-implantation</td><td>TE G28017</td><td>Yang et al., 2022</td></tr><tr><td>chr4:11400000-11401000</td><td>Intergenic*</td><td>Maternal</td><td>Pre-implantation</td><td>TE G27043</td><td>Yang et al., 2022</td></tr><tr><td>chr4:15341400-15344000</td><td>Intergenic*</td><td>Paternal</td><td>Pre-implantation</td><td>Tmen64</td><td>Yang et al., 2022</td></tr><tr><td>chr8:4044000-4047400</td><td>Intergenic*</td><td>Maternal</td><td>Pre-implantation</td><td>Map2K7</td><td>Yang et al., 2022</td></tr></tbody><tfoot><tr><td colspan="6">Table 2: Transient DMRs in mouse</td></tr></tfoot></table>

## ICR stores the methylation inherited from the germ cells

One of the most important features of the ICRs, and one that is fundamental to their function, is their ability to acquire a distinct methylation profile in each germ line and to maintain it throughout life. Historically, research into the underlying mechanisms has focused on how these regions could be specifically targeted by methylation in one of the two germ lines. However, the development of methods to determine methylation profiles genome-wide, in male and female gametes and at early stages of development, has changed this question considerably. These advances have shown that, to use the title of this review, the primary « superpower » of ICRs is not so much their ability to be specifically methylated in one of the germ lines, but rather their capacity to maintain this state of differential methylation during the peri-implantation phases.

In mammals, epigenetic patterns undergo significant modifications during two crucial developmental stages, involving the erasure of existing marks and the establishment of new ones. This process of epigenetic reprogramming takes place; first; during gametogenesis and later after fertilization, at the
peri-implantation stage (Reik et al. 2001). Gametogenesis reprogramming results in a global erasure of DNA methylation in primordial germ cells (PGCs), affecting virtually all epigenetic-based developmental processes, with only approximately 3.5% of CpG sites remaining methylated (Kobayashi et al. 2013). This includes genomic imprinting, as methylation is lost at all gDMR/ICR regions, ensuring the resetting of imprinting between generations. A new set of sex-specific methylation imprints is established in developing germ cells as part of a global program that asymmetrically methylates the sperm and oocyte genomes. In both humans and mice, the sperm genome is more methylated (80% in mice, 75% in humans) than the oocyte genome (40% in mice, 54% in humans) (Kobayashi et al. 2012; Okae et al. 2014).
* Oocyte-specific transcription and a distinct mechanism for recruiting DNMTs shape a widespread dimorphic methylation landscape between sperm and oocyte

The establishment of DNA methylation in the female mouse germline occurs postnatally in growing oocytes arrested at meiotic prophase. This process depends on the *de novo* methyltransferase DNMT3A and its non-catalytic cofactor DNMT3L (Bourc'his et al. 2001; Hata et al. 2002; Kaneda et al. 2004). Genome-wide transcriptome and methylome analyses in oocytes revealed that DNA methylation is primarily restricted to transcribed regions (Kobayashi et al. 2012). These transcription events reshape

<figure><img src="image_2.png" /><figcaption>**Figure 1:** Schematic representation of the genomic organization of the 10 largest imprinted loci. This illustration is not to scale and does not include non-imprinted genes of the locus. In addition, tissue-specific expression patterns and expression biases are not depicted. At the *Snrpn* domain, *Sngh14* initiates from both the ICR and regions upstream of the ICR. In addition, the existence of *Ube3a-ATS* as an independently processed transcript distinct from *Sngh14* has not been formally demonstrated.</figcaption></figure>
the histone methylation landscape, guiding the DNMT complex to specific genomic regions. Specifically, the transcription-coupled loss of H3K4me, an epigenetic mark that antagonizes DNA methylation (Ooi et al. 2007), at intragenic CpG islands, mediated by KDM1B, along with the deposition of H3K36me2 and H3K36me3 by SETDB2 in transcribed regions, creates a chromatin environment that facilitates DNA methylation (Ciccone et al. 2009; Veselovska et al. 2015; Stewart et al. 2015; Xu et al. 2019; Shirane et al. 2020). Accordingly, most, if not all, of the 17 ubiquitous maternal gDMRs/ICRs are covered by transcripts originating from upstream promoters in oocytes (Chotalia et al. 2009; Veselovska et al. 2015). These transcripts are essential for their *de novo* DNA methylation, as functionally demonstrated at *Gnasxl/Nespas*, *Snrpn*, *Plagl1*, *Kcnq1ot1*, and *Peg3*, among others (Chotalia et al. 2009; Smith et al. 2011; Veselovska et al. 2015; Singh et al. 2017; Bretz and Kim 2017). Beyond gDMRs/ICRs, this transcription-based mechanism also drives the methylation of several hundred intragenic CpG islands in oocytes (Figure 2). Overall, this mechanism is conserved in the human female germline, in terms of both the timing of methylation acquisition, which occurs in growing oocytes, and the fact that methylation is mainly deposited in actively transcribed regions (Okae et al. 2014; Anvar et al. 2021; Yan et al. 2021). However, the DNMTs involved may differ. Notably, DNMT3L is not expressed in human oocytes and is thought to be replaced by DNMT3B or an oocyte-specific isoform of DNMT3B as the partner of DNMT3A.

Interestingly, in both mice and humans, a fraction of the transcripts that shape the oocyte methylome originates from species-specific long terminal repeats (LTRs) of endogenous retroviruses (ERVs) (Veselovska et al. 2015; Brind'Amour et al. 2018). This phenomenon contributes to significant differences in the oocyte methylation pattern between the two species and is thus a driving force in species-specific imprinting. In particular, for two of the three ubiquitous mouse gDMR/ICRs that are not conserved in humans (*Impact* and *Slc38a4*; Table 1) the methylation is driven by transcription from a rodent-specific LTR. Accordingly, deletion of these LTRs prevents the acquisition of methylation at these regions in oocytes, resulting in the loss of imprinting for these two loci in the progeny (Bogutz et al. 2019). Similarly, oocyte transcripts covering two mouse and twelve human non-conserved transient gDMRs originate from rodent- and primate-specific LTRs, respectively (Bogutz et al. 2019).

Furthermore, the subcortical maternal complex (SCMC), a multiprotein complex localized in the oocyte cytoplasm, plays a crucial role in regulating DNA methylation both at the global genome level and at gDMR/ICRs (Monk et al. 2017). Notably, mutations in several SCMC proteins in humans, including NLRP7, KHDC3L, and PADI6, have been independently associated with the familial form of hydatidiform moles, an abnormal pregnancy characterized by excessive trophoblast proliferation and the absence of embryonic development, marked by a widespread loss of methylation at maternal gDMRs (Judson et al. 2002; El-Maarri et al. 2003; Kou et al. 2008; Soellner et al. 2017; Qian et al. 2018). A study on a patient carrying a *KHDC3L* mutation suggested that these defects are, at least in part, linked to impaired methylation establishment in oocytes (Demond et al. 2019). The underlying mechanism by which this cytoplasmic complex regulates oocyte methylation remains to be determined. However, a recent finding that the SCMC protein NLRP5 is required to stabilize the DNA methylation regulator UHRF1 in the oocyte cytoplasm provides an avenue for further investigation (Unoki et al. 2024).

Unlike the bimodal methylation pattern observed in oocytes, sperm exhibits widespread DNA methylation across most of the genome. This methylation process begins before birth in mitotically arrested pro-spermatogonia (~E14.5) and is completed in perinatal pro-spermatogonia, where it is maintained throughout meiotic and haploid stages (Davis et al. 2000; Kato et al. 2007; Ichiyanagi et al. 2011; Henckel et al. 2012). As in oocytes, this methylation pattern is established by DNMT3A and DNMT3L (Bourc'his and Bestor 2004; Kaneda et al. 2004), with an additional contribution from DNMT3C in rodents (Barau et al. 2016). However, a key difference lies in the guiding histone mark: while H3K36me3 serves this function in oocytes, sperm relies on H3K36me2, catalyzed by NSD1, to
direct the recruitment of the DNMT complex (Shirane et al. 2020). In pro-spermatogonia, NSD1 deposits H3K36me2 both within transcribed gene bodies (redundantly with SETD2) and in intergenic regions, resulting in broad H3K36me2 distribution to facilitate DNA methylation. The methylation of the three paternal gDMR/ICRs depends on NSD1, while complete methylation of the *Rasgrf1-DMR* additionally requires DNMT3C, guided by a PIWI-interacting RNA (piRNA) pathway (Watanabe et al. 2011; Barau et al. 2016; Shirane et al. 2020). Meanwhile, active promoters enriched in H3K4me3 in pro-spermatogonia, including maternal gDMR/ICRs, are devoid of NSD1 and thus protected from DNA methylation gain (Henckel et al. 2012; Shirane et al. 2020).

Thus, there is no germline-specific DNA methylation mechanism that exclusively targets gDMRs. Instead, oocyte-specific transcription and a distinct H3K36me-based mechanism for recruiting DNMTs contribute to the widespread asymmetrical methylation patterns observed between sperm and oocyte genomes (Figure 2).

<figure><img src="image_3.png" /><figcaption>Figure 2: ICRs stand out by preserving the DNA methylation patterns inherited from germ cells</figcaption></figure>

Oocyte-specific transcription, together with a distinct H3K36me-dependent mechanism for sex-specific DNMT recruitment and H3K4me3-mediated protection of active promoter regions in pro-spermatogonia, collectively contribute to the establishment of over a thousand germline differentially methylated regions (gDMRs) in the zygote. While most of these gDMRs are erased during the global DNA methylation reprogramming that occurs in preimplantation development (right), ICRs stand out by being protected through the recruitment of ZFP57/ZNF445 and the KRAB-ZNF co-repressor complex (left). This protection leads to the maintenance of 20 ubiquitous gDMRs/ICRs throughout mouse development. In addition to these, so-called transient gDMRs can also resist preimplantation reprogramming but lose their allele-specific methylation either upon implantation or, more rarely, in a tissue-specific manner.

* Maintenance of gamete-derived methylation at the peri-implantation stage specifies gDMRs/ICRs.

As a result of the asymmetrical methylation in parental gametes, more than 1,600 CpG islands, in addition to gDMRs/ICRs, are differentially methylated between oocytes and sperm (Kobayashi et al. 2012). However, while most of these differences are lost during the pre-implantation reprogramming step, gDMRs/ICRs stand out by being protected (Figure 2) (Smallwood et al. 2011; Kobayashi et al. 2012).
Following fertilization, a wave of DNA demethylation erases the gamete-specific DNA methylation pattern in the pre-implantation embryo, allowing the formation of totipotent cells. The paternal genome undergoes rapid demethylation before the first DNA replication, due, at least in part to the action of the TET3 methylcytosine dioxygenase (Shen et al. 2014). In contrast, the maternal genome undergoes passive demethylation during successive rounds of DNA replication leading up to the blastocyst stage. This occurs because most DNMT1 and the methylation maintenance factor UHRF1 are retained in the cytoplasm during pre-implantation (Maenohara et al. 2017). PGC7/Stella and the cytoplasmic SCMC complex, both maternal factors inherited from the oocyte, play key roles in this process. Stella appears to have a dual function: early studies suggested that it protects the maternal genome from TET-mediated demethylation (Nakamura et al. 2007; Wossidlo et al. 2011), while more recent studies indicate that it prevents methylation (Funaki et al. 2014; Du et al. 2019), specifically by excluding DNMT1 and UHRF1 from the nucleus, as observed in oocytes (Funaki et al. 2014; Li et al. 2018; Du et al. 2019). The retention of this complex in the cytoplasm of early embryos appears to be ensured by the SCMC, as mutations in *Padi6* and *Nlrp2*, both members of the murine SCMC, are associated with abnormal nuclear localization of the maintenance methylation machinery in pre-implantation embryos. Moreover, *Padi6* mutations lead to a failure of post-zygotic demethylation (Mahadevan et al. 2017; Giaccari et al. 2024).

Although its localization is primarily cytoplasmic, a small fraction of the methylation machinery targets gDMRs/ICRs in the nucleus, ensuring the maintenance of gamete-inherited methylation. Kruppel-associated box (KRAB)-containing zinc finger proteins (ZFPs), including ZFP57, play a crucial role in this process (Figure 2). ZFP57 recognizes a hexanucleotide sequence present in multiple copies within most ICRs and binds to this sequence only in its methylated form. ZFP57 then serves as a platform for assembling the KRAB repressor complex, which includes TRIM28 and SETDB1, a histone methyltransferase that deposits H3K9me3. These factors, in turn, facilitate the recruitment of the DNMT1/UHRF1 complex (Sharif et al. 2007; Quenneville et al. 2011; Strogantsev et al. 2015). A second KRAB-ZFP, ZNF445, is also involved in this process. In mice, it largely acts redundantly with ZFP57 to maintain methylation at gDMRs/ICRs, as double inactivation of *Zfp57* and *Znf445* leads to more severe methylation defects at ICRs than *Zfp57* inactivation alone (Takahashi et al. 2019). However, it is noteworthy that all ICRs but one, *Peg10*, are affected by this double inactivation, suggesting the existence of additional ZFPs involved in this process (Takahashi et al. 2019). In humans, the situation is different. Unlike in mice, ZFP57 is not expressed in oocytes and is only detected after embryonic genome activation. This suggests that, during the early stages of human development, ZNF445, who is highly expressed in human oocyte, is the major factor responsible for maintaining methylation at gDMRs/ICRs (Takahashi et al. 2019; Monteagudo-Sánchez et al. 2020). The involvement of these factors in imprinting-related methylation control is also evident in patients with imprinting disorders. In humans, mutations in *ZFP57* and *ZNF445* have been identified in a subgroup of patients presenting with methylation defects, primarily losses, at multiple imprinted loci, a condition known as multi-locus imprinting disturbance (MLID) (Touati et al. 2019; Eggermann et al. 2022).

The involvement of KRAB-ZFP proteins in the critical maintenance step that defines gDMRs/ICRs aligns with the hypothesis proposed by D. Barlow in 1993 (Barlow 1993), suggesting that genomic imprinting may have evolved as a host defence mechanism to silence foreign elements (Ondičová et al. 2020; Kaneko-Ishino and Ishino 2022). KRAB-ZFPs play a central role in repressing sequences derived from transposable elements, particularly endogenous retroviruses (ERVs). Notably, ZFP57 binds not only to imprinted gDMRs but also to numerous ERVs across the genome (Shi et al. 2019). This suggests that similar pathways may be involved in directing methylation at both ICRs and ERVs, though not in the germline, as initially proposed, but rather during the pre-implantation stage.

After implantation, the embryo undergoes a wave of remethylation primarily mediated by the *de novo* methyltransferase DNMT3B, and to a lesser extent DNMT3A. Subsequently, the maintenance DNA
methyltransferase DNMT1 ensures the faithful preservation of DNA methylation during each cell cycle, including at gDMRs/ICRs (Li et al. 1993). Conversely, the mechanisms that allow the opposite parental allele to resist the post-implantation methylation wave and remain unmethylated throughout life are less well documented. However, they likely follow similar principles to those governing CpG islands in general. Specifically, the presence of H3K4 methylation (H3K4me), which is anti-correlated with DNA methylation in mammalian genomes and marks CpG islands (Mikkelsen et al. 2007; Mohn et al. 2008), may play a crucial role. Indeed, the DNMT3 family interacts with histone H3 only when lysine 4 is unmethylated, suggesting that genomic regions enriched for H3K4me cannot recruit the *de novo* DNA methylation machinery (Ooi et al. 2007; Zhang et al. 2010; Dhayalan et al. 2010). This is consistent with the observation that H3K4me2/3 constitutively marks the unmethylated allele of ICRs, either alone at transcriptionally active ICRs or together with H3K27me3 at transcriptionally inactive ones (Sanz et al. 2008; Henckel et al. 2009; Maupetit-Méhouas et al. 2016).

* **When the maintenance of gamete-derived methylation asymmetry is temporary: the transient gDMRs.**

Along with ubiquitous gDMRs/ICRs, other regions that exhibit asymmetrical methylation in parental gametes can resist preimplantation reprogramming but lose their allele-specific methylation upon implantation or, more rarely, in a tissue-specific manner. These regions ultimately become either fully methylated or fully unmethylated, giving rise to the so-called transient gDMRs (Figure 2). More than twenty transient gDMRs have been characterized in mice (Table 2) (Proudhon et al. 2012; Duffié et al. 2014; Strogantsev et al. 2015; Yang et al. 2022). However, some of these were identified in mouse pre-implantation embryos with two maternal (gynogenetic) or two paternal (androgenetic) genomes (Yang et al. 2022), and may therefore require formal validation in biparental conceptuses. The majority of these gDMRs are maternally methylated and lose their gDMR status upon implantation. Notably, four transient gDMRs stand out by retaining their gDMR status in one or more tissues. For instance, *Cdh15*, which encodes the cell adhesion protein M-Cadherin, harbors a gDMR within its gene body, near the 3' end, that is maintained in mouse adult quadriceps, tail, and cerebellum but not in other analyzed tissues (Proudhon et al. 2012). Most transient gDMRs, though not all, are associated with the allele-specific expression of a nearby but isolated gene, suggesting a lack of cluster regulation, unlike ubiquitous gDMRs/ICRs. Additionally, six transient gDMRs are linked not to gene but to transposable elements (Yang et al. 2022). The precise functional impact of these transient allele-specific expression events remains to be determined. However, several observations suggest their importance for embryonic development (Yang et al. 2022). Functional studies have, in particular, revealed the critical role of a transient gDMR located in the promoter of *BC051142*, which, in embryonic development, regulates the paternal-specific expression of the distal *Bromodomain-containing protein 2 (Brd2)* gene (Yang et al. 2022). Strikingly, transient allele-specific expression can also have long-term effects, as illustrated by the *Zinc finger, DBF-type containing 2 (Zdbf2)* locus, whose regulatory mechanism is conserved between humans and mice. A transient gDMR modifies the epigenetic landscape upstream of the *Zdbf2* promoter, which is crucial for establishing paternal *Zdbf2* expression and its function throughout life (Duffié et al. 2014; Greenberg et al. 2017). Interestingly, embryos with disrupted regulation at this locus develop normally but exhibit postnatal growth reduction and fail to activate *Zdbf2* in the postnatal brain. Thus, the effects of pre-implantation paternal *Liz* expression manifest later in development (Greenberg et al. 2017).

In humans, tissue-specific maintenance of gDMRs is particularly prominent in the placenta, where several hundred of loci exhibit a maternal (oocyte-derived) DNA methylation bias (Court et al. 2014; Hanna et al. 2016; Sanchez-Delgado et al. 2016; Hamada et al. 2016; Daskeviciute et al. 2025b). This
phenomenon, which is not observed in mice, probably reflects the fact that pre-implantation
demethylation is less extensive in the human genome than in the mouse genome. This difference may
indeed contribute to an increased number of imprinted DMRs in humans (Okae et al. 2012). Notably,
not all of these loci regulate imprinted gene expression. Some are even located in the promoter regions
of biallelically expressed genes, raising questions about whether maternal methylation at these loci is
sufficient to recruit the repression machinery (Sanchez-Delgado et al. 2016; Hamada et al. 2016).
Establishing the function and significance of this phenomenon in pregnancy and development is further
complicated by its polymorphic nature among individuals.

ICR: a multifaceted cis-regulatory region

Once established and maintained, parental allele-specific DNA methylation at gDMRs/ICRs functions as a primary signal that governs imprinted gene expression across entire domains. This allelic signature, although constitutive, orchestrates the complex spatiotemporal expression patterns of imprinted genes by modulating a combination of regulatory mechanisms, some of which are tissue-specific. In embryonic stem (ES) cells, allelic expression is initially confined to genes in close proximity to the ICR, before extending to more distal genes following implantation. The most distal ICR-mediated effects are observed in extra-embryonic tissues, where imprinting domains tend to be more expansive (Andergassen et al. 2017). Nonetheless, the influence of the ICR on allelic gene expression decreases with genomic distance: genes positioned at the periphery of the clusters more frequently exhibit biased expression toward one of the two parental alleles, rather than strictly monoallelic expression (Perez et al. 2015).

Histone modifications, cis-spatial organization, long non-coding RNAs (lncRNAs), and tissue-specific regulatory regions have all been shown to contribute, alongside DNA methylation, to the long-range, ICR-mediated, tissue-specific regulation of imprinted domains. In some domains, these regulatory mechanisms lead to the formation of somatic DMRs that relay the action of the ICR.

Interactions between cis-regulatory regions and their target genes are facilitated by topologically associated domains (TADs), which are sub-chromosomal structural units (Dixon et al. 2012). Consistent with this organization, most imprinted domains are smaller than 1 Mb (Table 1), the average size of a TAD (Dixon et al. 2012), and are typically confined within a single TAD, with some even forming 'imprinted' sub-TADs (Llères et al. 2019; Cercy 2019). Nevertheless, the expansion of certain domains, up to 10 Mb for *Igf2r* in extra-embryonic tissues, suggests that part of the ICR-mediated regulation may occur across TAD boundaries.

To fulfil their role as central regulators of imprinted domains, ICRs integrate multiple cis-regulatory activities. Specifically, their function is mediated through three main types of regulatory activities: promoters, insulators, and enhancers, with some ICRs capable of combining several of these functions.

* ICR are promoters

Of the twenty ubiquitous gDMR/ICRs, the 17 with maternal methylation all overlap CpG island promoters that drive paternal expression of directly adjacent genes (Table 1). Three of these regions, located at the *Gnas*, *Peg3*, and *Peg10* domains, are bidirectional, while two others, at the *Snrpn* and *Plagl1* domains, regulate the expression of both a protein-coding gene (*Snrpn-Snurf* (*a bicistronic transcript*) and *Plagl1*, respectively) and a long non-coding RNA (*Snhg14* and *Hymai*, respectively). Most of these promoters display tissue-specific activity that appears to rely on the dynamics of the repressive H3K27me3 mark on the paternal allele. This allele, which is constitutively enriched in the permissive H3K4me3 mark, acquires H3K27me3 by default when ICRs are transcriptionally inactive, forming a bivalent chromatin signature. This allele-specific bivalency is thought to play a protective role by maintaining the paternal DNA in an unmethylated state while enabling tissue-specific promoter activity (Maupetit-Méhouas et al. 2016).

In addition to controlling the allelic expression of directly adjacent genes, the promoter function of ICRs
also induces distal *cis*-regulation through transcriptional interference and/or the production of lncRNAs.
o Transcription from the ICR interfere with the transcription of an overlapping gene

At several imprinted loci, transcription originating from the ICR overlaps with a neighbouring gene and modulates its expression. The impact of this overlap varies depending on factors such as the length of the overlap, the genomic regions involved and the orientation of the genes (Figure 3).

<figure><img src="image_4.png" /><figcaption>b) Promoter occlusion</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>c) Alternative polyadenylation</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>d) Alternative promoter usage</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 3 : Mechanism by which transcription initiated from the ICR interferes with the transcription of an overlapping gene.**</figcaption></figure>

a) **Transcriptional collision:** Transcriptional elongation from the ICR (gene B) is thought to interfere with the transcriptional machinery of the overlapping gene (gene A), potentially destabilizing its expression through collisions between converging RNA polymerases. b) **Promoter occlusion:** Transcription initiated from the ICR (gene B) overlaps with the promoter region of the associated gene (gene A). This readthrough transcription is thought to interfere with RNA polymerase II recruitment, thereby blocking transcriptional initiation of the overlapping gene. c) **Alternative polyadenylation:** Expression of an ICR-driven transcript (gene B) fully embedded within a host gene (gene A) is associated with premature polyadenylation of the host transcript. This likely results from transcriptional interference during elongation, promoting the use of upstream polyadenylation sites and leading to truncated isoforms. d) **Alternative promoter usage:** Intragenic ICRs can function as alternative promoters, driving the expression of shorter isoforms of the host gene (gene A).

In some cases, the overlap is restricted to the 3' end of the neighbouring gene in a convergent orientation (Figure 3a). This is observed at the *Mest* and *Snrpn* loci, where specific isoforms of the transcript initiated from the ICR (*MestXL* for *Mest* and *Snhg14* for *Snrpn*), overlaps the *Copg2* and *Ube3A* genes, respectively (Rougeulle et al. 1998; Chamberlain and Brannan 2001; MacIsaac et al. 2012). The paternal expression of these isoforms, in neural tissues, is associated with the repression of the overlapping gene, and results in its mono-allelic or preferential expression from the maternal allele (MacIsaac et al. 2012; Meng et al. 2012). Although the precise underlying mechanisms remain unclear, it is proposed that transcriptional interference during transcription elongation destabilises the expression of the overlapping gene. This hypothesis is supported by observations at *Ube3A*, where both the maternal and paternal alleles are bound by the transcription pre-initiation complex and produce nascent RNA, yet only the maternal allele yields mature transcripts (Meng et al. 2013).

At other loci, namely *Gnas* and *Igf2r*, the ICR resides within the gene body of *Nesp* and *Igf2r*, respectively (Figure 3b). In these cases, transcription from the ICR results in the production of an antisense lncRNA (*Nespas* for *Gnas* and *Ainr* for *Igf2r*) that overlaps the promoter of the associated gene (Hayward and Bonthron 2000; Lyle et al. 2000; Williamson et al. 2002). Paternal expression of these lncRNAs silences the associated gene on that allele, leading to maternal-only expression (Sleutels et al. 2002; Williamson et al. 2011). While the exact mechanisms are still under investigation, at least for *Ainr*, transcriptional repression appears to depend on readthrough transcription across the promoter, which interferes with RNA polymerase II recruitment and blocks transcriptional initiation (Latos et al.
2012). Furthermore, transcription trough the promoter also induces *de novo* DNA methylation on the paternal allele of the *Igf2r* and *Nesp* promoter and the establishment of a DMR. While this somatic DMR is not strictly required for the mono-allelic expression, it likely serves to reinforce the silencing (Williamson et al. 2011; Latos et al. 2012). A similar promoter-overlapping mechanism is proposed at the *Commd1* locus, where ICR-driven *Zrsr2-ps1* transcript overlaps the *Commd1* promoter on the paternal allele possibly interfering with transcriptional initiation (Wang et al. 2004; Joh et al. 2009). Notably, this overlap is brain-specific and shorter isoforms of *Zrsr2-ps1* that do not overlap the *Commd1* promoter seems to be expressed in other tissues (Joh et al. 2009). Additionally, unlike at *Gnas* and *Igf2r*, this event is not accompanied by the deposition of allelic DNA methylation at the *Commd1* promoter (Joh et al. 2009).

The most extreme cases of transcriptional overlap involve nested genes, where the transcript initiated at the ICR is fully contained within another gene (Figure 3c and d). This arrangement is found at five loci: *Mcts2*, *Nnat*, *Nap1L5*, *Kcnq1*, *Peg13*. In most of the cases (*Kcnq1* being an exception), paternal expression of the nested gene correlates with a loss of full-length isoforms and the generation of alternative isoforms of the genes hosting the ICR, typically *via* alternative polyadenylation or usage of alternative promoter. For example, *Mcts2* and *Nap1L5* nested genes are fully contained into an intron of their host gene, *H13* and *Herc3*, respectively. Paternal expression of these nested genes from the ICR correlates with early polyadenylation of the host transcripts upstream of the ICR, resulting in truncated host gene isoforms (Wood et al. 2008; Cowley et al. 2012). This is thought to result from elongation interference on the host caused by the expression of the nested transcript, promoting the use of upstream polyadenylation sites. A similar, though less well-characterised event may occur at the *Peg13* locus, where alternative polyadenylation of *Trappc9* on the paternal allele could be driven by *Peg13* expression from the ICR (Gregg et al. 2010). At *Nap1L5*, on the paternal allele, in addition to *Herc3* alternative polyadenylation, isoforms of *Herc3* gene initiating directly from the ICR have also been reported when *Nap1L5* is expressed (Cowley et al. 2012). A similar mechanism operates at *Nnat*: in the brain, expression of the nested gene *Nnat*, which resides in an intron of *Blcap*, is linked with the loss of full-length isoform of *Blcap* and the expression of a shorter isoform using the ICR as an alternative promoter (Schulz et al. 2009). At *Commd1*, brain-specific expression of *Zrsr2-ps1* (which is not completely nested, see previous paragraph) results also in the paternal expression of ICR-initiated *Commd1* isoforms (Schulz et al. 2009). The regulation of this alternative promoter usage remains unclear. One hypothesis proposes that while both the main and alternative promoters are active, transcripts from the main promoter are destabilised by nested gene expression, such that only transcripts initiated from the internal promoter (the ICR) are maintained. Supporting this, at the *Nnat* locus, on the paternal allele, as observed previously at the *Nap1L5* locus (Cowley et al. 2012), there are also signs of *Blcap* transcripts initiated at the main promoter and polyadenylated upstream of *Nnat* (Schulz et al. 2009). Finally, it is interesting to note that *Kcnq1* seems to deviates from this pattern. Despite the presence of the nested *Kcnq1ot1* transcript inside the *Kcnq1* gene, there is currently no evidence supporting isoform switching through alternative promoter usage or alternative polyadenylation. Instead, the lncRNA *Kcnq1ot1* contributes to the paternal repression of the *Kcnq1* gene by recruiting repressive chromatin-modifying complexes, similarly to what is observed for other genes in this domain (see paragraph below).

*o The ICR produces Lncrna mediating long-range repression*

In addition to silencing nearby genes through transcriptional interference, lncRNAs transcribed from ICRs can also recruit repressive chromatin-modifying complexes to induce *cis*-acting repression of distal, non-overlapping genes within the same domain. Among the six loci where the ICR functions as a promoter for a lncRNA, this mechanism has been described at two: *Igf2r* and *Kcnq1*, where the strictly nuclear lncRNAs, *Airn* and *Kcnq1ot1*, silence genes on the paternal allele over distances of approximately 10 Mb and 800 Kb, respectively, in extra-embryonic tissues (Andergassen et al. 2017). In addition to these two ICR-derived lncRNAs, the maternally expressed lncRNA *Meg3*, transcribed at the *Dlk1* locus and activated by the associated ICR (see section “ICR are enhancer”), also contributes to the repression of multiple distal protein-coding genes on the maternal chromosome (Sanli et al. 2018).

To mediate their long-range repressive effects in extra-embryonic tissues and silence distal genes within their respective domains, *Airn* and *Kcnq1ot1* recruit repressive histone-modifying complexes.
Descriptive and functional studies have highlighted the critical roles of Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2), which catalyze the deposition of H2AK119ub1 and H3K27me3, respectively, as well as the methyltransferase EHMT2, which deposits H3K9me2, in this silencing mechanism (Mager et al. 2003; Terranova et al. 2008; Nagano et al. 2008; Schertzer et al. 2019; Braceros et al. 2023). Accordingly, expression of a functional *Airn* or *Kcnq1ot1* transcript in trophoblast stem cells is required to induce the deposition of H2AK119ub1 and H3K27me3 across their respective domains, thereby repressing target gene across multiple TADs (Schertzer et al. 2019). However, this deposition is not uniform along the domain, suggesting that genomic features contribute to the mechanism by which these lncRNAs silence their target genes. The observation that *Airn* expression is accompanied by changes in 3D chromatin contacts further supports a role for chromatin conformation in the silencing process (Schertzer et al. 2019; Braceros et al. 2023). In the emerging model, based on studies at *Airn* in trophoblast stem cells (but may also apply to *Kcnq1ot1*), CpG islands located throughout the domain play a critical role (Figure 4). PRC1 and PRC2 are initially recruited on both alleles of these CpG islands that are in spatial proximity to the *Airn* locus independently of lncRNA expression. Upon *Airn* induction, and due to this pre-established proximity, these CpG islands act as nucleation centers from which H3K27me3 spreads. The accumulation of this mark reinforces contact with the *Airn* locus, reshaping 3D chromatin architecture and in turn facilitating the targeting of distal regions by *Airn* (Schertzer et al. 2019; Braceros et al. 2023). This model helps explain how *Airn*, a short-lived RNA that does not diffuse far from its transcription site (Seidl et al. 2006; Schertzer et al. 2019), can exert long-range silencing effects across multiple TADs.

<figure><img src="image_8.png" /><figcaption>**Figure 4: ICR-produced lncRNA mediates long-range repression**</figcaption></figure>

lncRNAs transcribed from ICRs recruit repressive chromatin-modifying complexes, such as PRC1 and PRC2, to mediate cis-repression of distal, non-overlapping genes within the same domain through deposition of H3K27me3 and H2AK119ub1 (pat allele). In the emerging model, PRC1 and PRC2 are initially recruited to CpG islands on both alleles independently of lncRNA expression. Upon lncRNA induction (pat allele), these CpG islands act as nucleation sites from which H3K27me3 spreads, reinforcing contacts with the lncRNA locus (gene B), reshaping 3D chromatin architecture and enabling the lncRNA to silence distal targets (genes A and D) without affecting all neighbouring genes (gene C). This model provides a mechanistic explanation for how short-lived, spatially restricted lncRNAs can exert long-range silencing effects.

The extent of *Kcnq1ot1*-mediated repression is lineage-specific. While multiple genes across the 800 kb of the *Kcnq1* domain are repressed on the paternal allele in extra-embryonic lineages, only a few genes located in the central portion of the domain, notably *Kcnq1*, *Cdkn1c*, *Slc22a18*, and *Phlda2*, which are considered ubiquitously imprinted genes, are paternally repressed in the embryo proper (Fitzpatrick et al. 2002; Mancini-DiNardo et al. 2006; Andergassen et al. 2017). These genes also require *Kcnq1ot1* transcription, although it acts through a mechanism distinct from that observed in the placenta (Pandey et al. 2008). A functional analysis of *Phlda2* regulation in the epiblast, where various epigenetic pathways were disrupted, supports the idea that this repression relies on the DNA methylation machinery (Andergassen et al. 2021). While the exact mechanism remains to be clarified, available data suggest that it involves a silencing domain located within the first 890 base pairs of the *Kcnq1ot1* transcript, which ensures cis-retention of the RNA at the locus and enables the paternal recruitment of the DNA methyltransferase DNMT1 to the ubiquitously imprinted genes (Mohammad et al. 2010). Furthermore, the observation that expression of a truncated form of *Kcnq1ot1* alters the chromatin conformation of the paternal allele in the mouse heart and brain (Korostowski et al. 2012) suggests that *Kcnq1ot1* may also act by modulating chromatin architecture. Notably, the imprinting patterns of ubiquitously
imprinted genes, and thus the underlying regulatory mechanisms, but not those of placenta-specific imprinted genes, are conserved between mouse and human (Monk et al. 2006).

### *The ICR produces a transcript that activates nearby genes*

By influencing the epigenetic landscape of its environment, the transcript derived from an ICR can also lead to the activation of adjacent genes. Although this atypical mechanism has not been observed from ubiquitous ICRs, it is well documented at the transiently imprinted *Zdbf2* locus (Figure 5).

At this locus, a transient gDMR located within an alternative promoter of *Zdbf2*, approximately 80 kb upstream of the canonical transcription start site, induces the transient expression of *Liz* (long isoform of *Zdbf2*) in early embryos until implantation, as well as in extra-embryonic tissues. While some isoforms of *Liz* may be classified as lncRNAs, the longest and most abundant isoform, spanning 114 kb, overlaps the transcription factor gene *Zdbf2* and displays coding potential (Duffié et al. 2014).

In early embryos, *Zdbf2* is maintained by default in a repressive state through a large block of H3K27me3 covering nearly 30 kb upstream of the promoter, present on both parental alleles (Greenberg et al. 2017). Transcription of *Liz*, specifically from the paternal allele, induces DNA methylation deposition in a portion of this upstream region, leading to the formation of a paternally methylated somatic DMR approximately 15 kb upstream of the canonical promoter. This DMR antagonizes the deposition of H3K27me3. Its maintenance in embryonic tissues protects the paternal allele from repressive H3K27me3 marking and enables paternal-specific expression of *Zdbf2* in embryonic lineages. The mechanism by which *Liz* transcription induces de novo methylation remains to be elucidated. It may involve direct recruitment of DNMTs by the transcript itself or may result from the formation of a chromatin structure permissive to DNMT recruitment, following RNA polymerase II passage (Greenberg et al. 2017). A genome-wide study conducted in embryonic stem cells exiting the naïve pluripotent state supports the idea that this switch from H3K27me3 to DNA methylation may be required for the activation of many genes (Richard Albert et al. 2025).

<figure><img src="image_9.png" /><figcaption>**Figure 5: Transient expression of the ICR-derived *Liz* transcript activates the nearby gene *Zdbf2***</figcaption></figure>

At the *Zdbf2* locus, a transient gDMR drives paternal-specific expression of *Liz* (a long *Zdbf2* isoform) in early embryos and extra-embryonic tissues. In embryos, *Zdbf2* is initially repressed on both alleles by a broad ~30 kb H3K27me3 domain upstream of the promoter. Paternal *Liz* transcription triggers DNA methylation in part of this region, forming a somatic DMR that antagonizes H3K27me3 deposition. Maintenance of this methylation in embryonic tissues protects the paternal allele from repression and permits *Zdbf2* expression specifically in embryonic lineages (adapted from Greenberg et al., 2017).

### *The ICR produces a transcript that hosts other imprinted transcripts*

The transcriptional unit initiated from the ICR region can undergo extensive processing to generate a variety of hosted transcripts. This is exemplified at the *Snrpn* locus. In addition to a bicistronic transcript that encodes the proteins SNRPN and SNURF (Gray et al. 1999), the ICR also serves as a promoter for a long paternally expressed non-coding RNA that acts as a host for numerous small nucleolar RNAs (snoRNAs) and several long non-coding RNAs (lncRNAs) (Runte et al. 2001). This transcript, known as *Snhg14*, is subject to complex processing including alternative splicing, alternative polyadenylation, and exonucleolytic cleavage of introns (Figure 1).

In mouse, *Snhg14* spans approximately 1.2 Mb and includes 172 exons. It hosts two lncRNAs, *Ipw* and *Ube3a-ATS*, as well as multiple box C/D snoRNAs (*Snord107*, *Snord64*, *Snord116*, and *Snord115*). Notably, *Snord116* and *Snord115* are present in high copy numbers (about 71 and 136 copies, respectively), with strong sequence homology. In humans, the *SHNG14* transcript is shorter, panning ~1 Mb and comprising 148 exons, but also hosts *SNORD108*, *SNORD109A*, and *SNORD109B* (Cavaillé 2017; Ariyanfar and Good 2023). The tissue-specific expression of *SNORD* genes differs markedly
between humans and mice (Chamberlain et al. 2010). In humans, the proximal 5' genes (*SNURF*, *SNRPN*, *SNORD107*, *SNORD108*, *SNORD109A*, *SNORD116*, and *IPW*) are broadly expressed across tissues, whereas the more distal 3' genes (*SNORD115*, *SNORD109B*, and *UBE3a-ATS*) are predominantly restricted to the brain. This differential expression pattern may be driven by a cis-regulatory element located at the 3' end of the *IPW* gene, acting as a transcriptional termination or boundary signal in non-neuronal tissues, or alternatively by unidentified neuron-specific promoters (Vitali et al. 2010; Martins-Taylor et al. 2014; Cavaillé 2017). Supporting the latter hypothesis, CAGE-seq analyses in human tissues have identified potential transcription start sites both upstream and downstream of the *SNORD115* repeat array (Galiveti et al. 2014). In mice, *Snurf-Snrpn* is ubiquitously expressed, while *Snords*, *Ipw*, and *Ube3a-ATS* are largely restricted to the brain (Cavaillé 2017).

Similarly, at the *Dlk1-Dio3* locus, although its transcription is not initiated from an ICR, a maternally expressed, single polycistronic transcription unit driven by the *Meg3* promoter also functions as a host gene for multiple non-coding RNAs, including the *Meg3* lncRNA, approximately 50 miRNAs (*Mirg* locus), and 22 copies of box C/D snoRNAs (*Rian* locus) (Figure 1) (Kota et al. 2014; Di Michele et al. 2023).

Unlike most box C/D snoRNAs, which are widely expressed and function post-transcriptionally through antisense base pairing to guide 2'-O-methylation of pre-rRNAs and the RNA polymerase III-transcribed *U6 snRNA*, the snoRNA arrays hosted by the paternally expressed *Snhg14* and the maternally expressed polycistron at *Dlk1-Dio3* locus lack complementarity to rRNAs and are predominantly expressed in the brain (Cavaillé 2017). Although their precise function remains unknown, increasing evidence from patient-derived and mouse models indicates that loss of paternal *SNORD116* expression is a primary cause of Prader-Willi syndrome, underscoring the urgent need to identify and validate their cellular RNA targets.

The protein-coding transcript produced by an ICR can also host other transcripts, as observed at the paternally expressed *Mest* gene, which harbors the microRNA *mir-335* within one of its introns. MicroRNAs are 18-25 nucleotide noncoding RNAs that regulate gene expression by binding to complementary sites in the 3'-untranslated regions (3'-UTRs) of target mRNAs. While several functions and targets of *mir-335* have been identified in various cancer types, where it is often misexpressed (Ye et al. 2021), its role during normal development remains less well understood. *Mir-335* expression depends on the activity of the *Mest* promoter (Kumar et al. 2023), suggesting co-regulation and convergence of *Mest* and *mir-335* in shared pathways. However, this association may not be universal, as skeletal muscle growth and regeneration appear to depend on *Mest* expression but not on *mir-335* (Hiramuki et al. 2015).

## ICR are enhancer

Enhancer activity is observed at the ICR of the *Dlk1-Dio3* locus, one of the three imprinted domains regulated by a paternally methylated ICR. This domain contains three protein-coding genes (*Dlk1*, *Rtl1*, and *Dio3*) expressed from the paternal chromosome, as well as several non-coding RNAs (*Meg3*, *Rtl1as*, *Rian*, and *Mirg*) expressed as a single polycistronic transcript from the maternal chromosome (Table 1, Figure 1). The ICR, known as the IG-DMR (intergenic differentially methylated region), is a 2.6 kb intergenic region located approximately 12 kb upstream of the *Meg3* polycistron's promoter. Several studies have demonstrated that the IG-DMR regulates *Meg3* expression through cis-acting enhancer activity. On the unmethylated maternal allele, several hallmarks of active enhancers are present, including the active histone mark H3K27ac, bidirectional transcription of a short non-coding enhancer RNA (eRNA), and a physical interaction with the *Meg3* promoter, as revealed by 3C-based approaches in embryonic stem (ES) cells (Kota et al. 2014; Das et al. 2015; Luo et al. 2016; Aronson et al. 2021). Functional studies, including targeted deletions of the IG-DMR region and disruption of eRNA production, have shown that the unmethylated maternal IG-DMR is essential for the activation of the *Meg3* polycistron (Lin et al. 2003; Kota et al. 2014). Conversely, methylation of the paternal allele suppresses this enhancer activity, thereby ensuring the allele-specific regulation necessary for proper expression of this imprinted domain (Kota et al. 2014; Das et al. 2015). Notably, allele-specific reporter assays have revealed that the IG-DMR is a bipartite ICR composed of two regions with distinct allele-specific functions. The enhancer activity is carried by the 1 kb region closest to the *Meg3* promoter, whereas the upstream portion of the IG-DMR is involved in the maintenance of paternal methylation (Aronson et al. 2021).

Enhancer activity has not been described at other ICRs beyond the IG-DMR. However, given that the mode of action of most ICRs has only been characterized for a limited number of their target genes (Table 1) this type of activity appears as an attractive possibility that warrants investigation at additional
loci. For instance, at the *Snrpn* locus, the unmethylated ICR allele is required for the activation of several distal genes (*Ndn*, *Magel2*, *Mkrn3*, and *Peg12*) on the paternal chromosome, through a mechanism that remains unknown. A study using 3C approach in human lymphoblastoid cells showed physical interactions between the ICR and these paternally expressed genes, supporting the possibility that the ICR may be a putative enhancer (Rabinovitz et al. 2012). Furthermore, studies in Drosophila have revealed that promoters with bidirectional transcriptional activity can often also function as enhancers (Mikhaylichenko et al. 2018). This raises the possibility that the three ICRs located within bidirectional promoters (*Peg3/Usp29*, *Peg10/Sgce*, and *Nespas/Gnasxl*) (Table 1) may possess enhancer-like functions and should be further explored in this context.

* ICR are insulator

The imprinting control region can regulate the expression of distal genes through an allele-specific
insulator function, mediated by the recruitment of the zinc finger protein CTCF (CCCTC-binding factor)
to its unmethylated allele (Figure 6). This mechanism is well illustrated at the *Igf2/H19* locus, located
on chromosome 7 in mice and 11p15 in humans. Spanning ~100 kb, this locus comprises *Igf2*, encoding
a growth factor, and *H19*, a long non-coding RNA. During embryonic development, both genes are co-
expressed mainly in endodermal and mesodermal tissues. While *Igf2* is expressed paternally, *H19* is
expressed maternally (Sasaki et al. 2000). This allele-specific expression is governed by an ICR located
~2 kb upstream of *H19*, which contains multiple CTCF binding sites. On the methylated paternal allele,
CTCF binding is blocked, whereas on the unmethylated maternal allele, CTCF binds and contributes to
maintaining the unmethylated state (Bell and Felsenfeld 2000; Kanduri et al. 2000; Hark et al. 2000;
Engel et al. 2006). CTCF binding promotes a parent-specific 3D chromatin architecture: the paternal
allele forms a single TAD, allowing enhancers to contact *Igf2*, while the maternal allele forms two sub-
TADs, insulating *Igf2* from enhancers and permitting their interaction with *H19* (Court et al. 2011;
Llères et al. 2019). This results in paternal *Igf2* and maternal *H19* expression. A similar CTCF-dependent
insulator function has been demonstrated at the *Peg13/Kcnk9* locus. Here, maternal CTCF binding to
the ICR within the *Peg13* promoter organizes allele-specific sub-TADs, enabling the enhancer-induced
differential expression of *Kcnk9* and *Peg13* during neural differentiation (Loftus et al. 2023; Rengifo
Rojas et al. 2024). Beyond ICRs, somatic DMRs (sDMRs) can also mediate insulator activity. At the
*Dlk1-Dio3* domain, maternal CTCF binding to a paternally methylated sDMR at the *Meg3* promoter is
essential for establishing 3D structure and regulating *Dlk1* imprinting during neural differentiation
(Sanli et al. 2018; Llères et al. 2019). Likewise, at the *Grb10* locus, a maternally methylated intragenic
sDMR recruits CTCF on its paternal allele, insulating the *Grb10* promoter from a heart-specific
enhancer (Juan et al. 2022). Deletion of this element leads to *Grb10* biallelic expression and loss of *Ddc*
expression from the paternal allele (Juan et al. 2022). Importantly, at these four loci CTCF-mediated 3D
organization precedes the onset of imprinted expression, suggesting that pre-established allele-specific
chromatin topology may prime the locus for later imprinted expression (Llères et al. 2019; Juan et al.
2022; Richer et al. 2023; Loftus et al. 2023; Rengifo Rojas et al. 2024; Moindrot et al. 2024).

Indirect evidence suggests an insulator model at the *Kcnq1* locus. Here, the ICR preferentially recruits CTCF to the paternal unmethylated allele (Fitzpatrick et al. 2007; Lin et al. 2011) and exhibits lineage-specific insulator activity (Kanduri et al. 2002; Fitzpatrick et al. 2007), potentially regulating *Cdkn1c* maternal expression in specific embryonic tissues (Shin et al. 2008). Similarly, analysis of a parent-specific deletion of a putative enhancer within the *Usp29* intron supports the notion that maternal expression of *Zim1* in the mouse neonatal brain may be regulated by the insulator activity of the *Peg3* ICR (Bakshi et al. 2018).

Besides *Igf2/H19*, *Peg13/Kcnk9*, and *Kcnq1*, several other ICRs, including those at *Peg10*, *Mest* and *Plagl1*, are known to recruit CTCF on their unmethylated allele in mice (Prickett et al. 2013). However, whether the insulator model applies broadly to these loci and whether it involves pre-established CTCF-mediated 3D architecture remains to be fully explored. Notably, recent analyses of allele-specific 3D chromatin organization in human cell lines suggest that ICRs may employ diverse mechanisms, beyond CTCF insulation alone, to establish allele-specific chromatin conformation at imprinted loci (Richer et al. 2023).
<figure><img src="image_10.png" /><figcaption>**Figure 6: ICR are insulator**</figcaption></figure>

The ICR can regulate distal gene expression through an allele-specific insulator function mediated by the zinc finger protein CTCF, which binds specifically to the unmethylated allele. In this example, CTCF binding is blocked on the maternally methylated allele, while on the unmethylated paternal allele, CTCF promotes the establishment of a parent-specific 3D chromatin architecture. On the maternal allele, a single TAD (red triangle) enables enhancers to interact with gene A. In contrast, the paternal allele forms two sub-TADs (blue triangles), insulating gene A from enhancers and redirecting their activity toward gene B. This configuration results in maternal expression of gene A and paternal expression of gene B. Notably, in loci analysed to date, CTCF-mediated chromatin architecture is established before imprinted gene expression, suggesting that allele-specific 3D topology primes the locus for later imprinted expression.

## A trans-regulatory role for the ICRs ?

Beyond the cis-acting regulation initiated by imprinting control regions (ICRs) within imprinted domains, an additional level of regulation has been documented, whereby tissue-specific expression of imprinted genes is coordinated across distinct imprinted domains. A pioneering study revealed that a subset of imprinted genes is part of a common transcriptional network during embryonic development (Varrault et al. 2006). This co-regulated network of imprinted genes has since been validated in various organs and cell types and shown to be dynamically modulated during processes such as cell proliferation, differentiation, and aging (Lui et al. 2008; Berg et al. 2011; Al Adhami et al. 2015). Such coordinated expression likely serves to synchronize the activity of imprinted genes located on different chromosomal domains but involved in related biological pathways, including insulin signalling and the transport of ions and nutrients.

The underlying mechanism may involve regulatory factors, either proteins or non-coding RNAs, produced at one imprinted locus that can influence gene expression at other, distant loci.

For example, the imprinted gene *Plagl1*, located on mouse chromosome 10, encodes a transcription factor that regulates several other imprinted genes located in distinct domains such as *Mest*, *Dlk1-Meg3*, *Igf2-H19*, and *Kcnq1*, on separate chromosomes (Varrault et al. 2006). Similarly, in both mice and humans, imprinted long non-coding RNAs (lncRNAs) *H19*, *IPW*, and *MEG3*, as well as the *Mirg* miRNA cluster within the *Dlk1-Dio3* domain, have been shown to act in *trans* to modulate the expression of other imprinted genes (Gabory et al. 2009; Stelzer et al. 2014; Abi Habib et al. 2019; Whipple et al. 2020).

This co-regulation may also be mediated, at least in part, by physical proximity between distinct imprinted domains in the nuclear space. Co-regulated genes and their regulatory elements have indeed been shown to physically associate within the nuclear space. For instance, KLF1 mediates physical interactions between co-regulated genes in erythroid lineages (Schoenfelder et al. 2010). Similarly, genes bound by pluripotency factors (OCT4, SOX2, NANOG) are known to cluster via inter-TAD contacts that are lost upon differentiation (Wei et al. 2013; de Wit et al. 2013; Apostolou and Hochedlinger 2013; Denholtz et al. 2013). Polycomb Repressive Complexes (PRC1 and PRC2) also contribute to the formation of spatial networks of co-repressed genes in embryonic stem cells (Denholtz et al. 2013; Schoenfelder et al. 2015).

Altogether, these findings support the notion that inter-chromosomal contacts between distant genomic regions may provide a structural framework for coordinated gene regulation. However, because such contacts are often stochastic and constrained by the global 3D architecture of the genome, their establishment may require initial interactions between transcription factors and “master” regulatory elements to stabilize multi-gene complexes (Fanucchi et al. 2013; Mueller et al. 2013). On this basis, it is tempting to speculate that the co-expression of imprinted genes could depend on physical interactions between imprinted loci, and more specifically between ICRs, given their central regulatory function. Supporting this hypothesis, a 4C (circular chromosome conformation capture) analysis demonstrated that the *H19* ICR engages in contacts with several other ICRs and imprinted loci located on different chromosomes (Zhao et al. 2006). However, as this remains an isolated study, further high-resolution and functional analyses are required to fully assess the extent to which ICRs can act as *trans*-regulatory hubs across the genome.
## Imprinting without ICR : Non-canonical Imprinting

As depicted above, germline-derived, parental allele-specific DNA methylation at gDMRs/ICRs serves as a necessary and primary signal governing imprinted gene expression across entire domains. However, early studies revealed that a subset of paternally expressed genes appeared to lack association with such germline DMRs and could maintain their imprinting in the placenta even in the absence of oocyte-derived DNA methylation (Chiba et al. 2008; Okae et al. 2012). Years later, the repressive histone mark H3K27me3 was identified as the underlying mechanism of this so-called non-canonical imprinting. Based on integrative analyses of DNA methylomes and H3K27me3 ChIP-seq data from mouse zygotes and pre-implantation embryos, Inoue and colleagues demonstrated that paternal expression of a subset of genes at these stages was independent of maternal DNA methylation and instead relied on maternal H3K27me3 (Inoue et al. 2017, 2018). This repressive mark is deposited by the Polycomb Repressive Complex 2 (PRC2), following PRC1-mediated monoubiquitination of histone H2A at lysine 119 (H2AK119ub1). Consistently, depletion of two essential components of PRC1 variant (Polycomb group ring finger proteins PCGF1 and PCGF6) leads to reduced H2AK119ub1 and subsequent loss of H3K27me3 at these loci, ultimately resulting in the loss of maternal non-canonical imprinting (Mei et al. 2021).

This non-canonical imprinting mediates maternal allele-specific silencing of approximately 30 genes in the post-implantation embryo, including *Jade1*, *Sfmbt2*, *Phf17*, *Gab1*, *Sall1*, *Platr20* (5133400J02Rik), *Smoc1*, and *Xist* (Inoue et al. 2018). At least nine of these genes maintain paternal-specific expression in extraembryonic tissues and the placenta. Nevertheless, maternal H3K27me3 is not maintained after implantation, suggesting that although it may serve as the initial imprinting mark, other mechanisms are responsible for maintaining imprinted expression in the placenta (Hanna et al. 2019). In particular, maternal H3K27me3 appears to be replaced by DNA methylation during placental development. While the precise mechanism remains unclear, studies on *Gab1* suggest a switch from H3K27me3 to DNA methylation at long terminal repeats (LTRs) of ERVK-family retrotransposons near the promoters of non-canonical imprinted genes. These unmethylated LTRs can act as alternative promoters, generating chimeric paternal transcripts with nearby genes (Hanna et al. 2019).

The functional relevance of this imprinting mechanism has been evaluated using embryos derived from oocytes deficient in PRC2 or PRC1 components. These embryos can survive to term but display reduced litter sizes, indicative of embryonic loss, and surviving offspring exhibit placental overgrowth at term (Inoue et al. 2018; Hanna et al. 2019; Inoue 2023). However, since *Xist* is also regulated by maternal H3K27me3, it remains challenging to distinguish the relative contributions of defective X-chromosome inactivation from those of disrupted non-canonical autosomal imprinting. Furthermore, whether this form of imprinting impacts late developmental processes remains to be determined.

Importantly, while this type of imprinting may be conserved in rat, current evidence suggests it is not conserved in humans (Inoue 2023). Specifically, a comprehensive analysis of whole-villi human placental samples failed to detect non-canonical imprinting (Daskeviciute et al. 2025a). Nonetheless, the question remains open to debate (Hanna and Kelsey 2021; Inoue 2023).

## Conclusion

Since the discovery of the first imprinted genes in 1991, significant progress has been made in understanding the mechanisms underlying their monoallelic expression. These advances have notably highlighted the central role of Imprinting Control Regions, which, through their ability to maintain the differential methylation profile inherited from the gametes and to exert various cis-regulatory functions, including promoter, enhancer, and/or insulator activities, influence allele-specific gene expression across large imprinted domains. Beyond their mechanistic interest, deciphering the molecular cascades initiated by the differential methylation of ICRs represents essential knowledge for improving the diagnosis and clinical management of patients with alterations in imprinted genes, particularly in the context of the 13 currently recognized imprinting disorders (Eggermann et al. 2023).

However, our synthesis (Table 1) highlights that understanding remains incomplete for many loci. For instance, little is known about the regulatory mechanisms operating within the large *Peg10* domain, despite the fact that the eponymous gene, an oncogene (Xie et al. 2018), has recently been linked to amyotrophic lateral sclerosis (Black et al. 2023). Similarly, within the *Peg13* domain, the mechanisms
responsible for the maternally biased expression of *Ago2* and *Trappc9* in the brain tissues, both implicated in various neurodevelopmental disorders (Liang et al. 2020; Lessel et al. 2020), remain to be elucidated.

More broadly, the data presented in the table necessarily reflect our knowledge in the tissues that have been studied, mainly the placenta and the brain, which represent the two principal targets of genomic imprinting. The example of *Cdkn1c*, whose paternal repression is mediated by the lncRNA *Kcnq1ot1* in the placenta and certain embryonic tissues (although through distinct mechanisms), while being regulated by the insulator activity of the ICR in other embryonic tissues, illustrates how a single ICR can adopt different cis-regulatory functions depending on tissue context. Furthermore, the observation that several imprinted genes display a parental-origin-specific allelic bias, rather than strictly monoallelic expression, raises important questions regarding their functional status and the precise mode of action of the ICR at the single-cell level.

The development of systematic analyses across all relevant tissues and cell types, combined with molecular studies at the single-cell level, will thus be crucial to further refine our understanding of the mechanisms by which ICRs regulate the genes within their domains. These mechanisms are expected to rely largely on cis-regulatory activities already attributed to ICRs, but deployed in a tissue- and possibly cell-specific manner. However, it is also conceivable that additional, as yet undocumented, functions, such as silencer activities, may be revealed.

Due to the multiple cis-regulatory functions exerted by ICRs, genomic imprinting, in which the two alleles of a given gene adopt opposing transcriptional states within the same nucleus, represents a particularly powerful paradigm for investigating the mechanisms of epigenetic regulation. It provides a unique framework to study how the epigenetic marking of a given region can influence the expression of distal genes. Research on imprinting has notably led to the identification of the insulator model in mammals and has raised key questions about how the presence or absence of DNA methylation at regulatory regions capable of recruiting CTCF influences the expression of neighbouring genes, both during normal development and in cancer cells (Flavahan et al. 2016), which exhibit profound alterations in their DNA methylation landscape. The study of allele-specific insulator regulation within imprinted loci also offers an especially relevant experimental context for exploring fundamental questions related to enhancer biology, such as the mechanisms through which enhancers activate their target genes, and the functional interplay between 3D genome organization and transcriptional activity at these loci.
Moreover, imprinting is emerging as a valuable model to dissect the mechanisms underlying various forms of transcriptional interference, a regulatory mode frequently employed by ICRs. This includes antisense and convergent transcription, whose functional impact remains poorly characterized in mammals, as well as the influence of nested genes on the expression and transcriptional diversity of their host genes, a "host-nested gene" configuration recently shown to be widespread in mammalian genomes (Montibus et al. 2024).

More than 35 years after the discovery of the first imprinted genes, these loci have revealed only a fraction of their secrets and remain an inexhaustible source of insight for deepening our understanding of the epigenetic mechanisms governing transcriptional programs during development.

## Acknowledgments

We thank all members of our laboratory for comments on the manuscript.

Work in P.A.'s laboratory is supported by grants from the Agence Nationale de la Recherche (ANR-23-CE12 CORGI), The Fondation pour la recherche Médicale (project FRM-MND202310017917) and the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25). B.M. is supported by a postdoctoral fellowship (I-SITE Clermont Auvergne Project - European Centre for Health and Human Mobility).

*Author Contribution:* Conceptualization: PA; Writing – original draft: BM, PA. Writing – review & editing: BM, FC, PA. Figures and Tables Conceptualization: FC, BM and PA. Figure original draft: FC. Figures and Table review and editing: BM, FC, PA.

**Competing Interests:** The authors declare no competing interests.
## Figure legends

**Figure 1: Schematic representation of the genomic organization of the 10 largest imprinted loci.**
This illustration is not to scale and does not include non-imprinted genes of the locus. In addition, tissue-specific expression patterns and expression biases are not depicted. At the *Snrpn* domain, *Sngh14* initiates from both the ICR and regions upstream of the ICR. In addition, the existence of *Ube3a-ATS* as an independently processed transcript distinct from *Sngh14* has not been formally demonstrated.

**Figure 2: ICRs stand out by preserving the DNA methylation patterns inherited from germ cells**
Oocyte-specific transcription, together with a distinct H3K36me-dependent mechanism for sex-specific DNMT recruitment and H3K4me3-mediated protection of active promoter regions in pro-spermatogonia, collectively contribute to the establishment of over a thousand germline differentially methylated regions (gDMRs) in the zygote. While most of these gDMRs are erased during the global DNA methylation reprogramming that occurs in preimplantation development (right), ICRs stand out by being protected through the recruitment of ZFP57/ZNF445 and the KRAB-ZNF co-repressor complex (left). This protection leads to the maintenance of 20 ubiquitous gDMRs/ICRs throughout mouse development. In addition to these, so-called transient gDMRs can also resist preimplantation reprogramming but lose their allele-specific methylation either upon implantation or, more rarely, in a tissue-specific manner.

**Figure 3: Mechanism by which transcription initiated from the ICR interferes with the transcription of an overlapping gene.**

a) **Transcriptional collision:** Transcriptional elongation from the ICR (gene B) is thought to interfere with the transcriptional machinery of the overlapping gene (gene A), potentially destabilizing its expression through collisions between converging RNA polymerases. b) **Promoter occlusion:** Transcription initiated from the ICR (gene B) overlaps with the promoter region of the associated gene (gene A). This readthrough transcription is thought to interfere with RNA polymerase II recruitment, thereby blocking transcriptional initiation of the overlapping gene. c) **Alternative polyadenylation:** Expression of an ICR-driven transcript (gene B) fully embedded within a host gene (gene A) is associated with premature polyadenylation of the host transcript. This likely results from transcriptional interference during elongation, promoting the use of upstream polyadenylation sites and leading to truncated isoforms. d) **Alternative promoter usage:** Intragenic ICRs can function as alternative promoters, driving the expression of shorter isoforms of the host gene (gene A).

## Figure 4: ICR-produced lncRNA mediate long-range repression

lncRNAs transcribed from ICRs recruit repressive chromatin-modifying complexes, such as PRC1 and PRC2, to mediate cis-repression of distal, non-overlapping genes within the same domain through deposition of H3K27me3 and H2AK119ub1 (pat allele). In the emerging model, PRC1 and PRC2 are initially recruited to CpG islands on both alleles independently of lncRNA expression. Upon lncRNA induction (pat allele), these CpG islands act as nucleation sites from which H3K27me3 spreads, reinforcing contacts with the lncRNA locus (gene B), reshaping 3D chromatin architecture and enabling the lncRNA to silence distal targets (genes A and D) without affecting all neighbouring genes (gene C). This model provides a mechanistic explanation for how short-lived, spatially restricted lncRNAs can exert long-range silencing effects.

## Figure 5: Transient expression of the ICR-derived Liz transcript activates the nearby gene Zdbf2

At the *Zdbf2* locus, a transient gDMR drives paternal-specific expression of *Liz* (a long *Zdbf2* isoform) in early embryos and extra-embryonic tissues. In embryos, *Zdbf2* is initially repressed on both alleles by a broad ~30 kb H3K27me3 domain upstream of the promoter. Paternal *Liz* transcription triggers DNA methylation in part of this region, forming a somatic DMR that antagonizes H3K27me3 deposition. Maintenance of this methylation in embryonic tissues protects the paternal allele from
repression and permits *Zdbf2* expression specifically in embryonic lineages (adapted from Greenberg *et al.*, 2017).

**Figure 6: ICR are insulator**

The ICR can regulate distal gene expression through an allele-specific insulator function mediated by the zinc finger protein CTCF, which binds specifically to the unmethylated allele. In this example, CTCF binding is blocked on the maternally methylated allele, while on the unmethylated paternal allele, CTCF promotes the establishment of a parent-specific 3D chromatin architecture. On the maternal allele, a single TAD (red triangle) enables enhancers to interact with gene A. In contrast, the paternal allele forms two sub-TADs (blue triangles), insulating gene A from enhancers and redirecting their activity toward gene B. This configuration results in maternal expression of gene A and paternal expression of gene B. Notably, in loci analysed to date, CTCF-mediated chromatin architecture is established before imprinted gene expression, suggesting that allele-specific 3D topology primes the locus for later imprinted expression.

**Table 1:** Summary of the 20 genomic domains regulated by ubiquitous ICRs and 3 loci controlled by transient DMRs in the mouse

**Table 2:** Transient DMRs in mouse

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