Angiogenesis is a crucial process for organ morphogenesis and growth during development, and it is especially relevant during the repair of wounded tissue in adults. It is coordinated by an equilibrium of pro- and anti-angiogenic factors; nevertheless, when affected, it promotes several diseases. Lately, a growing body of evidence is indicating that non-coding RNAs (ncRNAs), such as miRNAs, circRNAs, and lncRNAs, play critical roles in angiogenesis. These ncRNAs can act in cis or trans and alter gene transcription by several mechanisms including epigenetic processes. In the following pages, we will discuss the functions of ncRNAs in the regulation of angiogenesis and neovascularization, both in normal and disease contexts, from an epigenetic perspective. Additionally, we will describe the contribution of Next-Generation Sequencing (NGS) techniques to the discovery and understanding of the role of ncRNAs in angiogenesis.

Every cell of an organism shares the same genome; even so, each cellular lineage owns a different transcriptome and proteome. The Polycomb group proteins (PcG) are essential regulators of gene repression patterning during development and homeostasis. However, it is unknown how the repressive complexes, PRC1 and PRC2, identify their targets and elicit new Polycomb domains during cell differentiation. Classical recruitment models consider the pre-existence of repressive histone marks; still, de novo target binding overcomes the absence of both H3K27me3 and H2AK119ub. The CpG islands (CGIs), non-core proteins, and RNA molecules are involved in Polycomb recruitment. Nonetheless, it is unclear how de novo targets are identified depending on the physiological context and developmental stage and which are the leading players stabilizing Polycomb complexes at domain nucleation sites. Here, we examine the features of de novo sites and the accessory elements bridging its recruitment and discuss the first steps of Polycomb domain formation and transcriptional regulation, comprehended by the experimental reconstruction of the repressive domains through time-resolved genomic analyses in mammals.

Polycomb domains safeguard cell identity by maintaining lineage-specific chromatin states enriched in repressive histone modifications, preserving the epigenetic memory of cell lineages. While Polycomb Repressive Complex 2 (PRC2) can re-establish its occupancy after perturbation, the mechanisms that guide de novo Polycomb recruitment remain unclear. To address this, we engineered an auxin-inducible degradation system to reversibly deplete and reintroduce the endogenous PRC2 core subunit Suz12 in mouse embryonic stem cells (mESCs). Genome-wide profiling at an early recovery time point revealed ∼1,100 PRC2 nucleation sites, characterized by rapid Suz12 and histone H3K27me3 re-accumulation with strong signal, with minimal impact on gene expression. These sites were significantly enriched at bivalent promoters, coinciding with unmethylated CpG islands and chromatin states associated with developmental regulation, and were largely conserved in differentiated cells. Motif analysis identified G/C-rich DNA sequences associated with E2F and zinc-finger proteins, alongside strong co-occupancy with MTF2 and JARID2, two PRC2 cofactors previously implicated in Polycomb targeting. Notably, a subset of nucleation sites overlapped with long-range chromatin interaction anchors in histone H3K27me3 HiChIP datasets. These findings reveal that PRC2 de novo nucleation sites are associated with a combination of chromatin states, DNA sequence features, cofactor co-occupancy and spatial genome organization, suggesting that epigenetic memory can be re-established through defined genomic and chromatin features. Author summary: Polycomb group proteins are key epigenetic regulators that silence gene expression by establishing and dispersing repressive chromatin domains marked by histone modifications such as histone H3K27me3 and H2AK119ub1 and are critical for defining cell identity. During differentiation, Polycomb domains are dynamically redistributed, implying a mechanism for de novo targeting to specific loci. How the Polycomb Repressive Complex 2 (PRC2) is initially recruited to these nucleation sites and which features stabilize its binding remain poorly understood. To explore this, we studied the characteristics of the de novo recruitment sites of PRC2 in mouse embryonic stem cells (mESCs) using an auxin-inducible degradation (AID) system targeting PRC2 to deplete and reintroduce the core subunit Suz12. We identified recruitment sites after complete clearance of the histone H3K27me3 through ChIP-seq at a very early time point after Suz12 reintroduction. Most nucleation sites were located at bivalent promoters of developmental genes and correlated with unmethylated CpG islands. Motif analysis revealed over-represented sequences and accessory partners such as MTF2 and JARID2, along with long-range chromatin interactions. These nucleation sites were also conserved in differentiated cells, highlighting their potential role in developmental regulation. Our study provides insight into how Polycomb domains are established and how epigenetic memory is maintained in stem cells.