A significant portion of a phospholipid molecule is hydrophobic, and this part needs to be shielded from the aqueous cellular milieu either through embedment inside the lipid bilayer, forming an aggregated state, or enclosure within hydrophobic pockets of proteins. Most of the known functions of nuclear phospholipids, especially those involved in signaling events, depend upon the polar headgroups and their interactions with proteins and nucleic acids. While there has been limited exploration of the biology of the hydrophobic tails [13, 14], it is conceivable that, in addition to the widely investigated polar head groups, the acyl parts of nuclear phospholipids could have significant signaling potential (e.g., due to variations involving length/saturation of the acyl chain). While various aspects of nuclear phospholipid biology have been summarized in the recent literature [15,16,17,18,19,20], the functional domains in which phospholipids operate have rarely been addressed. We believe that a comprehensive understanding of the roles of nuclear phospholipids in the wide context of genome biology requires a closer examination of their functional domains within both the nuclear membrane and the nucleoplasm.

Fig. 3

Phosphatidyl inositol (PtdIns) and its derivatives inside the nucleus. PIP kinases and phosphatases control the phosphorylation states of the hydroxyl groups present on positions 3, 4, and 5 of the inositol head group giving rise to mono, di and tri-phosphorylated phosphoinositides. For abbreviations see text

Membrane-bound roles of nuclear phospholipids

Phospholipids are integral to the structural stability of the nuclear envelope, regulate nuclear pore functions, form lipid microdomains, and interact with nuclear membrane proteins or membrane-associated factors. Here, we focus on discussing the biological roles of nuclear phospholipids where a clear association with the nuclear membrane as a dynamic platform for genome regulation is apparent.

Phospholipid asymmetry of the nuclear membrane

The nuclear envelope functions as an adaptable barrier that safeguards the genome. In response to various nuclear processes, the nuclear envelope is remodeled via sealing, growth, reformation and breakdown. These processes require changes in the phospholipid composition, which are achieved in coordination with the endoplasmic reticulum, the cell’s primary production hub for membrane lipids (both cell and organelle membranes). The outer nuclear membrane is physically continuous with the endoplasmic reticulum, allowing free lateral diffusion of phospholipids between the two membrane systems. At sites where the nuclear envelope is perforated by nuclear pore complexes, the outer and inner nuclear membranes are joined. Despite this continuity and lateral lipid diffusion, the phospholipid composition of the outer and inner nuclear membranes remains asymmetric. The asymmetry is attributed to nuclear pore complexes topologically insulating the inner nuclear membrane [21]. The nuclear pore complexes not only sequester phospholipid-metabolizing enzymes (e.g., phospholipid synthetases, phospholipid hydrolases, transferases of fatty acids, phospholipids/lysophospholipids, sphingolipids, etc.) in the inner nuclear membrane [22], but also control the overall lipid traffic via a gating mechanism [21]. Since it faces the DNA, the inner nuclear membrane is enriched in proteins that are required for genome regulation, inheritance, and protection. This is achieved by accumulation of specific phospholipids and their derivatives that interact with a unique set of proteins, including LEM-domain factors (LAP2, Emerin, and MAN1) that bind chromatin-associated proteins and lamins [15, 21, 23]. Interaction of defined lamin attachment regions in the genome with their targets is essential for the establishment of heterochromatin and overall 3D genome organization [24].

While the compositional phospholipid asymmetry of the nuclear envelope in the context of genome regulation has not been fully explored, it is becoming clear that the inner nuclear membrane not only fulfils a structural role, but also a highly functional role. For example, the asymmetry between the inner and outer nuclear membranes is essential for the localization of enzymes that are key to nuclear lipid droplet biogenesis [25] and for inducing the recruitment of enzymes from the nucleoplasm to the membrane during mitosis or stress [26, 27].

Properties of the nuclear membrane and transcriptional sensors

The composition of phospholipids and their derivatives determine the membrane characteristics of the nuclear envelope, including fluidity/viscosity, packing, curvature and stiffness, as well as the stability of nuclear pore complexes [28, 29]. Studies, mostly carried out in budding yeast, have led to the identification of membrane-associated transcriptional regulators that sense the molecular properties of the nuclear envelope. These include the Opi1 repressor (OverProducer of Inositol) and the transcription factors Mga2 (Multicopy suppressor of GAm1 (snf2)) and Spt23 (SuPpressor of Ty), which link membrane stability to genome integrity. Mga2 and Spt23 form dimers that can sense lipid packing, in terms of tightness. High proportions of saturated acyl tails in phospholipids lead to tight lipid packing, which can trigger a conformational change in the membrane bound transcription factor dimers and result in their ubiquitination and subsequent proteolytic cleavage [30]. This process releases transcriptionally active N-terminal fragments of Mga2 or Spt23, which translocate to the nucleus to and initiate the transcription of lipid remodeling factors like Ole1 (Oleoyl-CoA desaturase gene 1), thereby reduce lipid packing by increasing the availability of unsaturated fatty acids [31].

In addition, Opi1 regulates membrane phospholipid biosynthesis as part of the Henry regulatory circuit [32]. When the levels of phosphatidic acid (PA) are high, Opi1 remains tethered to the nuclear envelope and interacts with an integral protein of the endoplasmic reticulum, Scs2 (Suppressor of Choline Sensitivity) [33, 34]. In this state, Opi1 promotes membrane lipid synthesis. However, when PA levels are low, Opi1 detaches from the nuclear envelope and represses genes involved in fatty acid and phospholipid biosynthesis. Moreover, Opi1 also senses membrane packing and curvature, showing a preference for positively curved and loosely packed membranes [31].

Regulation of target factors by phospholipids in the nuclear membrane

Proteins with well-known biology at the plasma membrane have recently been associated with nuclear functions in relation to phospholipids. MARCKS (myristoylated alanine-rich C-kinase substrate) is anchored to membranes by N-terminal myristoylation and has been implicated in cell adhesion, phagocytosis and inflammation. This protein was found to harbor a previously unrecognized nuclear localization signal in its effector domain. It binds PtdIns(4,5)P2 and appears to exert nuclear functions in terms of regulating nuclear PIP2 levels and target gene expression [35]. Similarly, GAP-43 (growth-associated protein 43), which is anchored to membranes by two saturated acyl chains and has been shown to cluster PtdIns(4,5)P2 in plasma membrane rafts during neurite formation, regeneration, and plasticity [36] was found to have a nuclear component in myoblasts and podocytes [37]. Lastly, GTPases of the Ras and Rho families possess a C-terminal polybasic region (protein motif rich in lysines and arginines) may harbor a nuclear localization signal and facilitates interactions with PtdIns(4,5)P2 and PtdIns(3,4,5)P3 on membranes [38, 39]. The above insights reveal that nuclear phospholipids do much more than just maintain the structure of the nuclear envelope. By creating an asymmetric and responsive membrane environment, they guide the activity of key transcriptional sensors and signaling proteins.

Membrane-derived roles of nuclear phospholipids as signal transducers

Several functions of nuclear phospholipids have been identified in analogy to the related canonical roles of phospholipids at the cell membrane and their contributions to well-characterized signaling pathways. In fact, these functions were initially thought to be cytoplasmic, until the nuclear biology of phospholipids was more fully appreciated. While intuitively membrane-derived, many of these biological roles continue to be difficult to assign precisely to the nuclear envelope or nucleoplasm-based functional domains due to the technical challenges of accurately localizing nuclear phospholipids within specific signaling pathways [40]. Also, many of these functions do not purely depend on the phospholipids, but are executed by their derivates, metabolites, or metabolizing enzymes. In the following section, we highlight the role of phospholipids and their metabolizing enzymes in various signalling events and how they shape up the nuclear physiology.

Phosphoinositide-specific phospholipase C (PI-PLC) signaling

The PI-PLC family constitutes an important group of enzymes that catalyze the hydrolysis of PtdIns(4,5)P2 into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in response to stimuli such as growth factors, hormones, and neurotransmitters. The PI-PLC family is comprised of 13 isoforms each bearing distinct functions, regulatory mechanisms, and tissue distributions [41]. Many isoforms of PI-PLC have been implicated in nuclear signaling. For example, activated PI-PLCβ1 facilitates the G1/S transition and G2/M progression during the cell cycle [41]. PI-PLCγ1 functions as a guanine nucleotide exchange factor for specific nuclear GTPases, including phosphoinositide 3-kinase enhancer and dynamin-1 [41]. In addition, the generally cytoplasmic PI-PLCδ translocates to the nucleus between the G1/S transition and towards the end of M phase, suggesting it is potentially involved in regulation of cell growth. Moreover, nuclear expression of PI-PLCδ4 is enhanced by mitogen stimulation, which presumably facilitates epidermal growth factor (EGF)-induced nuclear Ca2+ signaling [42]. Collectively, their dynamic nuclear localization indicates these isoforms of PI-PLC exert essential nuclear functions.

PI3 kinase/Akt/mTOR-mediated nuclear signaling

The nuclear presence and generation of PtdIns(3,4,5)P3 implies that the related lipid kinases function in the nuclear milieu [17, 43], either because they are constitutively present in the nucleus or shuttle between the cytoplasm and nucleus. Class I and II PI3Ks and IPMK/Ipk2 (Inositol Polyphosphate Multikinase) activities have been reported in the nucleus [44]. Indeed, nuclear p110β (catalytic isoform of class I PI3K), but not its cytosolic counterpart, is essential for the survival of mouse embryonic fibroblasts [45]. PI3K signaling, particularly through PtdIns(3,4,5)P3, modulates the activities of various checkpoint proteins to ensure that cells only progress through the cycle when conditions are favorable. For instance, the PI3K-Akt pathway can influence the G1/S checkpoint and help cells cope up with the DNA damage [46]. During the G1 phase, PtdIns(3,4,5)P3 is generated by PI3K, which promotes growth by enhancing gene regulation and protein synthesis to facilitate cell cycle entry and progression [47].

Akt, also known as protein kinase B (PKB), is a serine/threonine kinase that plays key roles in cell growth and metabolism and acts as the canonical downstream signaling effector of the PI3K pathway. Akt exists in three different isoforms in mammalian cells, two of which, Akt1 and Akt2 mainly localize to the cytosol or cell membrane but may shuttle to the nucleus upon growth factor stimulation [48]. The third isoform, Akt3 is primarily confined to the nucleus and the nuclear membrane [49]. The activity of nuclear Akt is tightly regulated by phospholipid messengers, such as PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in conjunction with complex upstream signals [50]. Various substrates of nuclear Akt activity have been identified, including the nucleolar phosphoprotein nucleophosmin (NPM)/B23. The flux of this phosphoprotein from the nucleus to cytoplasm and nucleolus to nucleoplasm is controlled during the S-phase of the cell cycle [51]. Another important effector of Akt is mTOR (Mammalian target of rapamycin), which is a highly conserved serine/threonine kinase and a component of the mTORC1 and mTORC2 complexes. mTORC1 controls the activity of several mRNA translation factors. A subunit of mTORC1, Raptor, associates firmly with PtdIns(3,5)P2 and weakly with PtdIns3P; this represents an additional mechanism of regulation of mTORC1 by phosphoinositides, in addition to its Akt-dependent phosphorylation [52].

Unclassified effects of nuclear phospholipids

Various experimental schemes set up to investigate the biology of nuclear phospholipids involved exogenous administration of different compounds to cells either directly, by transfection, or in form of membrane permeable esters that are intracellularly hydrolyzed [22]. While some nuclear roles of phospholipids have been derived using these approaches, the functional domains of action (membrane vs. nucleoplasm) are not yet clear. Exogenous incorporation of phospholipids, ceramides, and sphingosines has been found to impact replication and transcription in both prokaryotes and eukaryotes [53, 54]. Negatively charged phospholipid species such as phosphatidylinositol, cardiolipin, phosphatidylserine, and phosphatidylglycerol were generally found to stimulate RNA synthesis, while phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin inhibit this process [55, 56]. Studies in cell-free systems have demonstrated that exposure of isolated nuclei to phospholipid vesicles composed of phosphatidylserine or phosphatidylcholine can induce chromatin decondensation [9, 57, 58] and alterations in nucleosome structure, including loss of linker histones [59,60,61]. These lipid molecules can stimulate RNA synthesis and, in some cases, remove histones from chromatin; for example, phosphatidylserine [9, 62]. In another in vitro system, exposure to inositol lipids enhanced DNA polymerase α activity [63]. Thus, the genome generally seems to respond to changes in the abundance of phospholipids in the surrounding milieu; however, further work is required to decipher the exact effects and mechanisms.

Nuclear phospholipid signaling in the nucleoplasm

The functional domains of nuclear phospholipids extent beyond their traditional roles in lipid bilayers. In fact, a significant pool of nuclear phospholipids, in particular nuclear phosphoinositides, appear to be dissociated from the nuclear membrane [64]. In the nucleoplasm, phospholipids have been recognized to function either concealed within phase-separated structures or putatively solubilized in association with nuclear proteins.

Function of phospholipids within nuclear speckles and other phase-separated domains

Nuclear speckles are sub-nuclear membrane-free dynamic structures that house an array of pre-mRNA splicing factors, poly-adenylating polymerases, and enzymes that carry out post-transcriptional modifications [65]. These important functional structures are enriched in phosphoinositides, suggesting that phospholipids impact mRNA processing [43]. Phospholipids conceal their hydrophobic tails in these structures in order to stay functionally accessible in the nucleoplasm. Up to 40% of nuclear phosphoinositides are housed in nuclear speckles in the interchromatin regions of mammalian cell nuclei [66]. Specifically, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 have emerged as key players in mRNA export and within the pre-mRNA splicing machinery [67]. Aly/REF is a component of the TREX (TRanscription-EXport) complex that associates with spliced (but not unspliced) mRNA present in nuclear speckles (Fig. 4) [68,69,70]. Knockdown or mutation of Aly/REF inhibits mRNA export, but not splicing. Aly/REF interacts with PtdIns(4,5)P2, and mutation of residues that mediate phosphoinositide binding attenuates mRNA export [71]. Interestingly, Aly/REF is also a physiological substrate of Akt kinase (see above). Activation of Akt through PtdIns(3,4,5)P3 results in phosphorylation of Aly/REF that enhances mRNA export.

Separate from their roles in functionally well characterized nuclear speckles, phospholipids and in particular phosphatidylcholine have been proposed to arrange as micelles in the nucleoplasm, although this has not yet been demonstrated conclusively [16, 72]. A similar arrangement of somewhat phase separated phospholipid aggregates, known as nuclear lipid islets, has been described recently. For example, nuclear lipid islets of PtdIns(4,5)P2 aggregates, forming micelle-like structures, have been suggested to act as platforms for RNA polymerase II, thereby influencing mRNA transcription [73, 74]. Furthermore, nuclear lipid droplets derived from the inner nuclear membrane store phosphoinositides [25, 75] and serve as platforms for various enzymes implied in the biosynthesis of phospholipids [76].

Altogether, these findings highlight the emerging roles of nuclear phospholipids, particularly focusing on phosphoinositide within membrane-free compartments such as nuclear speckles and lipid islets. Their involvement in mRNA processing, export, and transcriptional regulation points to a broader functional landscape that needs to be further explored.

Fig. 4

Regulation of mRNA export by nuclear PI3K signaling. Export of mRNAs requires a series of events: pre-mRNA processing, ribonucleoprotein targeting to nuclear pore complexes, and translocation through nuclear pores to the cytoplasm. Aly/REF is a physiological target of PI3K signaling that regulates the protein’s localization and function through nuclear Akt-mediated phosphorylation and its association with the phosphoinositide PI(3,4,5)P3

Interactions of nuclear phospholipids with target proteins

Within the nucleus, phospholipids can engage in interactions with various soluble proteins. The formation of such complexes necessitates the shielding of the hydrophobic regions of the phospholipids. These hydrophobic regions are likely sequestered and complexed within the cavities and pockets of soluble nucleoplasmic proteins that allow the lipid-protein interaction to occur in a structurally favorable manner. However, due to significant technical challenges, particularly obtaining high-resolution structural data for these complexes, there is a scarcity of detailed molecular insights into how phospholipids interact with nuclear proteins [77]. This gap in understanding persists despite a few notable exceptions in the literature. Indeed, for several of the examples of signaling pathways discussed below, it remains a possibility that interactions are taking place between soluble proteins and nuclear phospholipids in their aggregated states. In the following, we discuss the interactions of nuclear phospholipids with proteins of the nucleoplasm within the aspects of control of gene expression, cell cycle regulation, programmed cell death, stress responses, and DNA damage repair.

Regulation of gene expression and chromatin states

PtdIns5P, a relatively less abundant nuclear phosphoinositide, has emerged as a key regulator of various factors involved in gene regulation that contain plant homeodomain (PHD) fingers [78]. Examples include ING2 (Inhibitor of Growth 2), a member of the ING family of tumor suppressors and a core component of the Sin3a-HDAC1 (switch-independent 3a/histone deacetylase 1) complex [79]. PtdIns5P directs ING2 towards specific chromatin regions where it functions as a histone code reader (recognizes histone 3 trimethylated at lysine 4, H3K4me3) and also facilitates the association between ING2 and chromatin-modifying enzymes, such as histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Fig. 5a) [80].

In addition, the PHD finger motif of ATX1 has high specificity for PtdIns5P [81]. ATX1 (Homolog of anti-oxidant 1) is a plant trithorax homologue that regulates flower development under drought stress conditions [81]. The ATX1 protein also contains a SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain that trimethylates histone 3 at lysine 4 [82]. Drought stress elevates the cellular levels of PtdIns5P, resulting in the displacement of ATX1 from the nucleus. This depletes the transcription factor from the promoter of the stress-regulated gene WRKY70 (WRKY transcription factor 70), which leads to altered transcriptional regulation of this drought-responsive gene [83].

Furthermore, PtdIns5P has a broader impact on transcriptional regulation through its interaction with components of the basal transcription factor complex TFIID, which binds to the promoter regions of multiple target genes and facilitates RNA polymerase positioning for transcription initiation. The TFIID complex includes the PHD finger protein TAF3 (TATA-binding protein-associated factor 3). PtdIns5P modulates the function of TAF3 by influencing its interaction with the H3K4me3 chromatin mark, and thereby regulates the expression of genes crucial for cellular differentiation and development (Fig. 5b) [84].

UHRF1 (Ubiquitin-like PHD and RING finger domain-containing protein 1) is an essential component of the DNA maintenance methylation machinery and is also implicated in the protection of chromatin structural integrity. PtdIns5P binds to UHRF1 and results in a conformational transition of the protein that connects a C-terminal region with a central PHD domain and enhances the affinity of UHRF1 for the repressive histone modification H3K9me3 (histone H3 trimethylated at lysine 9) (Fig. 5c) [14, 85]. Although the functional consequences of the interaction of PtdIns5P with UHRF1 in vivo are yet not fully clear, this interaction may regulate gene silencing and DNA damage response pathways.

Phospholipids can also directly influence chromatin structure and integrity. PtdIns(4,5)P2 can reverse transcriptional repression by interacting with the positively charged tails of histones H1 and H3 [17, 86]. The ceramide derivative sphingosine-1-phosphate (S1P) interacts with histone H3 to modulate chromatin structure and influencing gene expression [87]. S1P also acts as an inhibitor of the histone deacetylases HDAC1 and HDAC2, and thereby promotes histone acetylation at key lysine residues [88, 89]. Interestingly, sphingosine kinase 2 (SphK2), a key enzyme involved in S1P synthesis, binds directly to histone H3 and enhances acetylation of the H3 lysine 9, H4 lysine 5 and H2B lysine 12 residues, while also inhibiting HDAC activity [90] (Fig. 5d). In yeast and mammals, phosphatidylserine interacts with histones and protein kinases, and in turn impacts chromatin condensation and apoptosis signaling [91]. Furthermore, phosphatidic acid (PA) interacts with HDACs and transcription factors