The oily nucleus- role of phospholipids in genome biology: membrane-directed roles and signaling in the nucleoplasm.

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 – 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 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 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. 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 ]. 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 ]. 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. 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. 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 Ca 2+ signaling [ 42 ]. Collectively, their dynamic nuclear localization indicates these isoforms of PI-PLC exert essential nuclear functions. 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 ]. 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 – 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. 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. 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 – 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 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 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. 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.  5 a) [ 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.  5 b) [ 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.  5 c) [ 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.  5 d). 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, and thus contributes to DNA repair and chromatin decondensation [ 92 ] and sphingomyelin interacts with various kinases and transcription factors to regulate signal transduction and impact gene expression [ 87 ]. In addition, in a more direct role, phosphoinositides regulate the activity of the nuclear transcription factors SF-1/NR5A1(Steroidogenic Factor 1/Nuclear Receptor Subfamily 5 Group A Member 1) [ 77 , 93 ] and liver receptor homolog-1 (LRH-1/NR5A2) [ 94 ]. Binding of the hydrophobic part of phosphoinositides to a shielded pocket of the protein exposes the inositol head group. The phosphorylation state of phosphoinositides bound to SF-1 is regulated by the kinase IMPK and the phosphatase PTEN and determines the activity of the transcription factor (active: PtdIns(4,5)P2 vs. inactive: PtdIns(3,4,5)P3) [ 93 ]. Phospholipids have been shown to play a role in regulating the cell cycle, as is evident through the nuclear abundance of phosphoinositides, especially PtdIns4P, PtdIns(4,5)P2 and PtdIns5P, whose concentrations vary during different phases of the cell cycle [ 95 ]. Disruptions in the localization or levels of PtdIns(4,5)P2 can impair DNA synthesis. PtdIns(4,5)P2 is involved in the recruitment of replication factors to chromatin and is crucial for the proper loading of the PCNA (Proliferating cell nuclear antigen) sliding clamp [ 47 ]. PtdIns(3,4)P2 and PtdIns(3,4,5)P3 help regulate the mitotic spindle apparatus at the G2/M transition by influencing microtubule dynamics and spindle assembly. The proper functions of these pathways are essential for accurate chromosome segregation during mitosis [ 96 , 97 ]. In addition to phosphoinositides, nuclear diacylglycerol is important for the activation of nuclear PKC (Protein kinase C) [ 98 – 100 ], which in turn regulates the progression of cells through G1 and into S phase and through G2 to mitosis [ 101 – 106 ]. Fig. 5 Various phospholipid-chromatin and -chromatin binding protein interactions are crucial to stabilize nuclear architecture and function. ( a ) The ING2(PHD)-Sin3a-HDAC1 complex binds PtdIns(5)P, which facilitates its association with chromatin remodeling complexes. ( b ) PtdIns(5)P influences H3K4me3 and drives gene expression via its interaction with TAF3(PHD). ( c ) Binding of PtdIns(5)P to UHRF1 regulates its binding to the H3K9me3 chromatin mark. ( d ) S1P inhibits histone deacetylase HDAC1 and HDAC2. For abbreviations see text Various phospholipid-chromatin and -chromatin binding protein interactions are crucial to stabilize nuclear architecture and function. ( a ) The ING2(PHD)-Sin3a-HDAC1 complex binds PtdIns(5)P, which facilitates its association with chromatin remodeling complexes. ( b ) PtdIns(5)P influences H3K4me3 and drives gene expression via its interaction with TAF3(PHD). ( c ) Binding of PtdIns(5)P to UHRF1 regulates its binding to the H3K9me3 chromatin mark. ( d ) S1P inhibits histone deacetylase HDAC1 and HDAC2. For abbreviations see text Phospholipids, in particular phosphoinositides, have been implicated in programmed cell death and related pathways that culminate in apoptosis. Many studies have suggested that elevation of nuclear phosphoinositides affects transcriptional regulation, and influences the stabilization [ 107 ] and activation of p53 [ 108 ] and expression of the pro-apoptotic protein BIK (Bcl2-interacting killer) [ 109 ]. As previously discussed, PtdIns5P interacts with the PHD finger domain of ING2; this interaction not only affects chromatin but also triggers acetylation of p53, which leads to the induction of p21 and ultimately initiation of apoptosis [ 108 , 110 ]. Knockdown of ING2 or overexpression of PtdIns5P 4-kinase type IIβ (PIP4K2β) (which converts PtdIns5P to PtdIns(4,5)P2, thereby depleting the nuclear pool of PtdIns5P) inhibits apoptosis. This implies that the balance between PtdIns5P and PtdIns(4,5)P2 plays a critical role in modulating the apoptotic response. The nuclear level of PtdIns5P increases after inhibition of PIP4K2β, for example via UV irradiation or phosphorylation mediated by p38 stress-activated protein kinase [ 111 ]. Other mechanisms that alter the levels of PtdIns5P and affect apoptosis include Type I phosphatidylinositol (4,5) bisphosphate 4-phosphatase (Type I 4-ptase), which dephosphorylates PtdIns(4,5)P2 to PtdIns5P. In addition, DNA-damaging agents (etoposide or doxorubicin) induce translocation of type I 4-ptase to the nucleus which increases the nuclear level of PtdIns5P and enhances acetylation and stabilization of p53, subsequently triggering apoptosis [ 110 ]. PIP4K2β, which regulates the nuclear levels of PtdIns5P, becomes phosphorylated during oxidative stress. This triggers the interaction of PIP4K2β with Pin1 (Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1), which leads to inactivation of the enzyme [ 111 , 112 ]. The resulting nuclear surge of PtdIns5P induces the expression of genes involved in adaptation to oxidative stress. In contrast, knockout of Pin1 or overexpression of PIP4K reduces stress-induced nuclear accumulation of PtdIns5P, which diminishes the expression of target genes, and impairs cell survival under oxidative stress [ 112 ]. The precise mechanism by which PtdIns5P influences gene expression remains unclear, but may involve the NRF2 (Nuclear factor erythroid 2-related factor 2)/Keap1(Kelch-like ECH-associated protein 1) pathway, as many target genes downstream of this pathway are regulated by PtdIns5P. Early discoveries correlated changes in nuclear phosphoinositide levels with the cellular response to genotoxic stress. For example, the levels of nuclear PI(4,5)P2 were found to double within the first hour after ionizing radiation (IR) exposure, before gradually returning to normal over the next 18 h [ 113 ]. While we discuss the involvement of nuclear phosphoinositides in DNA damage repair below, a pathway initiated by activation of the nuclear PI3K-Akt pathway in response to genotoxic stress is worth mentioning in the discussion of stress responses. In response to cisplatin treatment, nuclear IPMK generates PtdIns(3,4,5)P3 by phosphorylation of PtdIns(3,4,5)P2. P53 in conjunction with PtdIns(3,4,5)P3 (referred to as p53-PIP signalosome) recruits Akt, PDK1, and mTORC2, which leads to activation of Akt and phosphorylation of FOXO (Forkhead Box O) proteins. Consequently, this leads to the inhibition of DNA damage-induced apoptosis, independent of the conventional membrane-localized PI3K-Akt pathway (Fig.  6 ) [ 114 ]. Changes in nuclear phosphoinositide levels have been associated with the repair of DNA damage at multiple instances, in terms of early, intermediate and late damage responses [ 95 ] – although the exact molecular mode of action of nuclear phosphoinositides in DNA repair has not yet been defined. Fig. 6 Phosphoinositide-p53 signalosome: p53-PI3K phosphorylates PtdIns(4,5)P2 to PtdIns(3,4,5)P3, which forms a complex with p53 that initiates a cascade involving activation of Akt and phosphorylation of FOXO to inhibit DNA damage-induced apoptosis Phosphoinositide-p53 signalosome: p53-PI3K phosphorylates PtdIns(4,5)P2 to PtdIns(3,4,5)P3, which forms a complex with p53 that initiates a cascade involving activation of Akt and phosphorylation of FOXO to inhibit DNA damage-induced apoptosis Early DNA damage responses, occurring within 1–3 min of damage induction, involve the rapid accumulation of nuclear phosphoinositides at sites of DNA damage, as indicated by UVA laser micro-irradiation and localization of GFP-tagged phosphoinositide-binding domains [ 45 ]. These phosphoinositides, including PtdIns(4,5)P2 and PtdIns(3,4,5)P3, play crucial roles in recruiting and activating ATR (Ataxia Telangiectasia And Rad3 Related), a PI3K enzyme implicated in their regulation and a key kinase in DNA damage repair pathways [ 45 , 96 ]. Class IA PI3Kβ (p110β), which has been identified as a key factor involved in the maintenance of genomic integrity, is also recruited at an early stage to sites of DNA damage, and associates with repair proteins such as Rad50 and Nbs1(nibrin) [ 45 , 96 , 115 – 117 ]. The intermediate DNA damage response occurs within tens of minutes after damage and involves significant changes in phosphoinositide metabolism through the modification and translocation of kinases and phosphatases. The levels of PtdIns5P increase significantly within 20–40 min post-damage, and these changes are driven by p38 MAPK-mediated inhibition of PIPK2β and nuclear import of TMEM55B (transmembrane protein 55B) [ 96 , 118 ]. Furthermore, PtdIns(4,5)P2 levels increase during the intermediate DNA damage response via processes mediated by activation of PIPK1α (phosphatidylinositol-4-phosphate 5-kinase) and inhibition of nuclear 5′-phosphatases, including SHIP2 (SH2-domain-containing inositol phosphatase 2) and INPP5E (Inositol polyphosphate-5-phosphatase E) [ 109 , 119 – 121 ]. Lastly, PtdIns(3,4,5)P3 is regulated in the intermediate DNA repair response by PI3K and the phosphatase PTEN (Phosphatase and tensin homolog), which exerts both lipid phosphatase-dependent and independent roles in DNA repair [ 96 ]. In late DNA damage responses, which occur hours to days after damage, PIPKIα stabilizes and activates p53, 24 h after cisplatin treatment [ 107 ]. Furthermore, PtdIns(3,4,5)P3 is required for the selective export of mRNAs encoding key homologous recombination (HR) repair proteins, such as RAD51 (RAD51 recombinase), CHK1 (Checkpoint kinase 1), and FANCD2 (Fanconi anemia complementation group D2). This is mediated by regulation/activation of the mRNA export factor Aly/REF (see above), which results in increased levels of the repair factors. In contrast, depletion of IPMK leads to reduced levels of PtdIns(3,4,5)P3 and thereby compromises the cell’s ability to repair DNA damage through homologous recombination [ 96 , 122 , 123 ]. These findings demonstrate that nuclear phospholipids are deeply woven into the cell’s regulatory fabric, from shaping gene expression and cell division to guiding stress responses and DNA repair.

While specific insights into the molecular functions of nuclear phospholipids have recently been obtained, our overall comprehension of the function and roles of nuclear phospholipids still remains in the early stages. From our perspective, this is not only due to the experimental difficulties in their study, but also because of conceptional challenges in defining their working modes (Fig.  7 ). In this context, it will be worth to revisit earlier, more general and partially generic findings and to look at new discoveries in the framework of membrane-directed vs. nucleoplasmic signaling and the related biochemistry of the nuclear phospholipids. After all, the detailed understanding of many biological pathways has been driven by characterizing and molecularly dissecting the functional domains these take place in. Fig. 7 The complexities of nuclear phospholipid biology. ( a ) Membrane-bound and/or -directed functions include effects driven by the hydrophobic tails. Phospholipid asymmetry in the outer vs. inner nuclear membrane affects (i) incorporation of specific factors and (ii) controls membrane curvature and fluidity that is sensed by specific proteins. The polar head groups of phospholipids are recognized by (iii) proteins likely anchored in the nuclear membrane or initiate downstream signaling via (iv) direct recruitment of factors or (v) by their metabolites (here membrane-associated or soluble effects are often unclear and difficult to resolve). ( b ) Some effects of nuclear phospholipids are still quite unclear. These include (i) impacts on chromatin structure/composition, (ii) effects on transcription and (iii) depletion of linker histones from chromatin. ( c ) Within the nucleoplasm, the hydrophobic tails of phospholipids need to be shielded from the aqueous milieu. It is conceivable that this occurs in (i) micelles (aka homogenous lipid composition), (ii) nuclear lipid droplets (aka heterogenous lipid composition), (iii) nuclear speckles or (iv) other aggregated/phase separated entities. In such scenario, the polar head groups would be available to initiate and control specific signaling events via recruitment of distinct proteins. (v) Alternatively, phospholipids can be fully accommodated by nuclear proteins simultaneously binding their hydrophobic and polar parts. (vi) In the case of PHD finger mediated events, it is yet unclear how the hydrophobic parts of nuclear phospholipids are dealt with biochemically. (vii) Specific signaling events can also be mediated by the exposed polar head groups of nuclear phospholipids when their acyl parts are bound in hydrophobic protein pockets. The question marks indicate aspects of nuclear phospholipid biology that are not fully resolved or hypothetical (order of events, localization of components, biochemistry of interactions, etc.) The complexities of nuclear phospholipid biology. ( a ) Membrane-bound and/or -directed functions include effects driven by the hydrophobic tails. Phospholipid asymmetry in the outer vs. inner nuclear membrane affects (i) incorporation of specific factors and (ii) controls membrane curvature and fluidity that is sensed by specific proteins. The polar head groups of phospholipids are recognized by (iii) proteins likely anchored in the nuclear membrane or initiate downstream signaling via (iv) direct recruitment of factors or (v) by their metabolites (here membrane-associated or soluble effects are often unclear and difficult to resolve). ( b ) Some effects of nuclear phospholipids are still quite unclear. These include (i) impacts on chromatin structure/composition, (ii) effects on transcription and (iii) depletion of linker histones from chromatin. ( c ) Within the nucleoplasm, the hydrophobic tails of phospholipids need to be shielded from the aqueous milieu. It is conceivable that this occurs in (i) micelles (aka homogenous lipid composition), (ii) nuclear lipid droplets (aka heterogenous lipid composition), (iii) nuclear speckles or (iv) other aggregated/phase separated entities. In such scenario, the polar head groups would be available to initiate and control specific signaling events via recruitment of distinct proteins. (v) Alternatively, phospholipids can be fully accommodated by nuclear proteins simultaneously binding their hydrophobic and polar parts. (vi) In the case of PHD finger mediated events, it is yet unclear how the hydrophobic parts of nuclear phospholipids are dealt with biochemically. (vii) Specific signaling events can also be mediated by the exposed polar head groups of nuclear phospholipids when their acyl parts are bound in hydrophobic protein pockets. The question marks indicate aspects of nuclear phospholipid biology that are not fully resolved or hypothetical (order of events, localization of components, biochemistry of interactions, etc.) Crucial questions that need to be addressed pertain to the nuclear sequestration, maintenance and regulation of nuclear phospholipids. There is not enough clarity on the mechanisms by which phospholipids, their precursors and metabolites are trafficked into the nuclear membrane and nucleoplasm and how they are regulated there. Are there differently regulated pools of nuclear phospholipids between the membrane and nucleoplasm, within the membrane or in the nucleoplasm? The immunostaining of certain phosphoinositides [ 17 ] and the discovery of phase separated entities of accumulated nuclear phospholipids at least would suggest so. In addition, it remains unclear how different pools of nuclear phospholipids are localized and physically separated, and what the biological and biochemical consequences of such localization and separation are. For example, are genome elements directed to the localized phospholipid pools or vice versa after specific stimuli and in specific physiological scenarios? Finally, further research is required to define the signaling contributions of the hydrophobic tail regions of nuclear phospholipids. Advances in experimental approaches in the detection and manipulation of nuclear phospholipids will be needed for addressing these issues, not only to advance fundamental understanding of their biology but also to further the translation of such knowledge. We look forward to future work that will expand the roles of nuclear phospholipids in disease mechanisms and define their position as potential targets for therapeutic intervention.

Lipids constitute a diverse group of hydrophobic and amphiphilic molecules that play essential roles in energy storage, cellular structure, and signaling. Phospholipids are an important class of complex lipids with relatively wide-reaching biological significance. These molecules possess a hydrophilic head group containing a phosphate moiety and two hydrophobic fatty acid chains, making them amphipathic in nature. The structural diversity of phospholipids is attributed to the variation in their length, the level of saturation in their acyl chains and the type of polar head groups they bear. Phospholipids can be classified as glycerophospholipids (phosphatic acid, phosphatidyl-ethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine and cardiolipins), which all carry a glycerol backbone, or sphingophospholipids, which possess a sphingosine (an amino alcohol) backbone (ceramide, sphingomyelin, cerebroside, ganglioside and sphingosine-1-phosphate) (Fig.  1 ). Due to their amphipathic nature, phospholipids spontaneously arrange into micelles when placed in an aqueous environment, and they form the basic structure of biological membranes – the lipid bilayer [ 1 ]. Membranes separate the extracellular milieu from the intracellular milieu and define the organelles inside eukaryotic cells. Apart from these structural roles, phospholipids are also involved in various other biological functions such as gene expression, cellular signaling, and chromatin modelling, both directly and through their metabolites [ 2 , 3 ]. Phospholipids were first identified in the cell nucleus in the 1960 s [ 4 , 5 ]. Although their existence and significance in this compartment was long considered spurious or anecdotal, accumulating evidence indicates nuclear phospholipids play crucial roles in maintaining nuclear architecture and function. This is largely because nuclear phospholipids are now known to be functionally active within the nucleoplasm and not just in the nuclear envelope. In this review, we explore the diverse biology of nuclear phospholipids, with a particular focus on their functional roles within the nuclear membrane and nucleoplasm in respect to their effects on the genome. Additionally, we aim to shed light on how nuclear phospholipids influence cellular physiology and disease, with an emphasis on the critical intersections between the metabolism of phospholipids and their biological functions.

The role of phospholipids in disease, particularly cancer, is receiving increased attention, not least due to the fact that many phospholipids are regulated by enzymes that are frequently mutated in transformed cells. These enzymes include kinases, phosphatases, lipases, and acyltransferases, which control the synthesis, modification, and degradation of phospholipids. While it remains an appealing possibility that specific diseases could be treated by delivering phospholipids or their derivates, it remains challenging to confirm the direct causal involvement of altered levels of phospholipids in malfunctioning cells. We believe that, at present, we are only beginning to see the tip of the iceberg in terms of the association between nuclear phospholipids and diseases. In the following, we describe key examples of links between disease and nuclear phospholipids aligned with our discussion of the functional working domains outlined in this review. Within the functional domain of the nuclear membrane, PI-PLCs is worth mentioning. This enzyme modulates the levels of inositol phosphates and DAG and has been implicated in tumorigenesis. The PI-PLCβ1 isoform, which is predominantly nuclear, has been linked to the pathogenesis of myelodysplastic syndrome (MDS), a precursor condition to acute myeloid leukemia (AML). PI-PLCβ1 is involved in hematopoietic differentiation that involves recruitment of Myeloid zinc finger-1 (MZF-1), suggesting a role in the initiation and progression of MSD to AML. Notably, monoallelic deletion of PLCβ1 is associated with an adverse prognosis and increased risk of AML progression. Furthermore, treatment with azacytidine, a DNA methylation inhibitor, alleviates the levels of nuclear PLCβ1 and promotes normal myeloid differentiation in MDS cells [ 41 , 124 ]. Beyond its role in hematological malignancies, PI-PLCβ1 serves as a biomarker for high-grade gliomas, with its expression inversely correlating with the malignant grade. Remarkably, silencing of PI-PLCβ1 increases activation of the Stat3 oncogenic signaling pathway and upregulates a variety of mesenchymal markers, including Slug, N-cadherin, and matrix metalloproteases (MMP-2, MMP-9) [ 125 ]. For phospholipid biology that cannot be clearly assigned a nuclear or cytoplasmic and membrane vs. nucleoplasm function we note that sphingolipids, particularly ceramide, induce apoptosis in leukemia cells, illustrating their metabolism may play a significant role in cancer therapy [ 126 ]. Furthermore, it was found that phosphatidic acid enhances mTOR signaling in prostate cancer, linking lipid metabolism to tumor growth [ 127 ]. Beyond cancer, PI-PLCβ1 also plays a crucial role in neural function. The nuclear isoform, PI-PLCβ1b, regulates endocannabinoid neuronal excitability through DAG synthesis, which influences depolarization and receptor activation while maintaining inhibitory pathways via 2-arachidonoylglycerol (2-AG) [ 128 ]. Additionally, PI-PLCβ1 signaling, which is activated through metabotropic glutamate receptors (mGluRs), is essential for cortical circuit development and activity-dependent maturation of the cerebral cortex [ 129 ]. Dysregulation of PI-PLCβ1 has been implicated in neurological disorders such as epilepsy, schizophrenia, and neuro-oncological conditions [ 130 , 131 ]. Furthermore, PI-PLCβ1 interacts with Lamin B1, a key component of the nuclear lamina, which highlights its link with Autosomal Dominant Leukodystrophy (ADLD)—a rare and fatal neurodegenerative disease characterized by LMNB1 gene duplications or deletions leading to Lamin B1 overexpression [ 132 ]. In the context of the nucleoplasm, phosphatidylinositol-5-phosphate 4-kinases (PI5P4Ks), which phosphorylate PtdIns5P to generate PtdIns(4,5)P2, a critical phosphoinositide implicated in cancer progression, merits further research [ 133 ]. Altered expression of PI5P4Ks has been observed across many types of cancer, including leukemia, glioblastoma, breast and prostate cancer. In mammals, there are three isoforms of the enzyme. The PI5P4Kβ isoform predominantly localizes to the nucleus [ 134 ]. Dysregulation of PI5P4Kβ has been implicated in many cancers through gene association studies [ 135 – 139 ]. This gene is located in proximity of the proto-oncogene ERBB2 , which often results in co-amplification in various malignant tumors [ 136 , 139 , 140 ]. However, the expression of PI5P4Kβ often seems to be independent of ERBB2 in breast cancer, leading to its therapeutic significance in patients that exhibit resistance to the prevalent HER-2 therapy [ 136 , 139 , 140 ]. Downregulation of PI5P4Kβ in cell culture models of triple negative breast cancer (TNBC) inhibits cell proliferation [ 141 ]. PI5P4Kβ has also been implicated in soft tissue sarcomas (SARC), which are a heterogeneous group of malignancies. In a panel of SARC cell lines, inhibition of PI5P4Kβ resulted in anti-proliferative effects [ 141 ]. Given their potentially crucial role in cancer, targeting phosphoinositide kinases could represent a promising therapeutic avenue. UNC3230, an inhibitor of PI4P5Kγ, and ISA-2011B, which targets PI4P5Kα, can selectively disrupt PtdIns(4,5)P2 signaling and have been shown to impair cell proliferation and increase survival rates in prostate, breast, and colorectal cancer cell lines [ 142 ]. Beyond these direct associations, various nuclear proteins that interact with phosphoinositides play pivotal roles in the etiology of diseases. The nuclear receptors Steroidogenic Factor-1 (SF-1; NR5A1) and its structural homolog Liver Receptor homolog-1 (LRH-1; NR5A2) bind PtdIns(4,5)P2 and PtdIns(3,4,5)P3 to form stable phosphoinositide/protein complexes. Dysregulation of these complexes has been implicated in several cancers (glioblastoma, hepatocellular carcinoma) and various other pathological states, including NAFLD/NASH (nonalcoholic fatty liver disease/steatohepatitis), endometriosis and obesity [ 66 , 143 ].

The nucleus is the distinguishing organelle of eukaryotic cells that houses the genetic blueprint of the organism. Lipids constitute a minor, although a vital component of the nucleus, which is defined by a lipid-rich nuclear envelope and has a lipid-poor nucleoplasm. Overall, phospholipids are the predominant nuclear lipids (Fig.  2 ). In rat liver nuclei, the total phospholipid content was reported to be 3.2% by weight, as compared to 74.6% for protein and 22.2% for DNA [ 6 , 7 ]. While the total lipid content of the nuclear envelope is ca. 50% of that of protein by weight, phospholipids make up 65% of these envelope lipids. Thus, the phospholipid content per mg of protein is approximately nine times higher in the nuclear envelope compared to whole nuclei [ 8 ]. Fig. 1 Phospholipids are classified into two major groups based on the presence of different backbones, and further subclassified based on the different head groups (R). ( a ) General classification of phospholipids. ( b ) Classification of glycerophospholipids. ( c ) Classification of sphingophospholipids Phospholipids are classified into two major groups based on the presence of different backbones, and further subclassified based on the different head groups (R). ( a ) General classification of phospholipids. ( b ) Classification of glycerophospholipids. ( c ) Classification of sphingophospholipids Phosphatidylcholine is the most abundant phospholipid in nuclei, followed by phosphatidylethanolamine and phosphatidylinositols, which are also present in significant amounts. In contrast, phosphatidylserine and sphingomyelin are found at lower levels [ 8 ]. Among the nuclear phospholipids, those containing phosphatidylinositol (PtdIn) are noteworthy due to their crucial biological roles. PtdIns consist of a hydrophilic myo-inositol head group linked to the sn-3 position of the glycerol group of a hydrophobic diacylglycerol (DAG) tail through a phosphodiester linkage. Phosphatidylinositol is the basic scaffold of phosphoinositides (PIPs). The hydroxyl groups on the myo-inositol ring can be reversibly phosphorylated at the C3, C4, and C5 positions, resulting in the formation of seven distinct poly-acidic phospholipids or phosphoinositides: PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Phosphatidylinositol and most of its derivatives except for PtdIns(3,5)P2 have been detected in both the nuclear envelope and nucleoplasm (Fig.  3 ) [ 9 – 12 ]. Fig. 2 Composition of the eukaryotic cell nucleus. ( a ) Total lipid, protein, and DNA contents of eukaryotic nuclei and ( b ) composition of the total nuclear lipid pool Composition of the eukaryotic cell nucleus. ( a ) Total lipid, protein, and DNA contents of eukaryotic nuclei and ( b ) composition of the total nuclear lipid pool