LAP2 Proteins Chaperone GLI1 Movement between the Lamina and Chromatin to Regulate Transcription.

Understanding transcription factor navigation through the nucleus remains critical for developing targeted therapeutics. The GLI1 transcription factor must maintain maximal Hedgehog pathway output in basal cell carcinomas (BCCs), and we have previously shown that resistant BCCs increase GLI1 deacetylation through atypical protein kinase Cι/λ (aPKC) and HDAC1. Here we identify a lamina-associated polypeptide 2 (LAP2) isoform-dependent nuclear chaperoning system that regulates GLI1 movement between the nuclear lamina and nucleoplasm to achieve maximal activation. LAP2ß forms a two-site interaction with the GLI1 zinc-finger domain and acetylation site, stabilizing an acetylation-dependent reserve on the inner nuclear membrane (INM). By contrast, the nucleoplasmic LAP2α competes with LAP2ß for GLI1 while scaffolding HDAC1 to deacetylate the secondary binding site. aPKC functions to promote GLI1 association with LAP2α, promoting egress off the INM. GLI1 intranuclear trafficking by LAP2 isoforms represents a powerful signal amplifier in BCCs with implications for zinc finger-based signal transduction and therapeutics.

Nuclear envelope assembly relies on CHMP-7 in the absence of BAF-LEM-mediated hole closure.

Barrier-to-autointegration factor (BAF) protein is a DNA-binding protein that crosslinks chromatin to allow mitotic nuclear envelope (NE) assembly. The LAP2-emerin-MAN1 (LEM)-domain protein LEMD2 and ESCRT-II/III hybrid protein CHMP7 close NE holes surrounding spindle microtubules (MTs). BAF binds LEM-domain family proteins to repair NE ruptures in interphase, but whether BAF-LEM binding participates in NE hole closure around spindle MTs is not known. Here, we took advantage of the stereotypical event of NE formation in fertilized Caenorhabditis elegans oocytes to show that BAF-LEM binding and LEM-2-CHMP-7 have distinct roles in NE closure around spindle MTs. LEM-2 and EMR-1 (homologs of LEMD2 and emerin) function redundantly with BAF-1 (the C. elegans BAF protein) in NE closure. Compromising BAF-LEM binding revealed an additional role for EMR-1 in the maintenance of the NE permeability barrier. In the absence of BAF-LEM binding, LEM-2-CHMP-7 was required for NE assembly and embryo survival. The winged helix domain of LEM-2 recruits CHMP-7 to the NE in C. elegans and a LEM-2-independent nucleoplasmic pool of CHMP-7 also contributes to NE stability. Thus, NE hole closure surrounding spindle MTs requires redundant mechanisms that safeguard against failure in NE assembly to support embryogenesis.

The role of inner nuclear membrane proteins in tumourigenesis and as potential targets for cancer therapy.

Despite significant advances in our understanding of tumourigenesis and cancer therapeutics, cancer continues to account for 30% of worldwide deaths. Therefore, there remains an unmet need for the development of cancer therapies to improve patient quality of life and survival outcomes. The inner nuclear membrane has an essential role in cell division, cell signalling, transcription, cell cycle progression, chromosome tethering, cell migration and mitosis. Furthermore, expression of several inner nuclear membrane proteins has been shown to be frequently altered in tumour cells, resulting in the dysregulation of cellular pathways to promote tumourigenesis. However, to date, minimal research has been conducted to investigate how targeting these dysregulated and variably expressed proteins may provide a novel avenue for cancer therapies. In this review, we present an overview of the involvement of the inner nuclear membrane proteins within the hallmarks of cancer and how they may be exploited as potent anti-cancer therapeutics.

Survival of Drosophila germline stem cells requires the chromatin-binding protein Barrier-to-autointegration factor.

The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear envelope. This network includes LAP2-emerin-MAN1 domain (LEM-D) proteins that associate with the chromatin and DNA-binding protein Barrier-to-autointegration factor (BAF). Here, we investigate the partnership between three NL Drosophila LEM-D proteins and BAF. In most tissues, only Emerin/Otefin is required for NL enrichment of BAF, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, we studied BAF contributions in the ovary, a tissue where Emerin/Otefin function is essential. We show that germ cell-specific BAF knockdown causes phenotypes that mirror emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that, similar to emerin/otefin mutants, BAF depletion activates the NL checkpoint that causes germ cell loss. Taken together, our findings provide evidence for a prominent NL partnership between the LEM-D protein Emerin/Otefin and BAF, revealing that BAF functions with this partner in the maintenance of an adult stem cell population.

The Discovery of a LEMD2-Associated Nuclear Envelopathy with Early Progeroid Appearance Suggests Advanced Applications for AI-Driven Facial Phenotyping.

Over a relatively short period of time, the clinical geneticist's "toolbox" has been expanded by machine-learning algorithms for image analysis, which can be applied to the task of syndrome identification on the basis of facial photographs, but these technologies harbor potential beyond the recognition of established phenotypes. Here, we comprehensively characterized two individuals with a hitherto unknown genetic disorder caused by the same de novo mutation in LEMD2 (c.1436C>T;p.Ser479Phe), the gene which encodes the nuclear envelope protein LEM domain-containing protein 2 (LEMD2). Despite different ages and ethnic backgrounds, both individuals share a progeria-like facial phenotype and a distinct combination of physical and neurologic anomalies, such as growth retardation; hypoplastic jaws crowded with multiple supernumerary, yet unerupted, teeth; and cerebellar intention tremor. Immunofluorescence analyses of patient fibroblasts revealed mutation-induced disturbance of nuclear architecture, recapitulating previously published data in LEMD2-deficient cell lines, and additional experiments suggested mislocalization of mutant LEMD2 protein within the nuclear lamina. Computational analysis of facial features with two different deep neural networks showed phenotypic proximity to other nuclear envelopathies. One of the algorithms, when trained to recognize syndromic similarity (rather than specific syndromes) in an unsupervised approach, clustered both individuals closely together, providing hypothesis-free hints for a common genetic etiology. We show that a recurrent de novo mutation in LEMD2 causes a nuclear envelopathy whose prognosis in adolescence is relatively good in comparison to that of classical Hutchinson-Gilford progeria syndrome, and we suggest that the application of artificial intelligence to the analysis of patient images can facilitate the discovery of new genetic disorders.

ESCRT recruitment by the S. cerevisiae inner nuclear membrane protein Heh1 is regulated by Hub1-mediated alternative splicing.

Misassembled nuclear pore complexes (NPCs) are removed by sealing off the surrounding nuclear envelope (NE), which is conducted by the endosomal sorting complexes required for transport (ESCRT) machinery. Recruitment of ESCRT proteins to the NE is mediated by the interaction between the ESCRT member Chm7 and the inner nuclear membrane protein Heh1, which belongs to the conserved LEM family. Increased ESCRT recruitment results in excessive membrane scission at damage sites but its regulation remains poorly understood. Here, we show that Hub1-mediated alternative splicing of HEH1 pre-mRNA, resulting in production of its shorter form Heh1-S, is critical for the integrity of the NE in Saccharomyces cerevisiae ESCRT-III mutants lacking Hub1 or Heh1-S display severe growth defects and accumulate improperly assembled NPCs. This depends on the interaction of Chm7 with the conserved MSC domain, which is only present in the longer variant Heh1-L. Heh1 variants assemble into heterodimers, and we demonstrate that a unique splice segment in Heh1-S suppresses growth defects associated with the uncontrolled interaction between Heh1-L and Chm7. Together, our findings reveal that Hub1-mediated splicing generates Heh1-S to regulate ESCRT recruitment to the NE.This article has an associated First Person interview with the first author of the paper.

Collective analysis of the expression and prognosis for LEM-domain proteins in prostate cancer.

BACKGROUND: Mammalian LEM-domain proteins (LEMs) are encoded by seven genes, including LAP2, EMD, LEMD1, LEMD2, LEMD3, ANKLE1, and ANKLE2. Though some LEMs were involved in various tumor progression, the expression and prognostic values of LEMs in prostate adenocarcinoma (PRAD) have yet to be analyzed. METHODS: Herein, we investigated the expression, survival data, and immune infiltration levels of LEMs in PRAD patients from ATCG, TIMER, LinkedOmics, and TISIDB databases. We also further validated the mRNA and protein expression levels of ANKLE1, EMD, and LEMD2 in human prostate tumor specimens by qPCR, WB, and IHC. RESULTS: We found that all LEM expressions, except for that of LAP2, were markedly altered in PRAD compared to the normal samples. Among all LEMs, only the expressions of ANKLE1, EMD, and LEMD2 were correlated with advanced tumor stage and survival prognosis in PRAD. Consistent with the predicted computational results, the mRNA and protein expression levels of these genes were markedly increased in the PRAD group. We then found that ANKLE1, EMD, and LEMD2 expressions were markedly correlated with immune cell infiltration levels. High ANKLE1, EMD, and LEMD2 expressions predicted a worse prognosis in PRAD based on immune cells. DNA methylation or/and copy number variations may contribute to the abnormal upregulation of ANKLE1, EMD, and LEMD2 in PRAD. CONCLUSIONS: Taken together, this study implied that ANKLE1, EMD, and LEMD2 were promising prognosis predictors and potential immunotherapy targets for PRAD patients.

Forever young: the key to rejuvenation during gametogenesis.

Cell aging is the result of deteriorating competence in maintaining cellular homeostasis and quality control. Certain cell types are able to rejuvenate through asymmetric cell division by excluding aging factors, including damaged cellular compartments and extrachromosomal rDNA circles, from entering the daughter cell. Recent findings from the budding yeast S. cerevisiae have shown that gametogenesis represents another type of cellular rejuvenation. Gametes, whether produced by an old or a young mother cell, are granted a renewed replicative lifespan through the formation of a fifth nuclear compartment that sequesters the harmful senescence factors accumulated by the mother. Here, we describe the importance and mechanism of cellular remodeling at the nuclear envelope mediated by ESCRT-III and the LEM-domain proteins, with a focus on nuclear pore biogenesis and chromatin interaction during gamete rejuvenation.

Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing.

The integrity of the nuclear envelope barrier relies on membrane remodeling by the ESCRTs, which seal nuclear envelope holes and contribute to the quality control of nuclear pore complexes (NPCs); whether these processes are mechanistically related remains poorly defined. Here, we show that the ESCRT-II/III chimera, Chm7, is recruited to a nuclear envelope subdomain that expands upon inhibition of NPC assembly and is required for the formation of the storage of improperly assembled NPCs (SINC) compartment. Recruitment to sites of NPC assembly is mediated by its ESCRT-II domain and the LAP2-emerin-MAN1 (LEM) family of integral inner nuclear membrane proteins, Heh1 and Heh2. We establish direct binding between Heh2 and the "open" forms of both Chm7 and the ESCRT-III, Snf7, and between Chm7 and Snf7. Interestingly, Chm7 is required for the viability of yeast strains where double membrane seals have been observed over defective NPCs; deletion of CHM7 in these strains leads to a loss of nuclear compartmentalization suggesting that the sealing of defective NPCs and nuclear envelope ruptures could proceed through similar mechanisms.

Drosophila male and female germline stem cell niches require the nuclear lamina protein Otefin.

The nuclear lamina is an extensive protein network that underlies the inner nuclear envelope. This network includes the LAP2-emerin-MAN1-domain (LEM-D) protein family, proteins that share an association with the chromatin binding protein Barrier-to-autointegration factor (BAF). Loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Mechanisms associated with laminopathies are not yet understood. Here we present our studies of one of the Drosophila nuclear lamina LEM-D proteins, Otefin (Ote), a homologue of emerin. Previous studies have shown that Ote is autonomously required for the survival of female germline stem cells (GSCs). We demonstrate that Ote is also required for survival of somatic cells in the ovarian niche, with loss of Ote causing a decrease in cap cell number and altered signal transduction. We show germ cell-restricted expression of Ote rescues these defects, revealing a non-autonomous function for Ote in niche maintenance and emphasizing that GSCs contribute to the maintenance of their own niches. Further, we investigate the requirement of Ote in the male fertility. We show that ote mutant males become prematurely sterile as they age. Parallel to observations in females, this sterility is associated with GSC loss and changes in somatic cells of the niche, phenotypes that are largely rescued by germ cell-restricted Ote expression. Taken together, our studies demonstrate that Ote is required autonomously for survival of two stem cell populations, as well as non-autonomously for maintenance of two somatic niches. Finally, our data add to growing evidence that LEM-D proteins have critical roles in stem cell survival and tissue homeostasis.

The molecular basis of emerin-emerin and emerin-BAF interactions.

Emerin is a conserved membrane component of nuclear lamina structure. Here, we report an advance in understanding the molecular basis of emerin function: intermolecular emerin-emerin association. There were two modes: one mediated by association of residues 170-220 in one emerin molecule to residues 170-220 in another, and the second involving residues 170-220 and 1-132. Deletion analysis showed residues 187-220 contain a positive element essential for intermolecular association in cells. By contrast, deletion of residues 168-186 inactivated a proposed negative element, required to limit or control association. Association of GFP-emerin with nuclear BAF in cells required the LEM domain (residues 1-47) and the positive element. Emerin peptide arrays revealed direct binding of residues 170-220 to residues 206-225 (the proposed positive element), residues 147-174 (particularly P(153)MYGRDSAYQSITHYRP(169)) and the LEM domain. Emerin residues 1-132 and 159-220 were each sufficient to bind lamin A or B1 tails in vitro, identifying two independent regions of molecular contact with lamins. These results, and predicted emerin intrinsic disorder, support the hypothesis that there are multiple 'backbone' and LEM-domain configurations in a proposed intermolecular emerin network at the nuclear envelope.

The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth.

Background: Triple-negative breast cancer (TNBC) is a sub-classification of breast carcinomas, which leads to poor survival outcomes for patients. TNBCs do not possess the hormone receptors that are frequently targeted as a therapeutic in other cancer subtypes and, therefore, chemotherapy remains the standard treatment for TNBC. Nuclear envelope proteins are frequently dysregulated in cancer cells, supporting their potential as novel cancer therapy targets. The Lem-domain (Lem-D) (LAP2, Emerin, MAN1 domain, and Lem-D) proteins are a family of inner nuclear membrane proteins, which share a ~45-residue Lem-D. The Lem-D proteins, including Ankle2, Lemd2, TMPO, and Emerin, have been shown to be associated with many of the hallmarks of cancer. This study aimed to define the association between the Lem-D proteins and TNBC and determine whether these proteins could be promising therapeutic targets. Methods: GENT2, TCGA, and KM plotter were utilized to investigate the expression and prognostic implications of several Lem-D proteins: Ankle2, TMPO, Emerin, and Lemd2 in publicly available breast cancer patient data. Immunoblotting and immunofluorescent analysis of immortalized non-cancerous breast cells and a panel of TNBC cells were utilized to establish whether protein expression of the Lem-D proteins was significantly altered in TNBC. SiRNA was used to decrease individual Lem-D protein expression, and functional assays, including proliferation assays and apoptosis assays, were conducted. Results: The Lem-D proteins were generally overexpressed in TNBC patient samples at the mRNA level and showed variable expression at the protein level in TNBC cell lysates. Similarly, protein levels were generally negatively correlated with patient survival outcomes. siRNA-mediated depletion of the individual Lem-D proteins in TNBC cells induced aberrant nuclear morphology, decreased proliferation, and induced cell death. However, minimal effects on nuclear morphology or cell viability were observed following Lem-D depletion in non-cancerous MCF10A cells. Conclusion: There is evidence to suggest that Ankle2, TMPO, Emerin, and Lemd2 expressions are correlated with breast cancer patient outcomes, but larger patient sample numbers are required to confirm this. siRNA-mediated depletion of these proteins was shown to specifically impair TNBC cell growth, suggesting that the Lem-D proteins may be a specific anti-cancer target.

Developmental changes in nuclear lamina components during germ cell differentiation.

The nuclear lamina (NL) changes composition for regulation of nuclear events. We investigated changes that occur in Drosophila oogenesis, revealing switches in NL composition during germ cell differentiation. Germline stem cells (GSCs) express only LamB and predominantly emerin, whereas differentiating nurse cells predominantly express LamC and emerin2. A change in LamC-specific localization also occurs, wherein phosphorylated LamC redistributes to the nuclear interior only in the oocyte, prior to transcriptional reactivation of the meiotic genome. These changes support existing concepts that LamC promotes differentiation, a premise that was tested. Remarkably ectopic LamC production in GSCs did not promote premature differentiation. Increased LamC levels in differentiating germ cells altered internal nuclear structure, increased RNA production, and reduced female fertility due to defects in eggshell formation. These studies suggest differences between Drosophila lamins are regulatory, not functional, and reveal an unexpected robustness to level changes of a major scaffolding component of the NL.

Mechanisms by which barrier-to-autointegration factor regulates dynamics of nucleocytoplasmic leakage and membrane repair following nuclear envelope rupture.

The nuclear envelope (NE) creates a barrier between the cytosol and nucleus during interphase that is key for cellular compartmentalization and protecting genomic DNA. NE rupture can expose genomic DNA to the cytosol and allow admixture of the nuclear and cytosolic constituents, a proposed mechanism of cancer and NE-associated diseases. Barrier-to-autointegration factor (BAF) is a DNA-binding protein that localizes to NE ruptures where it recruits LEM-domain proteins, A-type lamins, and participates in rupture repair. To further reveal the mechanisms by which BAF responds to and aids in repairing NE ruptures, we investigated known properties of BAF including LEM domain binding, lamin binding, compartmentalization, phosphoregulation of DNA binding, and BAF dimerization. We demonstrate that it is the cytosolic population of BAF that functionally repairs NE ruptures, and phosphoregulation of BAF's DNA-binding that enables its ability to facilitate that repair. Interestingly, BAF's LEM or lamin binding activity appears dispensable for its role in functional repair. Furthermore, we demonstrate that BAF functions to reduce the extent of leakage though NE ruptures, suggesting that BAF effectively forms a diffusion barrier prior to NE repair. Collectively, these results enhances our knowledge of the mechanisms by which BAF responds to NE ruptures and facilitates their repair.

Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes.

The Drosophila ovary represents an outstanding model for investigating tissue homeostasis. Females continuously produce oocytes throughout their lifetime. However, as females age, fecundity declines, in part, due to changes in ovarian niche function and germline stem cell (GSC) homeostasis. Understanding the dynamics of GSC maintenance will provide needed insights into how coordinated tissue homeostasis is lost during aging. Critical regulators of GSC maintenance are proteins that reside in the nuclear lamina (NL), including the NL proteins emerin and Barrier-to-Autointegration Factor (BAF). Continued investigation of how emerin, BAF, and other NL proteins contribute to GSC function depends upon the availability of antibodies for NL proteins, a limiting resource. In this chapter, we discuss strategies for using clustered regularly interspaced short palindromic repeats (CRISPR) genomic editing to produce endogenously tagged NL genes to circumvent this obstacle, using the generation of the gfp-baf allele as an example. We describe strategies for validation of tagged alleles. Finally, we outline methods for immunohistochemical analysis of resulting tagged-NL proteins.

Dictyostelium spastin is involved in nuclear envelope dynamics during semi-closed mitosis.

Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae.

Advances in understanding the mechanisms of repairing damaged nuclear envelop.

The nuclear envelope (NE) separates genomic DNA from the cytoplasm in eukaryotes. The structure of the NE is dynamically altered not only in mitotic disassembly and reassembly but also during interphase. Recent studies have shown that the NE is frequently damaged by various cellular stresses that degenerate NE components and/or disrupt their functional interactions. These stresses are referred to as 'NE stress'. Accumulating evidence has demonstrated that NE stress potentially causes severe cellular dysfunctions, such as cell death and genome instability. In this review, the concept of NE stress, the processes repairing damage of the NE caused by NE stress, and the molecular mechanisms by which NE stress contributes to disease pathogenesis are introduced.

The ESCRT machinery directs quality control over inner nuclear membrane architecture.

The late-acting endosomal sorting complex required for transport (ESCRT) machinery has been implicated in facilitating the resealing of the nuclear envelope (NE) after mitosis, enabling compartmentalization of the genome away from the cytoplasm. Here, we leverage the stereotypic first division of the C. elegans embryo to identify additional functions of the ESCRT machinery in maintaining the structure of the inner nuclear membrane. Specifically, impaired ESCRT function results in a defect in the pruning of inner nuclear membrane invaginations, which arise normally during NE reformation and expansion. Additionally, in combination with a hypomorphic mutation that interferes with assembly of the underlying nuclear lamina, inhibition of ESCRT function significantly perturbs NE architecture and increases chromosome segregation defects, resulting in penetrant embryonic lethality. Our findings highlight links between ESCRT-mediated inner nuclear membrane remodeling, maintenance of nuclear envelope morphology, and the preservation of the genome during early development.

Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants.

The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication, and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin-binding protein Barrier-to-Autointegration Factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to the deformation of NL structure, and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate the contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf misregulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored the expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained misexpressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in the repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global misexpression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found upregulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

Disruption of nuclear envelope integrity as a possible initiating event in tauopathies.

The microtubule-associated protein tau is an abundant component of neurons of the central nervous system. In Alzheimer's disease and other neurodegenerative tauopathies, tau is found hyperphosphorylated and aggregated in neurofibrillary tangles. To obtain a better understanding of the cellular perturbations that initiate tau pathogenesis, we performed a CRISPR-Cas9 screen for genetic modifiers that enhance tau aggregation. This initial screen yielded three genes, BANF1, ANKLE2, and PPP2CA, whose inactivation promotes the accumulation of tau in a phosphorylated and insoluble form. In a complementary screen, we identified three additional genes, LEMD2, LEMD3, and CHMP7, that, when overexpressed, provide protection against tau aggregation. The proteins encoded by the identified genes are mechanistically linked and recognized for their roles in the maintenance and repair of the nuclear envelope. These results implicate the disruption of nuclear envelope integrity as a possible initiating event in tauopathies and reveal targets for therapeutic intervention.