Purine-Rich Element Binding Protein Alpha, a Nuclear Matrix Protein, Has a Role in Prostate Cancer Progression.

Solid tumors as well as leukemias and lymphomas show striking changes in nuclear structure including nuclear size and shape, the number and size of nucleoli, and chromatin texture. These alterations have been used in cancer diagnosis and might be related to the altered functional properties of cancer cells. The nuclear matrix (NM) represents the structural composition of the nucleus and consists of nuclear lamins and pore complexes, an internal ribonucleic protein network, and residual nucleoli. In the nuclear microenvironment, the NM is associated with multi-protein complexes, such as basal transcription factors, signaling proteins, histone-modifying factors, and chromatin remodeling machinery directly or indirectly through scaffolding proteins. Therefore, alterations in the composition of NM could result in altered DNA topology and changes in the interaction of various genes, which could then participate in a cascade of the cancer process. Using an androgen-sensitive prostate cancer cell line, LNCaP, and its androgen-independent derivative, LN96, conventional 2D-proteomic analysis of the NM proteins revealed that purine-rich element binding protein alpha (PURα) was detected in the NM proteins and differentially expressed between the cell lines. In this article, we will review the potential role of the molecule in prostate cancer.

MATR3's Role beyond the Nuclear Matrix: From Gene Regulation to Its Implications in Amyotrophic Lateral Sclerosis and Other Diseases.

Matrin-3 (MATR3) was initially discovered as a component of the nuclear matrix about thirty years ago. Since then, accumulating studies have provided evidence that MATR3 not only plays a structural role in the nucleus, but that it is also an active protein involved in regulating gene expression at multiple levels, including chromatin organization, DNA transcription, RNA metabolism, and protein translation in the nucleus and cytoplasm. Furthermore, MATR3 may play a critical role in various cellular processes, including DNA damage response, cell proliferation, differentiation, and survival. In addition to the revelation of its biological role, recent studies have reported MATR3's involvement in the context of various diseases, including neurodegenerative and neurodevelopmental diseases, as well as cancer. Moreover, sequencing studies of patients revealed a handful of disease-associated mutations in MATR3 linked to amyotrophic lateral sclerosis (ALS), which further elevated the gene's importance as a topic of study. In this review, we synthesize the current knowledge regarding the diverse functions of MATR3 in DNA- and RNA-related processes, as well as its involvement in various diseases, with a particular emphasis on ALS.

FLN-2 functions in parallel to linker of nucleoskeleton and cytoskeleton complexes and CDC-42/actin pathways during P-cell nuclear migration through constricted spaces in Caenorhabditis elegans.

Nuclear migration through narrow constrictions is important for development, metastasis, and proinflammatory responses. Studies performed in tissue culture cells have implicated linker of nucleoskeleton and cytoskeleton (LINC) complexes, microtubule motors, the actin cytoskeleton, and nuclear envelope repair machinery as important mediators of nuclear movements through constricted spaces. However, little is understood about how these mechanisms operate to move nuclei in vivo. In Caenorhabditis elegans larvae, six pairs of hypodermal P cells migrate from lateral to ventral positions through a constricted space between the body wall muscles and the cuticle. P-cell nuclear migration is mediated in part by LINC complexes using a microtubule-based pathway and by an independent CDC-42/actin-based pathway. However, when both LINC complex and actin-based pathways are knocked out, many nuclei still migrate, suggesting the existence of additional pathways. Here, we show that FLN-2 functions in a third pathway to mediate P-cell nuclear migration. The predicted N-terminal actin-binding domain in FLN-2 that is found in canonical filamins is dispensable for FLN-2 function; this and structural predictions suggest that FLN-2 does not function as a filamin. The immunoglobulin-like repeats 4-8 of FLN-2 were necessary for P-cell nuclear migration. Furthermore, in the absence of the LINC complex component unc-84, fln-2 mutants had an increase in P-cell nuclear rupture. We conclude that FLN-2 functions to maintain the integrity of the nuclear envelope in parallel with the LINC complex and CDC-42/actin-based pathways to move P-cell nuclei through constricted spaces.

Life outside the LINC complex - Do SUN proteins have LINC-independent functions?

Sad1 and UNC84 (SUN) and Klarsicht, ANC-1, and Syne homology (KASH) proteins interact at the nuclear periphery to form the linker of nucleoskeleton and cytoskeleton (LINC) complex, spanning the nuclear envelope (NE) and connecting the cytoskeleton with the nuclear interior. It is now well-documented that several cellular functions depend on LINC complex formation, including cell differentiation and migration. Intriguingly, recent studies suggest that SUN proteins participate in cellular processes where their association with KASH proteins may not be required. Building on this recent research, we elaborate on the hypothesis that SUN proteins may perform LINC-independent functions and discuss the modalities that may allow SUN proteins to function at the INM when they are not forming LINC complex.

Chromatin attachment to the nuclear matrix represses hypocotyl elongation in Arabidopsis thaliana.

The nuclear matrix is a nuclear compartment that has diverse functions in chromatin regulation and transcription. However, how this structure influences epigenetic modifications and gene expression in plants is largely unknown. In this study, we show that a nuclear matrix binding protein, AHL22, together with the two transcriptional repressors FRS7 and FRS12, regulates hypocotyl elongation by suppressing the expression of a group of genes known as SMALL AUXIN UP RNAs (SAURs) in Arabidopsis thaliana. The transcriptional repression of SAURs depends on their attachment to the nuclear matrix. The AHL22 complex not only brings these SAURs, which contain matrix attachment regions (MARs), to the nuclear matrix, but it also recruits the histone deacetylase HDA15 to the SAUR loci. This leads to the removal of H3 acetylation at the SAUR loci and the suppression of hypocotyl elongation. Taken together, our results indicate that MAR-binding proteins act as a hub for chromatin and epigenetic regulators. Moreover, we present a mechanism by which nuclear matrix attachment to chromatin regulates histone modifications, transcription, and hypocotyl elongation.

SUMOylation is enriched in the nuclear matrix and required for chromosome segregation.

As an important posttranslational modification, SUMOylation plays critical roles in almost all biological processes. Although it has been well-documented that SUMOylated proteins are mainly localized in the nucleus and have roles in chromatin-related processes, we showed recently that the SUMOylation machinery is actually enriched in the nuclear matrix rather than chromatin. Here, we provide compelling biochemical, cellular imaging and proteomic evidence that SUMOylated proteins are highly enriched in the nuclear matrix. We demonstrated that inactivation of SUMOylation by inhibiting SUMO-activating E1 enzyme or KO of SUMO-conjugating E2 enzyme UBC9 have only mild effect on nuclear matrix composition, indicating that SUMOylation is neither required for nuclear matrix formation nor for targeting proteins to nuclear matrix. Further characterization of UBC9 KO cells revealed that loss of SUMOylation did not result in significant DNA damage, but led to mitotic arrest and chromosome missegregation. Altogether, our study demonstrates that SUMOylated proteins are selectively enriched in the nuclear matrix and suggests a role of nuclear matrix in mediating SUMOylation and its regulated biological processes.

Force transmission and SUN-KASH higher-order assembly in the LINC complex models.

The linkers of the nucleoskeleton and cytoskeleton (LINC) complex comprises Sad-1 and UNC-84 (SUN) and Klarsicht, ANC-1, SYNE homology (KASH) domain proteins, whose conserved interactions provide a physical coupling between the cytoskeleton and the nucleoskeleton, thereby mediating the transfer of physical forces across the nuclear envelope. The LINC complex can perform distinct cellular functions by pairing various KASH domain proteins with the same SUN domain protein. Recent studies have suggested a higher-order assembly of SUN and KASH instead of a more widely accepted linear trimer model for the LINC complex. In the present study, we use molecular dynamics simulations to investigate the mechanism of force transfer across the two proposed models of LINC complex assembly, namely the 3:3 linear trimer model and the 6:6 higher-order model. Employing steered molecular dynamics simulations with various structures using forces at different rates and directions, we examine the structural stability of the two models under various biologically relevant conditions. Our results suggest that both models can withstand and transfer significant levels of force while retaining their structural integrity. However, the force response of various SUN/KASH assemblies depend on the force direction and pulling rates. Slower pulling rates result in higher mean square fluctuations of the 3:3 assembly compared to the fast pulling. Interestingly, the 6:6 assembly tends to provide an additional range of motion flexibility and might be more advantageous to the structural rigidity and pliability of the nuclear envelope. These findings offer insights into how the SUN and KASH proteins maintain the structural integrity of the nuclear membrane.

Biochemical Deconstruction and Reconstruction of Nuclear Matrix Reveals the Layers of Nuclear Organization.

Nuclear matrix (NuMat) is the fraction of the eukaryotic nucleus insoluble to detergents and high-salt extractions that manifests as a pan-nuclear fiber-granule network. NuMat consists of ribonucleoprotein complexes, members of crucial nuclear functional modules, and DNA fragments. Although NuMat captures the organization of nonchromatin nuclear space, very little is known about components organization within NuMat. To understand the organization of NuMat components, we subfractionated it with increasing concentrations of the chaotrope guanidinium hydrochloride (GdnHCl) and analyzed the proteomic makeup of the fractions. We observe that the solubilization of proteins at different concentrations of GdnHCl is finite and independent of the broad biophysical properties of the protein sequences. Looking at the extraction pattern of the nuclear envelope and nuclear pore complex, we surmise that this fractionation represents easily solubilized/loosely bound and difficultly solubilized/tightly bound components of NuMat. Microscopic analyses of the localization of key NuMat proteins across sequential GdnHCl extractions of in situ NuMat further elaborate on the divergent extraction patterns. Furthermore, we solubilized NuMat in 8M GdnHCl and upon removal of GdnHCl through dialysis, en masse renaturation leads to RNA-dependent self-assembly of fibrous structures. The major proteome component of the self-assembled fibers comes from the difficultly solubilized, tightly bound component. This fractionation of the NuMat reveals different organizational levels within it which may reflect the structural and functional organization of nuclear architecture.

Dynamic regulation of LINC complex composition and function across tissues and contexts.

The concept of mechanotransduction to the nucleus through a direct force transmission mechanism has fascinated cell biologists for decades. Central to such a mechanism is the linker of nucleoskeleton and cytoskeleton (LINC) complex, which spans the nuclear envelope to couple the cytoplasmic cytoskeleton to the nuclear lamina. In reality, there is not one LINC complex identity, but instead, a family of protein configurations of varied composition that exert both shared and unique functions. Regulated expression of LINC complex components, splice variants, and mechanoresponsive protein turnover mechanisms together shape the complement of LINC complex forms present in a given cell type. Disrupting specific gene(s) encoding LINC complex components therefore gives rise to a range of organismal defects. Moreover, evidence suggests that the mechanical environment remodels LINC complexes, providing a feedback mechanism by which cellular context influences the integration of the nucleus into the cytoskeleton. In particular, evidence for crosstalk between the nuclear and cytoplasmic intermediate filament networks communicated through the LINC complex represents an emerging theme in this active area of ongoing investigation.

Evolutionarily conserved protein motifs drive interactions between the plant nucleoskeleton and nuclear pores.

The nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear shape and gene expression. To understand how the Arabidopsis (Arabidopsis thaliana) nucleoskeleton physically connects to the nuclear periphery in plants, we investigated the Arabidopsis nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. We identified 3 conserved peptide motifs within the N-terminal region of KAKU4, which are required for intermolecular interactions of KAKU4 with itself, interaction with the nucleoskeleton protein CROWDED NUCLEI (CRWN), localization at the nuclear periphery, and nuclear elongation in differentiated tissues. Unexpectedly, we find these motifs to be present also in NUP82 and NUP136, 2 plant-specific nucleoporins from the nuclear pore basket. We further show that NUP82, NUP136, and KAKU4 have a common evolutionary history predating nonvascular land plants with KAKU4 mainly localizing outside the nuclear pore suggesting its divergence from an ancient nucleoporin into a new nucleoskeleton component. Finally, we demonstrate that both NUP82 and NUP136, through their shared N-terminal motifs, interact with CRWN and KAKU4 proteins revealing the existence of a physical continuum between the nuclear pore and the nucleoskeleton in plants.

The LINC Complex Inhibits Excessive Chromatin Repression.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. We show here that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, we suggest that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.

Nuclear architecture and the structural basis of mitotic memory.

The nucleus is a complex organelle that hosts the genome and is essential for vital processes like DNA replication, DNA repair, transcription, and splicing. The genome is non-randomly organized in the three-dimensional space of the nucleus. This functional sub-compartmentalization was thought to be organized on the framework of nuclear matrix (NuMat), a non-chromatin scaffold that functions as a substratum for various molecular processes of the nucleus. More recently, nuclear bodies or membrane-less subcompartments of the nucleus are thought to arise due to phase separation of chromatin, RNA, and proteins. The nuclear architecture is an amalgamation of the relative organization of chromatin, epigenetic landscape, the nuclear bodies, and the nucleoskeleton in the three-dimensional space of the nucleus. During mitosis, the nucleus undergoes drastic changes in morphology to the degree that it ceases to exist as such; various nuclear components, including the envelope that defines the nucleus, disintegrate, and the chromatin acquires mitosis-specific epigenetic marks and condenses to form chromosome. Upon mitotic exit, chromosomes are decondensed, re-establish hierarchical genome organization, and regain epigenetic and transcriptional status similar to that of the mother cell. How this mitotic memory is inherited during cell division remains a puzzle. NuMat components that are a part of the mitotic chromosome in the form of mitotic chromosome scaffold (MiCS) could potentially be the seeds that guide the relative re-establishment of the epigenome, chromosome territories, and the nuclear bodies. Here, we synthesize the advances towards understanding cellular memory of nuclear architecture across mitosis and propose a hypothesis that a subset of NuMat proteome essential for nucleation of various nuclear bodies are retained in MiCS to serve as seeds of mitotic memory, thus ensuring the daughter cells re-establish the complex status of nuclear architecture similar to that of the mother cells, thereby maintaining the pre-mitotic transcriptional status.

The linker of nucleoskeleton and cytoskeleton complex is required for X-ray-induced epithelial-mesenchymal transition.

The linker of nucleoskeleton and cytoskeleton (LINC) complex has been implicated in various functions of the nuclear envelope, including nuclear migration, mechanotransduction and DNA repair. We previously revealed that the LINC complex component Sad1 and UNC84 domain containing 1 (SUN1) is required for sublethal-dose X-ray-enhanced cell migration and invasion. This study focused on epithelial-mesenchymal transition (EMT), which contributes to cell migration. Hence, the present study aimed to examine whether sublethal-dose X-irradiation induces EMT and whether LINC complex component SUN1 is involved in low-dose X-ray-induced EMT. This study showed that low-dose (0.5 Gy or 2 Gy) X-irradiation induced EMT in human breast cancer MDA-MB-231 cells. Additionally, X-irradiation increased the expression of SUN1. Therefore, SUN1 was depleted using siRNA. In SUN1-depleted cells, low-dose X-irradiation did not induce EMT. In addition, although the SUN1 splicing variant SUN1_916-depleted cells (containing 916 amino acids [AA] of SUN1) were induced EMT by low-dose X-irradiation like as non-transfected control cells, SUN1_888-depleted cells (which encodes 888 AA) were not induced EMT by low-dose X-irradiation. Moreover, since the Wnt/ß-catenin signaling pathway regulates E-cadherin expression via the expression of the E-cadherin repressor Snail, the expression of ß-catenin after X-irradiation was examined. After 24 hours of irradiation, ß-catenin expression increased in non-transfected cells or SUN1_916-depleted cells, whereas ß-catenin expression remained unchanged and did not increase in SUN1- or SUN1_888-depleted cells. Therefore, in this study, we found that low-dose X-irradiation induces EMT, and LINC complex component SUN1, especially SUN1_888, is required for X-ray-induced EMT via activation of the Wnt/ß-catenin signaling pathway.

SUN2 regulates mitotic duration in response to extracellular matrix rigidity.

How cells adjust their growth to the spatial and mechanical constraints of their surrounding environment is central to many aspects of biology. Here, we examined how extracellular matrix (ECM) rigidity affects cell division. We found that cells divide more rapidly when cultured on rigid substrates. While we observed no effect of ECM rigidity on rounding or postmitotic spreading duration, we found that changes in matrix stiffness impact mitosis progression. We noticed that ECM elasticity up-regulates the expression of the linker of nucleoskeleton and cytoskeleton (LINC) complex component SUN2, which in turn promotes metaphase-to-anaphase transition by acting on mitotic spindle formation, whereas when cells adhere to soft ECM, low levels of SUN2 expression perturb astral microtubule organization and delay the onset of anaphase.

The LINC Complex Assists the Nuclear Import of Mechanosensitive Transcriptional Regulators.

Mechanical forces play pivotal roles in directing cell functions and fate. To elicit gene expression, either intrinsic or extrinsic mechanical information are transmitted into the nucleus beyond the nuclear envelope via at least two distinct pathways, possibly more. The first and well-known pathway utilizes the canonical nuclear transport of mechanoresponsive transcriptional regulators through the nuclear pore complex, which is an exclusive route for macromolecular trafficking between the cytoplasm and nucleoplasm. The second pathway depends on the linker of the nucleoskeleton and cytoskeleton (LINC) complex, which is a molecular bridge traversing the nuclear envelope between the cytoskeleton and nucleoskeleton. This protein complex is a central component in mechanotransduction at the nuclear envelope that transmits mechanical information from the cytoskeleton into the nucleus to influence the nuclear structure, nuclear stiffness, chromatin organization, and gene expression. Besides the mechanical force transducing function, recent increasing evidence shows that the LINC complex plays a role in controlling nucleocytoplasmic transport of mechanoresponsive transcriptional regulators. Here we discuss recent findings regarding the contribution of the LINC complex to the regulation of intracellular localization of the most-notable mechanosensitive transcriptional regulators, ß-catenin, YAP, and TAZ.

Short-term hypoxia triggers ROS and SAFB mediated nuclear matrix and mRNA splicing remodeling.

The cellular response to hypoxia, in addition to HIF-dependent transcriptional reprogramming, also involves less characterized transcription-independent processes, such as alternative splicing of the VEGFA transcript leading to the production of the proangiogenic VEGF form. We now show that this event depends on reorganization of the splicing machinery, triggered after short-term hypoxia by ROS production and intranuclear redistribution of the nucleoskeletal proteins SAFB1/2. Exposure to low oxygen causes fast dissociation of SAFB1/2 from the nuclear matrix, which is reversible, inhibited by antioxidant treatment, and also observed under normoxia when the mitochondrial electron transport chain is blocked. This is accompanied by altered interactions between SAFB1/2 and the splicing machinery, translocation of kinase SRPK1 to the cytoplasm, and dephosphorylation of RS-splicing factors. Depletion of SAFB1/2 under normoxia phenocopies the hypoxic and ROS-mediated switch in VEGF mRNA splicing. These data suggest that ROS-dependent remodeling of the nuclear architecture can promote production of splicing variants that facilitate adaptation to hypoxia.

Structural and developmental dynamics of Matrix associated regions in Drosophila melanogaster genome.

BACKGROUND: Eukaryotic genome is compartmentalized into structural and functional domains. One of the concepts of higher order organization of chromatin posits that the DNA is organized in constrained loops that behave as independent functional domains. Nuclear Matrix (NuMat), a ribo-proteinaceous nucleoskeleton, provides the structural basis for this organization. DNA sequences located at base of the loops are known as the Matrix Attachment Regions (MARs). NuMat relates to multiple nuclear processes and is partly cell type specific in composition. It is a biochemically defined structure and several protocols have been used to isolate the NuMat where some of the steps have been critically evaluated. These sequences play an important role in genomic organization it is imperative to know their dynamics during development and differentiation. RESULTS: Here we look into the dynamics of MARs when the preparation process is varied and during embryonic development of D. melanogaster. A subset of MARs termed as "Core-MARs" present abundantly in pericentromeric heterochromatin, are constant unalterable anchor points as they associate with NuMat through embryonic development and are independent of the isolation procedure. Euchromatic MARs are dynamic and reflect the transcriptomic profile of the cell. New MARs are generated by nuclear stabilization, and during development, mostly at paused RNA polymerase II promoters. Paused Pol II MARs depend on RNA transcripts for NuMat association. CONCLUSIONS: Our data reveals the role of MARs in functionally dynamic nucleus and contributes to the current understanding of nuclear architecture in genomic context.

The ribonucleoprotein network of the nucleus: a historical perspective.

There is a long experimental history supporting the principle that RNA is essential for normal nuclear and chromatin architecture. Most of the genome is transcribed into RNA but only 2% of the sequence codes for proteins. In the nucleus, most non-coding RNA, packaged in proteins, is bound into structures including chromatin and a non-chromatin scaffolding, the nuclear matrix, which was first observed by electron microscopy. Removing nuclear RNA or inhibiting its transcription causes the condensation of chromatin, showing the importance of RNA in spatially and functionally organizing the genome. Today, powerful techniques for the molecular characterization of RNA and for mapping its spatial organization in the nucleus have provided molecular detail to these principles.

Actin nucleoskeleton in embryonic development and cellular differentiation.

Dynamic assembly and disassembly of actin proteins play a key role in the cytoskeleton, but the cellular functions of actin are not only restricted to the cytoplasmic compartment. Recent studies have shown that actin spatiotemporally changes its polymerized state in the nucleus as well and such dynamic nature of actin is relevant to key nuclear events including gene expression, DNA damage response and chromatin organization. In this review, we highlight emerging roles of actin in the nuclear compartment especially in the context of embryonic development and cellular differentiation. We first explain how the actin nucleoskeleton can be formed and function in cells. Notably, nuclear actin dynamics are greatly altered when cell fates change, such as after fertilization and T cell differentiation. We discuss how the dynamic actin nucleoskeleton contributes to accomplishing developmental programs.

Nuclear organization by satellite DNA, SAF-A/hnRNPU and matrix attachment regions.

The need of large-scale chromatin organization in the nucleus has become more and more appreciated. The higher order nuclear organization ultimately regulate a plethora of biological processes including transcription, DNA replication, and DNA repair. In this context, it is of critical importance to understand the mechanisms that allow higher order nuclear organization. Scaffold Attachment Factor A (SAF-A/hnRNPU), which was originally identified as the component of nuclear matrix, has emerged as an important regulator of higher order nuclear organization. It is shown that SAF-A/hnRNPU binds to tandem repeats (TRs) and scaffold/matrix attachment regions (S/MAR) in a sequence-non-specific, but structure-specific manner (e.g. DNA curvature). Recent studies showed that SAF-A interacts with chromatin-associated RNAs (caRNAs) to regulate interphase chromatin structures in a transcription-dependent manner. It is proposed that SAF-A/hnRNPU and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes chromatin in a large scale. The common structural features of S/MAR and pericentromeric (periCEN) TR promotes SAF-A-mediated association with each other. Collectively a model is presented wherein SAF-A/hnRNPU and periCEN TR are the key players in large-scale nuclear organization that supports general transcription.