CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses.

Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in heat shock protein (hsp) transcription in fly ovaries, but the mechanisms remain unclear. Here we demonstrate that in mammalian cells, MRTFs are dispensable for hsp gene induction. However, the widely used small molecule inhibitors of MRTF/SRF transcription pathway, derived from CCG-1423, efficiently inhibit hsp gene transcription in both fly and mammalian cells also in the absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF/SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb.

Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9, and is not involved with releasing positive transcription elongation factor b from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

Chromatin Immunoprecipitation Experiments from Drosophila Ovaries.

Chromatin is composed of DNA and its associated proteins, and has an essential role in all cellular processes, including those taking place during Drosophila oogenesis. In order to understand the molecular basis of chromatin-based processes, such as transcription, it is essential to be able to study how and when different proteins, such as transcription factors, histones and RNA polymerases, interact with chromatin. One of the most popular methods to study this is chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Here, we describe a ChIP-seq protocol that has been optimized for Drosophila ovaries, focusing on sample preparation through preliminary data processing.

Regulation of nuclear actin levels and MRTF/SRF target gene expression during PC6.3 cell differentiation.

Actin has important functions in both cytoplasm and nucleus of the cell, with active nuclear transport mechanisms maintaining the cellular actin balance. Nuclear actin levels are subject to regulation during many cellular processes from cell differentiation to cancer. Here we show that nuclear actin levels increase upon differentiation of PC6.3 cells towards neuron-like cells. Photobleaching experiments demonstrate that this increase is due to decreased nuclear export of actin during cell differentiation. Increased nuclear actin levels lead to decreased nuclear localization of MRTF-A, a well-established transcription cofactor of SRF. In line with MRTF-A localization, transcriptomics analysis reveals that MRTF/SRF target gene expression is first transiently activated, but then substantially downregulated during PC6.3 cell differentiation. This study therefore describes a novel cellular context, where regulation of nuclear actin is utilized to tune MRTF/SRF target gene expression during cell differentiation.

Nuclear translocation of vitellogenin in the honey bee (Apis mellifera).

Vitellogenin (Vg) is a conserved protein used by nearly all oviparous animals to produce eggs. It is also pleiotropic and performs functions in oxidative stress resistance, immunity, and, in honey bees, behavioral development of the worker caste. It has remained enigmatic how Vg affects multiple traits. Here, we asked whether Vg enters the nucleus and acts via DNA-binding. We used cell fractionation, immunohistology, and cell culture to show that a structural subunit of honey bee Vg translocates into cell nuclei. We then demonstrated Vg-DNA binding theoretically and empirically with prediction software and chromatin immunoprecipitation with sequencing (ChIP-seq), finding binding sites at genes influencing immunity and behavior. Finally, we investigated the immunological and enzymatic conditions affecting Vg cleavage and nuclear translocation and constructed a 3D structural model. Our data are the first to show Vg in the nucleus and suggest a new fundamental regulatory role for this ubiquitous protein. Supplementary information: The online version contains supplementary material available at 10.1007/s13592-022-00914-9.

Lamina-associated polypeptide 2α is required for intranuclear MRTF-A activity.

Myocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for efficient recruitment of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.

SARS-CoV-2-host proteome interactions for antiviral drug discovery.

Treatment options for COVID-19, caused by SARS-CoV-2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS-CoV-2-host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS-CoV-2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP-MS) and the complementary proximity-based labeling MS method (BioID-MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS-CoV-2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image-based drug screen with infectious SARS-CoV-2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein-protein interactions.

ß2-Integrin Adhesion Regulates Dendritic Cell Epigenetic and Transcriptional Landscapes to Restrict Dendritic Cell Maturation and Tumor Rejection.

Dendritic cells (DC), the classic antigen-presenting cells of the immune system, switch from an adhesive, phagocytic phenotype in tissues, to a mature, nonadhesive phenotype that enables migration to lymph nodes to activate T cells and initiate antitumor responses. Monocyte-derived DCs are used in cancer immunotherapy, but their clinical efficacy is limited. Here, we show that cultured bone marrow-derived DCs (BM-DC) expressing dysfunctional ß2-integrin adhesion receptors displayed enhanced tumor rejection capabilities in B16.OVA and B16-F10 melanoma models. This was associated with an increased CD8+ T-cell response. BM-DCs expressing dysfunctional ß2-integrins or manipulated to disrupt integrin adhesion or integrin/actin/nuclear linkages displayed spontaneous maturation in ex vivo cultures (increased costimulatory marker expression, IL12 production, and 3D migration capabilities). This spontaneous maturation was associated with an altered DC epigenetic/transcriptional profile, including a global increase in chromatin accessibility and H3K4me3/H3K27me3 histone methylation. Genome-wide analyses showed that H3K4me3 methylation was increased on DC maturation genes, such as CD86, Il12, Ccr7, and Fscn1, and revealed a role for a transcription factor network involving Ikaros and RelA in the integrin-regulated phenotype of DCs. Manipulation of the integrin-regulated epigenetic landscape in wild-type ex vivo-cultured BM-DCs enhanced their functionality in tumor rejection in vivo. Thus, ß2-integrin-mediated adhesion to the extracellular environment plays an important role in restricting DC maturation and antitumor responses through regulation of the cellular epigenetic and transcriptional landscape. Targeting ß2-integrins could therefore be a new strategy to improve the performance of current DC-based cancer immunotherapies.

RTN4B interacting protein FAM134C promotes ER membrane curvature and has a functional role in autophagy.

The endoplasmic reticulum (ER) is composed of a controlled ratio of sheets and tubules, which are maintained by several proteins with multiple functions. Reticulons (RTNs), especially RTN4, and DP1/Yop1p family members are known to induce ER membrane curvature. RTN4B is the main RTN4 isoform expressed in nonneuronal cells. In this study, we identified FAM134C as a RTN4B interacting protein in mammalian, nonneuronal cells. FAM134C localized specifically to the ER tubules and sheet edges. Ultrastructural analysis revealed that overexpression of FAM134C induced the formation of unbranched, long tubules or dense globular structures composed of heavily branched narrow tubules. In both cases, tubules were nonmotile. ER tubulation was dependent on the reticulon homology domain (RHD) close to the N-terminus. FAM134C plays a role in the autophagy pathway as its level elevated significantly upon amino acid starvation but not during ER stress. Moreover, FAM134C depletion reduced the number and size of autophagic structures and the amount of ER as a cargo within autophagic structures under starvation conditions. Dominant-negative expression of FAM134C forms with mutated RHD or LC3 interacting region also led to a reduced number of autophagic structures. Our results suggest that FAM134C provides a link between regulation of ER architecture and ER turnover by promoting ER tubulation required for subsequent ER fragmentation and engulfment into autophagosomes.

Emerging Properties and Functions of Actin and Actin Filaments Inside the Nucleus.

Recent years have provided considerable insights into the dynamic nature of the cell nucleus, which is constantly reorganizing its genome, controlling its size and shape, as well as spatiotemporally orchestrating chromatin remodeling and transcription. Remarkably, it has become clear that the ancient and highly conserved cytoskeletal protein actin plays a crucial part in these processes. However, the underlying mechanisms, regulations, and properties of actin functions inside the nucleus are still not well understood. Here we summarize the diverse and distinct roles of monomeric and filamentous actin as well as the emerging roles for actin dynamics inside the nuclear compartment for genome organization and nuclear architecture.

Regulation of nuclear actin dynamics in development and disease.

Actin has essential functions both in the cytoplasm and in the nucleus, where it has been linked to key nuclear processes, from transcription to DNA damage response. The multifunctional nature of actin suggests that the cell must contain mechanisms to accurately control the cellular actin balance. Indeed, recent results have demonstrated that nuclear actin levels fluctuate to regulate the transcriptional activity of the cell and that controlled nuclear actin polymerization is required for transcription activation, cell cycle progression, and DNA repair. Intriguingly, aberrant nuclear actin regulation has been observed, for example, in cancer, signifying the importance of this process for cellular homeostasis. This review discussed the latest research on how nuclear actin is regulated, and how this influences actin-dependent nuclear processes.

Optogenetic regulation of endogenous proteins.

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.

Nuclear actin dynamics in gene expression and genome organization.

Although best known from its functions in the cytoplasm, actin also localizes to the cell nucleus, where it has been linked to many essential functions from regulation of gene expression to maintenance of genomic integrity. While majority of cytoplasmic functions of actin depend on controlled actin polymerization, in the nucleus both actin monomers and filaments have their own specific roles. Actin monomers are core components of several chromatin remodeling and modifying complexes and can also regulate the activity of specific transcription factors, while actin filaments have been linked to DNA damage response and cell cycle progression. Consequently the balance between monomeric and filamentous actin must be precise controlled also in the nucleus, since their effects are dynamically coupled. In this review, we discuss the recent data on how actin dynamics is regulated within the nucleus and how this influences the different nuclear processes dependent on actin.

Cytoskeletal proteins in the cell nucleus: a special nuclear actin perspective.

The emerging role of cytoskeletal proteins in the cell nucleus has become a new frontier in cell biology. Actin and actin-binding proteins regulate chromatin and gene expression, but importantly they are beginning to be essential players in genome organization. These actin-based functions contribute to genome stability and integrity while affecting DNA replication and global transcription patterns. This is likely to occur through interactions of actin with nuclear components including nuclear lamina and subnuclear organelles. An exciting future challenge is to understand how these actin-based genome-wide mechanisms may regulate development and differentiation by interfering with the mechanical properties of the cell nucleus and how regulated actin polymerization plays a role in maintaining nuclear architecture. With a special focus on actin, here we summarize how cytoskeletal proteins operate in the nucleus and how they may be important to consolidate nuclear architecture for sustained gene expression or silencing.

Nuclear actin interactome analysis links actin to KAT14 histone acetyl transferase and mRNA splicing.

In addition to its essential functions within the cytoskeleton, actin also localizes to the cell nucleus, where it is linked to many important nuclear processes from gene expression to maintenance of genomic integrity. However, the molecular mechanisms by which actin operates in the nucleus remain poorly understood. Here, we have used two complementary mass spectrometry (MS) techniques, AP-MS and BioID, to identify binding partners for nuclear actin. Common high-confidence interactions highlight the role of actin in chromatin-remodeling complexes and identify the histone-modifying complex human Ada-Two-A-containing (hATAC) as a novel actin-containing nuclear complex. Actin binds directly to the hATAC subunit KAT14, and modulates its histone acetyl transferase activity in vitro and in cells. Transient interactions detected through BioID link actin to several steps of transcription as well as to RNA processing. Alterations in nuclear actin levels disturb alternative splicing in minigene assays, likely by affecting the transcription elongation rate. This interactome analysis thus identifies both novel direct binding partners and functional roles for nuclear actin, as well as forms a platform for further mechanistic studies on how actin operates during essential nuclear processes.This article has an associated First Person interview with the first author of the paper.

Nuclear Actin Is Required for Transcription during Drosophila Oogenesis.

Actin has been linked to processes spanning the whole gene expression cascade, from regulating specific transcription factors, such as myocardin-related transcription factor, to chromatin remodeling and RNA polymerase function. However, whether actin controls the transcription of only specific genes or has a global role in gene expression has remained elusive. Our genome-wide analysis reveals, for the first time, that actin interacts with essentially all transcribed genes in Drosophila ovaries. Actin co-occupies the majority of gene promoters together with Pol II, and on highly expressed genes, these two proteins also associate with gene bodies. Mechanistically, actin is required for Pol II recruitment to gene bodies, and manipulation of nuclear transport factors for actin leads to the decreased expression of eggshell genes. Collectively, these results uncover a global role for actin in transcription and demonstrate the in vivo importance of balanced nucleocytoplasmic shuttling of actin in the transcriptional control of a developmental process.

Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization.

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms.

F-actin organizes the nucleus.

After mitosis, the nucleus must be rebuilt and chromatin decondensed to permit interphase genomic functions, but decondensation mechanisms are poorly understood. Now, the traditional cytoskeletal protein actin is shown to form transient nuclear filaments that are required for chromatin decondensation and nuclear expansion at mitotic exit.

The actin binding cytoskeletal protein Moesin is involved in nuclear mRNA export.

Current models imply that the evolutionarily conserved, actin-binding Ezrin-Radixin-Moesin (ERM) proteins perform their activities at the plasma membrane by anchoring membrane proteins to the cortical actin network. Here we show that beside its cytoplasmic functions, the single ERM protein of Drosophila, Moesin, has a novel role in the nucleus. The activation of transcription by heat shock or hormonal treatment increases the amount of nuclear Moesin, indicating biological function for the protein in the nucleus. The distribution of Moesin in the nucleus suggests a function in transcription and the depletion of mRNA export factors Nup98 or its interacting partner, Rae1, leads to the nuclear accumulation of Moesin, suggesting that the nuclear function of the protein is linked to mRNA export. Moesin localizes to mRNP particles through the interaction with the mRNA export factor PCID2 and knock down of Moesin leads to the accumulation of mRNA in the nucleus. Based on our results we propose that, beyond its well-known, manifold functions in the cytoplasm, the ERM protein of Drosophila is a new, functional component of the nucleus where it participates in mRNA export.