Tumor-induced reshuffling of lipid composition on the endoplasmic reticulum membrane sustains macrophage survival and pro-tumorigenic activity.

Tumor-associated macrophages (TAMs) display pro-tumorigenic phenotypes for supporting tumor progression in response to microenvironmental cues imposed by tumor and stromal cells. However, the underlying mechanisms by which tumor cells instruct TAM behavior remain elusive. Here, we uncover that tumor-cell-derived glucosylceramide stimulated unconventional endoplasmic reticulum (ER) stress responses by inducing reshuffling of lipid composition and saturation on the ER membrane in macrophages, which induced IRE1-mediated spliced XBP1 production and STAT3 activation. The cooperation of spliced XBP1 and STAT3 reinforced the pro-tumorigenic phenotype and expression of immunosuppressive genes. Ablation of XBP1 expression with genetic manipulation or ameliorating ER stress responses by facilitating LPCAT3-mediated incorporation of unsaturated lipids to the phosphatidylcholine hampered pro-tumorigenic phenotype and survival in TAMs. Together, we uncover the unexpected roles of tumor-cell-produced lipids that simultaneously orchestrate macrophage polarization and survival in tumors via induction of ER stress responses and reveal therapeutic targets for sustaining host antitumor immunity.

Approaching Protein Aggregation and Structural Dynamics by Equilibrium and Nonequilibrium Paramagnetic Perturbation.

PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation) is the presented nuclear magnetic resonance (NMR) approach to identify at once the location of proteins' exposed surface, hindered accessibility, and exchange processes occurring on a µs-ms time scale. In addition to mapping the protein surface accessibility, the application of this method under specific conditions makes it possible to distinguish conformational mobility and chemical exchange processes, thereby providing an alternative to characterization by more demanding techniques (transverse relaxation dispersion, saturation transfer, and high-pressure NMR). Moreover, its high sensitivity enables studying samples at low, physiologically more relevant concentrations. Association, dynamics, and oligomerization are addressed by PENELOP for a component of SARS-CoV-2 replication transcription complex and an amyloidogenic protein.

ß-actin regulates a heterochromatin landscape essential for optimal induction of neuronal programs during direct reprograming.

During neuronal development, ß-actin serves an important role in growth cone mediated axon guidance. Consistent with this notion, in vivo ablation of the ß-actin gene leads to abnormalities in the nervous system. However, whether ß-actin is involved in the regulation of neuronal gene programs is not known. In this study, we directly reprogramed ß-actin+/+ WT, ß-actin+/- HET and ß-actin-/- KO mouse embryonic fibroblast (MEFs) into chemically induced neurons (CiNeurons). Using RNA-seq analysis, we profiled the transcriptome changes among the CiNeurons. We discovered that induction of neuronal gene programs was impaired in KO CiNeurons in comparison to WT ones, whereas HET CiNeurons showed an intermediate levels of induction. ChIP-seq analysis of heterochromatin markers demonstrated that the impaired expression of neuronal gene programs correlated with the elevated H3K9 and H3K27 methylation levels at gene loci in ß-actin deficient MEFs, which is linked to the loss of chromatin association of the BAF complex ATPase subunit Brg1. Together, our study shows that heterochromatin alteration in ß-actin null MEFs impedes the induction of neuronal gene programs during direct reprograming. These findings are in line with the notion that H3K9Me3-based heterochromatin forms a major epigenetic barrier during cell fate change.

ß-Actin-dependent global chromatin organization and gene expression programs control cellular identity.

During differentiation and development, cell fate and identity are established by waves of genetic reprogramming. Although the mechanisms are largely unknown, during these events, dynamic chromatin reorganization is likely to ensure that multiple genes involved in the same cellular functions are coregulated, depending on the nuclear environment. In this study, using high-content screening of embryonic fibroblasts from a ß-actin knockout (KO) mouse, we found major chromatin rearrangements and changes in histone modifications, such as methylated histone (H)3-lysine-(K)9. Genome-wide H3K9 trimethylation-(Me)3 landscape changes correlate with gene up- and down-regulation in ß-actin KO cells. Mechanistically, we found loss of chromatin association by the Brahma-related gene ( Brg)/Brahma-associated factor (BAF) chromatin remodeling complex subunit Brg1 in the absence of ß-actin. This actin-dependent chromatin reorganization was concomitant with the up-regulation of sets of genes involved in angiogenesis, cytoskeletal organization, and myofibroblast features in ß-actin KO cells. Some of these genes and phenotypes were gained in a ß-actin dose-dependent manner. Moreover, reintroducing a nuclear localization signal-containing ß-actin in the knockout cells affected nuclear features and gene expression. Our results suggest that, by affecting the genome-wide organization of heterochromatin through the chromatin-binding activity of the BAF complex, ß-actin plays an essential role in the determination of gene expression programs and cellular identity.-Xie, X., Almuzzaini, B., Drou, N., Kremb, S., Yousif, A., Östlund Farrants, A.-K., Gunsalus, K., Percipalle, P. ß-Actin-dependent global chromatin organization and gene expression programs control cellular identity.

Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

Elevated transforming growth factor ß signaling activation in ß-actin-knockout mouse embryonic fibroblasts enhances myofibroblast features.

Signaling by the transforming growth factor-ß (TGF-ß) is an essential pathway regulating a variety of cellular events. TGF-ß is produced as a latent protein complex and is required to be activated before activating the receptor. The mechanical force at the cell surface is believed to be a mechanism for latent TGF-ß activation. Using ß-actin null mouse embryonic fibroblasts as a model, in which actin cytoskeleton and cell-surface biophysical features are dramatically altered, we reveal increased TGF-ß1 activation and the upregulation of TGF-ß target genes. In ß-actin null cells, we show evidence that the enhanced TGF-ß signaling relies on the active utilization of latent TGF-ß1 in the cell culture medium. TGF-ß signaling activation contributes to the elevated reactive oxygen species production, which is likely mediated by the upregulation of Nox4. The previously observed myofibroblast phenotype of ß-actin null cells is inhibited by TGF-ß signaling inhibition, while the expression of actin cytoskeleton genes and angiogenic phenotype are not affected. Together, our study shows a scenario that the alteration of the actin cytoskeleton and the consequent changes in cellular biophysical features lead to changes in cell signaling process such as TGF-ß activation, which in turn contributes to the enhanced myofibroblast phenotype.

An actin-based nucleoskeleton involved in gene regulation and genome organization.

In eukaryotic cells gene regulation is dependent on global genome organization. This is achieved, in response to favorable environmental conditions, through spatial redistribution of chromatin and changes in global epigenetic levels. This eventually drives movement of gene-rich chromatin loops and formation of DNA loops, consolidating neighborhoods of gene expression and silencing. One of the challenges for future work is to examine how these neighborhoods are formed and whether they host genes involved in the same cellular functions for sustained expression or silencing over time. In the present review, we summarize evidence that actin and actin-associated proteins regulate gene activity. Furthermore we discuss how these specific nuclear tasks in which actin is engaged are important to organize and consolidate the mammalian genome, ensuring gene activation and repression of gene programs important to establish cellular identity. We propose that these mechanisms are essential to control cellular development and differentiation.

The relative composition of actin isoforms regulates cell surface biophysical features and cellular behaviors.

BACKGROUND: Cell surface mechanics is able to physically and biomechanically affect cell shape and motility, vesicle trafficking and actin dynamics. The biophysical properties of cell surface are strongly influenced by cytoskeletal elements. In mammals, tissue-specific expression of six actin isoforms is thought to confer differential biomechanical properties. However, the relative contribution of actin isoforms to cell surface properties is not well understood. Here, we sought to investigate whether and how the composition of endogenous actin isoforms directly affects the biomechanical features of cell surface and cellular behavior. METHODS: We used fibroblasts isolated from wild type (WT), heterozygous (HET) and from knockout (KO) mouse embryos where both ß-actin alleles are not functional. We applied a combination of genome-wide analysis and biophysical methods such as RNA-seq and atomic force microscopy. RESULTS: We found that endogenous ß-actin levels are essential in controlling cell surface stiffness and pull-off force, which was not compensated by the up-regulation of other actin isoforms. The variations of surface biophysical features and actin contents were associated with distinct cell behaviors in 2D and 3D WT, HET and KO cell cultures. Since ß-actin in WT cells and smooth muscle α-actin up-regulated in KO cells showed different organization patterns, our data support the differential localization and organization as a mechanism to regulate the biophysical properties of cell surface by actin isoforms. CONCLUSIONS: We propose that variations in actin isoforms composition impact on the biophysical features of cell surface and cause the changes in cell behavior.

In ß-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects.

Actin and nuclear myosin 1 (NM1) are regulators of transcription and chromatin organization. Using a genome-wide approach, we report here that ß-actin binds intergenic and genic regions across the mammalian genome, associated with both protein-coding and rRNA genes. Within the rDNA, the distribution of ß-actin correlated with NM1 and the other subunits of the B-WICH complex, WSTF and SNF2h. In ß-actin(-/-) mouse embryonic fibroblasts (MEFs), we found that rRNA synthesis levels decreased concomitantly with drops in RNA polymerase I (Pol I) and NM1 occupancies across the rRNA gene. Reintroduction of wild-type ß-actin, in contrast to mutated forms with polymerization defects, efficiently rescued rRNA synthesis underscoring the direct role for a polymerization-competent form of ß-actin in Pol I transcription. The rRNA synthesis defects in the ß-actin(-/-) MEFs are a consequence of epigenetic reprogramming with up-regulation of the repressive mark H3K4me1 (monomethylation of lys4 on histone H3) and enhanced chromatin compaction at promoter-proximal enhancer (T0 sequence), which disturb binding of the transcription factor TTF1. We propose a novel genome-wide mechanism where the polymerase-associated ß-actin synergizes with NM1 to coordinate permissive chromatin with Pol I transcription, cell growth, and proliferation.-Almuzzaini, B., Sarshad, A. A. , Rahmanto, A. S., Hansson, M. L., Von Euler, A., Sangfelt, O., Visa, N., Farrants, A.-K. Ö., Percipalle, P. In ß-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects.

Glycogen synthase kinase (GSK) 3ß phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells.

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3ß phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3ß selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3ß-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3ß directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3ß-mediated phosphorylation of NM1 is required for pol I transcription activation.

New insights into co-transcriptional sorting of mRNA for cytoplasmic transport during development.

During mRNA biogenesis regulation of mRNA transport and localization is an essential step. It guarantees asymmetric protein distribution across the cell necessary for specialized cellular functions, and it is a driving force for cellular differentiation and development. mRNA transport and localization depends on the interactions between cis-acting elements located across the transcript with cellular transacting factors, and emerging data supports also the involvement of structural proteins such as actin. These synergies are believed to occur co-transcriptionally when the nascent transcript is packaged into ribonucleoprotein complexes, and to determine a translationally repressed form of the mRNA only compatible with transport. The aim of this review is to highlight part of the molecular circuitry behind the decisions that control assembly of translationally repressed and de-repressed ribonucleoprotein complexes, compatible with mRNA transport or localization. I will specifically place the impact of these mechanisms in the context of spermatogenesis.

The transacting factor CBF-A/Hnrnpab binds to the A2RE/RTS element of protamine 2 mRNA and contributes to its translational regulation during mouse spermatogenesis.

During spermatogenesis, mRNA localization and translation are believed to be regulated in a stage-specific manner. We report here that the Protamine2 (Prm2) mRNA transits through chromatoid bodies of round spermatids and localizes to cytosol of elongating spermatids for translation. The transacting factor CBF-A, also termed Hnrnpab, contributes to temporal regulation of Prm2 translation. We found that CBF-A co-localizes with the Prm2 mRNA during spermatogenesis, directly binding to the A2RE/RTS element in the 3' UTR. Although both p37 and p42 CBF-A isoforms interacted with RTS, they associated with translationally repressed and de-repressed Prm2 mRNA, respectively. Only p42 was found to interact with the 5'cap complex, and to co-sediment with the Prm2 mRNA in polysomes. In CBF-A knockout mice, expression of protamine 2 (PRM2) was reduced and the Prm2 mRNA was prematurely translated in a subset of elongating spermatids. Moreover, a high percentage of sperm from the CBF-A knockout mouse showed abnormal DNA morphology. We suggest that CBF-A plays an important role in spermatogenesis by regulating stage-specific translation of testicular mRNAs.

Nuclear myosin 1c facilitates the chromatin modifications required to activate rRNA gene transcription and cell cycle progression.

Actin and nuclear myosin 1c (NM1) cooperate in RNA polymerase I (pol I) transcription. NM1 is also part of a multiprotein assembly, B-WICH, which is involved in transcription. This assembly contains the chromatin remodeling complex WICH with its subunits WSTF and SNF2h. We report here that NM1 binds SNF2h with enhanced affinity upon impairment of the actin-binding function. ChIP analysis revealed that NM1, SNF2h, and actin gene occupancies are cell cycle-dependent and require intact motor function. At the onset of cell division, when transcription is temporarily blocked, B-WICH is disassembled due to WSTF phosphorylation, to be reassembled on the active gene at exit from mitosis. NM1 gene knockdown and motor function inhibition, or stable expression of NM1 mutants that do not interact with actin or chromatin, overall repressed rRNA synthesis by stalling pol I at the gene promoter, led to chromatin alterations by changing the state of H3K9 acetylation at gene promoter, and delayed cell cycle progression. These results suggest a unique structural role for NM1 in which the interaction with SNF2h stabilizes B-WICH at the gene promoter and facilitates recruitment of the HAT PCAF. This leads to a permissive chromatin structure required for transcription activation.

Nuclear myosin 1 contributes to a chromatin landscape compatible with RNA polymerase II transcription activation.

BACKGROUND: Nuclear myosin 1c (NM1) is emerging as a regulator of transcription and chromatin organization. RESULTS: Using chromatin immunoprecipitation and deep sequencing (ChIP-Seq) in combination with molecular analyses, we investigated the global association of NM1 with the mammalian genome. Analysis of the ChIP-Seq data demonstrates that NM1 binds across the entire mammalian genome with occupancy peaks correlating with distributions of RNA Polymerase II (Pol II) and active epigenetic marks at class II gene promoters. In mouse embryonic fibroblasts subjected to RNAi mediated NM1 gene silencing, we show that NM1 synergizes with polymerase-associated actin to maintain active Pol II at the promoter. NM1 also co-localizes with the nucleosome remodeler SNF2h at class II promoters where they assemble together with WSTF as part of the B-WICH complex. A high resolution micrococcal nuclease (MNase) assay and quantitative real time PCR shows that this mechanism is required for local chromatin remodeling. Following B-WICH assembly, NM1 mediates physical recruitment of the histone acetyl transferase PCAF and the histone methyl transferase Set1/Ash2 to maintain and preserve H3K9acetylation and H3K4trimethylation for active transcription. CONCLUSIONS: We propose a novel genome-wide mechanism where myosin synergizes with Pol II-associated actin to link the polymerase machinery with permissive chromatin for transcription activation.

Assessing nanobody interaction with SARS-CoV-2 Nsp9.

The interaction between SARS-CoV-2 non-structural protein Nsp9 and the nanobody 2NSP90 was investigated by NMR spectroscopy using the paramagnetic perturbation methodology PENELOP (Paramagnetic Equilibrium vs Nonequilibrium magnetization Enhancement or LOss Perturbation). The Nsp9 monomer is an essential component of the replication and transcription complex (RTC) that reproduces the viral gRNA for subsequent propagation. Therefore preventing Nsp9 recruitment in RTC would represent an efficient antiviral strategy that could be applied to different coronaviruses, given the Nsp9 relative invariance. The NMR results were consistent with a previous characterization suggesting a 4:4 Nsp9-to-nanobody stoichiometry with the occurrence of two epitope pairs on each of the Nsp9 units that establish the inter-dimer contacts of Nsp9 tetramer. The oligomerization state of Nsp9 was also analyzed by molecular dynamics simulations and both dimers and tetramers resulted plausible. A different distribution of the mapped epitopes on the tetramer surface with respect to the former 4:4 complex could also be possible, as well as different stoichiometries of the Nsp9-nanobody assemblies such as the 2:2 stoichiometry suggested by the recent crystal structure of the Nsp9 complex with 2NSP23 (PDB ID: 8dqu), a nanobody exhibiting essentially the same affinity as 2NSP90. The experimental NMR evidence, however, ruled out the occurrence in liquid state of the relevant Nsp9 conformational change observed in the same crystal structure.

Chromatin remodelling and transcription: be-WICHed by nuclear myosin 1.

Transcription in eukaryotic cells requires dynamic changes of chromatin structure to facilitate or prevent RNA polymerase access to active genes. These structural modifications rely on the concerted action of ATP-dependent chromatin-remodelling complexes and histone-modifying enzymes, which generate a chromatin configuration that is either compatible with transcription (euchromatin) or incompatible (heterochromatin). Insights into how these structural changes might be coordinated for RNA polymerase I (pol I) genes come from the discoveries of the nucleolar-remodelling complex (NoRC) and B-WICH--a high molecular weight fraction of the WSTF/SNF2h chromatin-remodelling complex. NoRC produces a repressive chromatin state; B-WICH, together with nuclear myosin 1, activates pol I transcription directly on chromatin templates and might also function in the maintenance of ribosomal chromatin structure.

ß-actin mediated H3K27ac changes demonstrate the link between compartment switching and enhancer-dependent transcriptional regulation.

BACKGROUND: Recent work has demonstrated that three-dimensional genome organization is directly affected by changes in the levels of nuclear cytoskeletal proteins such as ß-actin. The mechanisms which translate changes in 3D genome structure into changes in transcription, however, are not fully understood. Here, we use a comprehensive genomic analysis of cells lacking nuclear ß-actin to investigate the mechanistic links between compartment organization, enhancer activity, and gene expression. RESULTS: Using HiC-Seq, ATAC-Seq, and RNA-Seq, we first demonstrate that transcriptional and chromatin accessibility changes observed upon ß-actin loss are highly enriched in compartment-switching regions. Accessibility changes within compartment switching genes, however, are mainly observed in non-promoter regions which potentially represent distal regulatory elements. Our results also show that ß-actin loss induces widespread accumulation of the enhancer-specific epigenetic mark H3K27ac. Using the ABC model of enhancer annotation, we then establish that these epigenetic changes have a direct impact on enhancer activity and underlie transcriptional changes observed upon compartment switching. A complementary analysis of fibroblasts undergoing reprogramming into pluripotent stem cells further confirms that this relationship between compartment switching and enhancer-dependent transcriptional change is not specific to ß-actin knockout cells but represents a general mechanism linking compartment-level genome organization to gene expression. CONCLUSIONS: We demonstrate that enhancer-dependent transcriptional regulation plays a crucial role in driving gene expression changes observed upon compartment-switching. Our results also reveal a novel function of nuclear ß-actin in regulating enhancer function by influencing H3K27 acetylation levels.

Axonal mRNA binding of hnRNP A/B is crucial for axon targeting and maturation of olfactory sensory neurons.

Spatiotemporal control of gene expression is important for neural development and function. Here, we show that heterogeneous nuclear ribonucleoprotein (hnRNP) A/B is highly expressed in developing olfactory sensory neurons (OSNs), and its knockout results in reduction in mature OSNs and aberrant targeting of OSN axons to the olfactory bulb. RNA immunoprecipitation analysis reveals that hnRNP A/B binds to a group of mRNAs that are highly related to axon projections and synapse assembly. Approximately 11% of the identified hnRNP A/B targets, including Pcdha and Ncam2, encode cell adhesion molecules. In Hnrnpab knockout mice, PCDHA and NCAM2 levels are significantly reduced at the axon terminals of OSNs. Furthermore, deletion of the hnRNP A/B-recognition motif in the 3' UTR of Pcdha leads to impaired PCDHA expression at the OSN axon terminals. Therefore, we propose that hnRNP A/B facilitates OSN maturation and axon projection by regulating the local expression of its target genes at axon terminals.

Positive regulation of oxidative phosphorylation by nuclear myosin 1 protects cells from metabolic reprogramming and tumorigenesis in mice.

Metabolic reprogramming is one of the hallmarks of tumorigenesis. Here, we show that nuclear myosin 1 (NM1) serves as a key regulator of cellular metabolism. NM1 directly affects mitochondrial oxidative phosphorylation (OXPHOS) by regulating mitochondrial transcription factors TFAM and PGC1α, and its deletion leads to underdeveloped mitochondria inner cristae and mitochondrial redistribution within the cell. These changes are associated with reduced OXPHOS gene expression, decreased mitochondrial DNA copy number, and deregulated mitochondrial dynamics, which lead to metabolic reprogramming of NM1 KO cells from OXPHOS to aerobic glycolysis.This, in turn, is associated with a metabolomic profile typical for cancer cells, namely increased amino acid-, fatty acid-, and sugar metabolism, and increased glucose uptake, lactate production, and intracellular acidity. NM1 KO cells form solid tumors in a mouse model, suggesting that the metabolic switch towards aerobic glycolysis provides a sufficient carcinogenic signal. We suggest that NM1 plays a role as a tumor suppressor and that NM1 depletion may contribute to the Warburg effect at the onset of tumorigenesis.

Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells.

The next steps of deep space exploration are manned missions to Moon and Mars. For safe space missions for crew members, it is important to understand the impact of space flight on the immune system. We studied the effects of 21 days dry immersion (DI) exposure on the transcriptomes of T cells isolated from blood samples of eight healthy volunteers. Samples were collected 7 days before DI, at day 7, 14, and 21 during DI, and 7 days after DI. RNA sequencing of CD3+ T cells revealed transcriptional alterations across all time points, with most changes occurring 14 days after DI exposure. At day 21, T cells showed evidence of adaptation with a transcriptional profile resembling that of 7 days before DI. At 7 days after DI, T cells again changed their transcriptional profile. These data suggest that T cells adapt by rewiring their transcriptomes in response to simulated weightlessness and that remodeling cues persist when reexposed to normal gravity.