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.

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.

The Role of Nuclear Actin in Genome Organization and Gene Expression Regulation During Differentiation.

In the cell nucleus, actin participates in numerous essential processes. Actin is involved in chromatin as part of specific ATP-dependent chromatin remodeling complexes and associates with the RNA polymerase machinery to regulate transcription at multiple levels. Emerging evidence has also shown that the nuclear actin pool controls the architecture of the mammalian genome playing an important role in its hierarchical organization into transcriptionally active and repressed compartments, contributing to the clustering of RNA polymerase II into transcriptional hubs. Here, we review the most recent literature and discuss how actin involvement in genome organization impacts the regulation of gene programs that are activated or repressed during differentiation and development. As in the cytoplasm, we propose that nuclear actin is involved in key nuclear tasks in complex with different types of actin-binding proteins that regulate actin function and bridge interactions between actin and various nuclear components.

A dynamic actin-dependent nucleoskeleton and cell identity.

Actin is an essential regulator of cellular functions. In the eukaryotic cell nucleus, actin regulates chromatin as a bona fide component of chromatin remodelling complexes, it associates with nuclear RNA polymerases to regulate transcription and is involved in co-transcriptional assembly of nascent RNAs into ribonucleoprotein complexes. Actin dynamics are, therefore, emerging as a major regulatory factor affecting diverse cellular processes. Importantly, the involvement of actin dynamics in nuclear functions is redefining the concept of nucleoskeleton from a rigid scaffold to a dynamic entity that is likely linked to the three-dimensional organization of the nuclear genome. In this review, we discuss how nuclear actin, by regulating chromatin structure through phase separation may contribute to the architecture of the nuclear genome during cell differentiation and facilitate the expression of specific gene programs. We focus specifically on mitochondrial genes and how their dysregulation in the absence of actin raises important questions about the role of cytoskeletal proteins in regulating chromatin structure. The discovery of a novel pool of mitochondrial actin that serves as 'mitoskeleton' to facilitate organization of mtDNA supports a general role for actin in genome architecture and a possible function of distinct actin pools in the communication between nucleus and mitochondria.

ß-actin dependent chromatin remodeling mediates compartment level changes in 3D genome architecture.

ß-actin is a crucial component of several chromatin remodeling complexes that control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes (PRCs). While previous work has shown that ß-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in ß-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of ß-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that ß-actin levels can induce changes in chromatin structure by affecting the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2. Our results show that changes in ß-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversible changes in 3D genome architecture. Our findings reveal that ß-actin-dependent chromatin remodeling plays a role in shaping the chromatin landscape and influences the regulation of genes involved in development and differentiation.

Transcriptional Profiling Reveals Ribosome Biogenesis, Microtubule Dynamics and Expression of Specific lncRNAs to be Part of a Common Response to Cell-Penetrating Peptides.

Cell-penetrating peptides (CPPs) are short peptides that are able to efficiently penetrate cellular lipid bilayers. Although CPPs have been used as carriers in conjugation with certain cargos to target specific genes and pathways, how rationally designed CPPs per se affect global gene expression has not been investigated. Therefore, following time course treatments with 4 CPPs-penetratin, PepFect14, mtCPP1 and TP10, HeLa cells were transcriptionally profiled by RNA sequencing. Results from these analyses showed a time-dependent response to different CPPs, with specific sets of genes related to ribosome biogenesis, microtubule dynamics and long-noncoding RNAs being differentially expressed compared to untreated controls. By using an image-based high content phenotypic profiling platform we confirmed that differential gene expression in CPP-treated HeLa cells strongly correlates with changes in cellular phenotypes such as increased nucleolar size and dispersed microtubules, compatible with altered ribosome biogenesis and cell growth. Altogether these results suggest that cells respond to different cell penetrating peptides by alteration of specific sets of genes, which are possibly part of the common response to such stimulus.

Loss of ß-Actin Leads to Accelerated Mineralization and Dysregulation of Osteoblast-Differentiation Genes during Osteogenic Reprogramming.

Actin plays fundamental roles in both the cytoplasm and the cell nucleus. In the nucleus, ß-actin regulates neuronal reprogramming by consolidating a heterochromatin landscape required for transcription of neuronal gene programs, yet it remains unknown whether it has a role in other differentiation models. To explore the potential roles of ß-actin in osteogenesis, ß-actin wild-type (WT) and ß-actin knockout (KO) mouse embryonic fibroblasts (MEFs) are reprogrammed to osteoblast-like cells using small molecules in vitro. It is discovered that loss of ß-actin leads to an accelerated mineralization phenotype (hypermineralization), accompanied with enhanced formation of extracellular hydroxyapatite microcrystals, which originate in the mitochondria in the form of microgranules. This phenotype is a consequence of rapid upregulation of mitochondrial genes including those involved in oxidative phosphorylation (OXPHOS) in reprogrammed KO cells. It is further found that osteogenic gene programs are differentially regulated between WT and KO cells, with clusters of genes exhibiting different temporal expression patterns. A novel function for ß-actin in osteogenic reprogramming through a mitochondria-based mechanism that controls cell-mediated mineralization is proposed.