Abstractβ-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 β-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in β-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of β-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that β-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 β-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 β-actin-dependent chromatin remodeling plays a role in shaping the chromatin landscape and influences the regulation of genes involved in development and differentiation.
Actin is an essential regulator of cellular functions. In the eukaryotic cell nucleus, actin regulates chromatin as a bona fide component of chromatin remodeling 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 3 D 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.
Adipogenesis is regulated by a cascade of signals that drive transcriptional reprogramming in adipocytes. Here, we report that nuclear actin regulates the chromatin states that establish tissue specific expression during adipogenesis. To study the role of β-actin in adipocyte differentiation we conducted RNA-Sequencing on wild-type (WT) and β-actin knockout (KO) mouse embryonic fibroblasts (MEFs) after reprograming to adipocytes. We found β-actin depletion affects induction of several adipogenic genes during transcriptional reprograming. This impaired regulation of adipogenic genes is linked to reduced expression of the pioneer factor Cebpa and is rescued by reintroducing NLS-tagged β-actin. ATAC-Seq in KO MEFs revealed that actin-dependent reduction in Cebpa expression correlates with decreased chromatin accessibility and loss of chromatin association of the ATPase Brg1. This, in turn, impairs CEBPB's association with its Cebpa promoter-proximal binding site during adipogenesis. We propose a role for the nuclear β-actin pool in maintaining open chromatin for transcriptional reprogramming during adipogenic differentiation.
Actin plays fundamental roles in both the cytoplasm and the cell nucleus. In the nucleus,-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-actin in osteogenesis,-actin wild-type (WT) and-actin knockout (KO) mouse embryonic fibroblasts (MEFs) are reprogrammed to osteoblast-like cells using small molecules in vitro. It is discovered that loss of-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-actin in osteogenic reprogramming through a mitochondria-based mechanism that controls cell-mediated mineralization is proposed.
β-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 β-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in β-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of β-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that β-actin levels can affect the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2 in a dosage-dependent manner. Our results show that changes in β-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 support a novel role for β-actin-dependent chromatin remodeling in shaping the chromatin landscape and regulating genes involved in development and differentiation. METHODSCell culture: WT, KO and HET mouse embryonic fibroblasts (MEFs) (from the lab of Dr. Christophe Ampe, University of Gent, Belgium) were maintained and cultured with Dulbecco's modified Eagle medium (DMEM) with high glucose, 10% fetal bovine serum (FBS) and 100 units/mL penicillin and 100 μg/mL streptomycin, in a humidified incubator with 5% CO2 at 37⁰C. The KO(Mm) and KO(Hs) cells with GFP and NLS-β actin re-introduced into KO cells were generated by retroviral transduction as described in (2). ATAC-Seq:Samples with 50,000 cells per condition were shipped in frozen medium (DMEM with 50 % FBS and 10% DMSO) on dry ice to Novogene (Beijing, China). All subsequent processing was performed by Novogene using standard DNA extraction and library preparation protocols. Cell nuclei were isolated, mixed with Tn5 Transposase with two adapters and tagmentation was performed for 30 minutes at 37⁰C. The fragmented DNA was purified and amplified with limited PCR cycle using index primers. Libraries were prepared according to recommended Illumina NovaSeq6000 protocols. All ATAC-Seq processing was performed by Novogene (Beijing, China). ChIP-Seq:ChIP-Seq for BRG1, H3K9me3 and H3K27me3 was performed as described in (2). For EZH2 ChIP-Seq, Mouse Embryonic fibroblasts were crosslinked using 1% formaldehyde (Sigma cat. No. F8775) for 10 minutes followed by quenching with 0.125 M Glycine for 5 minutes and lysis with lysis buffer 1 -LB1-(50 mM Hepes KOH pH 7.5, 10 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100). Nuclei were pelleted, collected and washed using lysis buffer 2 -LB2-(10 mM Tris HCl pH 8, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA). This was followed by lysis using lysis buf...
In the eukaryotic cell nucleus, cytoskeletal proteins are emerging as essential players in nuclear function. In particular, actin regulates chromatin as part of ATP-dependent chromatin remodeling complexes, it modulates transcription and it is incorporated into nascent ribonucleoprotein complexes, accompanying them from the site of transcription to polyribosomes. The nuclear actin pool is undistinguishable from the cytoplasmic one in terms of its ability to undergo polymerization and it has also been implicated in the dynamics of chromatin, regulating heterochromatin segregation at the nuclear lamina and maintaining heterochromatin levels in the nuclear interiors. One of the next frontiers is, therefore, to determine a possible involvement of nuclear actin in the functional architecture of the cell nucleus by regulating the hierarchical organization of chromatin and, thus, genome organization. Here, we discuss the repertoire of these potential actin functions and how they are likely to play a role in the context of cellular differentiation. ARTICLE HISTORY
Background Recent work has demonstrated that three-dimensional genome organization is directly affected by changes in the levels of nuclear cytoskeletal proteins such as β-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 β-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 β-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 β-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 β-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 β-actin in regulating enhancer function by influencing H3K27 acetylation levels.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.