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 β‐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 β‐actin KO cells. Mechanistically, we found loss of chromatin association by the Brahma‐related gene ( Brg )/Brahma‐associated factor (BAF) chromatin remodeling complex subunit Brgl in the absence of β‐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 β‐actin KO cells. Some of these genes and phenotypes were gained in a β‐actin dose‐dependent manner. Moreover, reintroducing a nuclear localization signal‐containing β‐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, β‐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. β‐Actin‐dependent global chromatin organization and gene expression programs control cellular identity. FASEB J. 32,1296‐1314 (2018). www.fasebj.org

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 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.

During neuronal development, β-actin serves an important role in growth cone mediated axon guidance. Consistent with this notion, in vivo ablation of the β-actin gene leads to abnormalities in the nervous system. However, whether β-actin is involved in the regulation of neuronal gene programs is not known. In this study, we directly reprogramed β-actin^+/+ WT, β-actin^+/- HET and β-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 β-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 β-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. Although β-actin plays an important role in growth cone mediated axon guidance in neurons, the potential role of β-actin in controlling neuron differentiation remains unknown. Here, we converted β-actin^+/+ WT, β-actin^+/- HET and β-actin^-/- KO mouse embryonic fibroblast (MEFs) into chemically induced neurons (CiNeurons) by direct reprograming. We found that the up-regulation of neuronal programs was impaired in β-actin^-/- CiNeurons in comparison to WT ones. β-actin^+/- HET CiNeurons showed an intermediate level of neuronal program expression, suggesting that β-actin dosage plays an important role during direct neuronal reprograming. Importantly, the impaired up-regulation of neuron-related genes was associated with the elevated H3K9 and H3K27 methylation levels at gene loci in KO MEFs. These epigenetic changes were accompanied by the impaired chromatin association of Brg1-containing chromatin remodeling BAF complex in β-actin null cells. Together our study demonstrates that β-actin is required for the optimal induction of neuronal gene programs during direct reprograming by presetting a favorable chromatin status.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.

In eukaryotic cells, actin regulates both cytoplasmic and nuclear functions. However, whether actin-based structures are present in the mitochondria and are involved in mitochondrial functions has not been investigated. Here, using wild-type ?-actin +/+ and knockout (KO) ?-actin ?/? mouse embryonic fibroblasts we show evidence for the defect in maintaining mitochondrial membrane potential (MMP) in ?-actin-null cells. MMP defects were associated with impaired mitochondrial DNA (mtDNA) transcription and nuclear oxidative phosphorylation (OXPHOS) gene expression. Using super-resolution microscopy we provided direct evidence on the presence of ?-actin-containing structures inside mitochondria. Large aggregates of TFAM-stained nucleoids were observed in bulb-shaped mitochondria in KO cells, suggesting defects in mitochondrial nucleoid segregation without ?-actin. The observation that mitochondria-targeted ?-actin rescued mtDNA transcription and MMP suggests an indispensable functional role of a mitochondrial ?-actin pool necessary for mitochondrial quality control.

Actin and nuclear myosin 1 (NM1) are regulators of transcription and chromatin organization. Using a genome‐wide approach, we report here that β‐actin binds intergenic and genic regions across the mammalian genome, associated with both protein‐coding and rRNA genes. Within the rDNA, the distribution of β‐actin correlated with NM1 and the other subunits of the B‐WICH complex, WSTF and SNF2h. In β‐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 β‐actin, in contrast to mutated forms with polymerization defects, efficiently rescued rRNA synthesis underscoring the direct role for a polymerization‐competent form of β‐actin in Pol I transcription. The rRNA synthesis defects in the β‐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 β‐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 β‐actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects. FASEB J. 30, 2860‐2873 (2016). www.fasebj.org