INO80 Chromatin Remodeling Complex Research Articles

The mechanism of INO80D involved in chromatin remodeling regulating spermatogenesis in Chinese mitten crab (Eriocheir sinensis).

The INO80D protein, a component of the INO80 chromatin remodeling complex, plays a pivotal role in chromatin remodeling, gene expression, and DNA repair within mammalian sperm. In contrast to the condensed nuclear structure of mammalian sperm, Chinese mitten crab, Eriocheir sinensis, exhibits a distinctively decondensed sperm nucleus. The distribution and function of INO80D during the E. sinensis spermatogenesis were previously enigmatic. Our research endeavored to elucidate the distribution and function of INO80D, thereby enhancing our comprehension of sperm decondensation and the process of spermatogenesis in this species. Employing transcriptome sequencing, RT-qPCR, western blot analysis, and immunofluorescence techniques, we observed a pronounced upregulation of INO80D in the adult E. sinensis in comparison to the juvenile. The protein predominantly resides in the cellular nucleus, with high levels in spermatogonia and spermatocytes, less in stage I and III spermatids, and lowest in mature sperm. The results indicated that INO80D is initially instrumental in chromatin decondensation to facilitate gene accessibility and DNA repair during the early phases of spermatogenesis. Its role subsequently shifts to maintaining decondensed chromatin stability and genetic integrity during spermiogenesis. The sustained presence of INO80D during spermiogenesis is essential for the ultimate maturation of the decondensed sperm nucleus, imperative for preserving the unique decondensed state and the protection of genetic material in E. sinensis. Our study concludes that INO80D exerts a multifaceted influence on the spermatogenesis of E. sinensis, impacting chromatin decondensation, genetic integrity, and the regulation of early gene expression. This understanding could potentially improve crab breeding in aquaculture.

Abstract 1696: ACTL6A deficiency drives cell death through KLF4-involved chromatin accessibility changes in colorectal cancer

Abstract ACTL6A is a subunit of the SWI/SNF and INO80 chromatin remodeling complexes and plays roles in neural development and cancer cell proliferation. Additionally, it is known to be upregulated in various types of cancer and is associated with a decrease in patient survival rates. However, the mechanism of how chromatin remodelers including ACTL6A cause cell death has yet to be defined. Therefore, we would like to investigate how the depletion of ACTL6A promotes cell growth reduction by mediating changes in the epigenetic landscape in colorectal cancer cells. We engineered ACTL6A knockout by CRISPR-Cas9. We performed Assay for transposase-accessible chromatin using sequencing (ATAC-seq), RNA sequencing (RNA-seq), and Chromatin Immunoprecipitation sequencing (ChIP-seq) for SMARCB1 and INO80, as representative members of their respective families, and KLF4. Moreover, HCT116 cells stably transduced with doxycycline-inducible shGFP or shACTL6A were implanted into the flank of nude mice. One week after tumor implantation, mice were randomly allocated to 4 groups (n=13-15 per group) and doxycycline treated group was administered with doxycycline (1mg/ml) and 5% sucrose in the drinking water. Tumor volume was measured with calipers. Upon depletion of ACTL6A, the regions of increased chromatin accessibility were correlated with the upregulated genes. After the loss of ACTL6A, there was a significant increase in KLF4 expression and genomic binding largely increased, coinciding with an increase in newly accessible regions containing KLF4 motifs. Interestingly, there is a strong positive correlation between changes in chromatin accessibility and overall changes in the binding of KLF4, SWI/SNF, and INO80 complexes. We find that changes in chromatin accessibility are co-regulated by KLF4, the SWI/SNF and the INO80 complex, while SWI/SNF and INO80 complex have a low correlation in binding between the SWI/SNF and INO80 complexes at newly accessible regions, indicating no physical interactions between these two chromatin complexes. Overall, we demonstrated that the redistribution of ACTL6A-deficient two chromatin remodeling complexes and KLF4 is associated with changes in chromatin accessibility, which are also linked to upregulated genes that promote the p53 pathway. These findings revealed the role of KLF4 along with the SWI/SNF and INO80 complexes, in regulating the chromatin landscape and gene expression in ACTL6A-depleted colorectal cancer cells. Citation Format: Hye-Ju Yang, Eun-Ju Kim, Sang-Hyun Song, Tae-You Kim. ACTL6A deficiency drives cell death through KLF4-involved chromatin accessibility changes in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1696.

Exploring Transcriptional Regulation of Beta Cell SASP by Brd4-Associated Proteins and Cell Cycle Control Protein p21.

Type 1 diabetes (T1D) is a metabolic disease resulting from progressive autoimmune destruction of insulin-producing pancreatic beta cells. Although the majority of beta cells are lost in T1D, a small subset undergoes senescence, a stress response involving growth arrest, DNA damage response, and activation of a senescence-associated secretory phenotype (SASP). SASP in beta cells of the nonobese diabetic (NOD) mouse model of T1D and primary human islets is regulated at the level of transcription by bromodomain extra-terminal (BET) proteins, but the mechanisms remain unclear. To explore how SASP is transcriptionally regulated in beta cells, we used the NOD beta cell line NIT-1 to model beta cell SASP and identified binding partners of BET protein Brd4 and explored the role of the cyclin-dependent kinase inhibitor p21. Brd4 interacted with a variety of proteins in senescent NIT-1 cells including subunits of the Ino80 chromatin remodeling complex, which was expressed in beta cells during T1D progression in NOD mice and in human beta cells of control, autoantibody-positive, and T1D donors as determined from single-cell RNA-seq data. RNAi knockdown of p21 during senescence in NIT-1 cells did not significantly impact viability or SASP. Taken together, these results suggest that Brd4 interacts with several protein partners during senescence in NIT-1 cells, some of which may play roles in SASP gene activation and that p21 is dispensable for the SASP in this beta cell model.

INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined. In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction. The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of Ino80, its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of Ino80 in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function. In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target.

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency.

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned+1 and+2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.

Feedback Modulation between Human INO80 Chromatin Remodeling Complex and miR-372 in HCT116 Cells.

Human INO80 chromatin remodeling complex (INO80 complex) as a transcription cofactor is widely involved in gene transcription regulation and is frequently highly expressed in tumor cells. However, few reports exist on the mutual regulatory mechanism between INO80 complex and non-coding microRNAs. Herein, we showed evidence that the INO80 complex transcriptionally controls microRNA-372 (miR-372) expression through RNA-Seq analysis and a series of biological experiments. Knocking down multiple subunits in the INO80 complex, including the INO80 catalytic subunit, YY1, Ies2, and Arp8, can significantly increase the expression level of miR-372. Interestingly, mimicking miR-372 expression in HCT116 cells, in turn, post-transcriptionally suppressed INO80 and Arp8 expression at both mRNA and protein levels, indicating the existence of a mutual regulatory mechanism between the INO80 complex and miR-372. The target relationship between miR-372 and INO80 complex was verified using luciferase assays in HCT116 colon cancer cells. As expected, miR-372 mimics significantly suppressed the luciferase activity of pMIR-luc/INO80 and pMIR-luc/Arp8 3'-UTR in cells. In contrast, the miR-372 target sites in the 3'-UTRs linked to the luciferase reporter were mutagenized, and both mutant sites lost their response to miR-372. Furthermore, the mutual modulation between the INO80 complex and miR-372 was involved in cell proliferation and the p53/p21 signaling pathway, suggesting the synergistic anti-tumor role of the INO80 complex and miR372. Our results will provide a solid theoretical basis for exploring miR-372 as a biological marker of tumorigenesis.

An essential role for the Ino80 chromatin remodeling complex in regulation of gene expression during cellular quiescence

Cellular quiescence is an important physiological state both in unicellular and multicellular eukaryotes. Quiescent cells are halted for proliferation and stop the cell cycle at the G0 stage. Using fission yeast as a model organism, we have previously found that several subunits of a conserved chromatin remodeling complex, Ino80C (INOsitol requiring nucleosome remodeling factor), are required for survival in quiescence. Here, we demonstrate that Ino80C has a key function in the regulation of gene expression in G0 cells. We show that null mutants for two Ino80C subunits, Iec1 and Ies2, a putative subunit Arp42, a null mutant for the histone variant H2A.Z, and a null mutant for the Inositol kinase Asp1 have very similar phenotypes in quiescence. These mutants show reduced transcription genome-wide and specifically fail to activate 149 quiescence genes, of which many are localized to the subtelomeric regions. Using spike in normalized ChIP-seq experiments, we show that there is a global reduction of H2A.Z levels in quiescent wild-type cells but not in iec1∆ cells and that a subtelomeric chromosome boundary element is strongly affected by Ino80C. Based on these observations, we propose a model in which Ino80C is evicting H2A.Z from chromatin in quiescent cells, thereby inactivating the subtelomeric boundary element, leading to a reorganization of the chromosome structure and activation of genes required to survive in quiescence.

ACTR5 controls CDKN2A and tumor progression in an INO80-independent manner.

Epigenetic dysregulation of cell cycle is a hallmark of tumorigenesis in multiple cancers, including hepatocellular carcinoma (HCC). Nonetheless, the epigenetic mechanisms underlying the aberrant cell cycle signaling and therapeutic response remain unclear. Here, we used an epigenetics-focused CRISPR interference screen and identified ACTR5 (actin-related protein 5), a component of the INO80 chromatin remodeling complex, to be essential for HCC tumor progression. Suppression of ACTR5 activated CDKN2A expression, ablated CDK/E2F-driven cell cycle signaling, and attenuated HCC tumor growth. Furthermore, high-density CRISPR gene tiling scans revealed a distinct HCC-specific usage of ACTR5 and its interacting partner IES6 compared to the other INO80 complex members, suggesting an INO80-independent mechanism of ACTR5/IES6 in supporting the HCC proliferation. Last, our study revealed the synergism between ACTR5/IES6-targeting and pharmacological inhibition of CDK in treating HCC. These results indicate that the dynamic interplay between epigenetic regulators, tumor suppressors, and cell cycle machinery could provide novel opportunities for combinational HCC therapy.

Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence

T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of Tcell exhaustion, which identified an enrichment of epigenetic factors. Invivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved Tcell persistence in tumors. Invivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating Tcells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of Tcell exhaustion and demonstrate that modulation of epigenetic state can improve Tcell responsesin cancer immunotherapy.

Abstract 1517: Molecular architecture of replication stress response to oncogene deregulation

Abstract Oncogene deregulation triggers replication stress, which can be detected in precancerous lesions and is a source of subsequent genomic instability in cancer. Studies on the mechanisms underlying DNA replication stress caused by frequently deregulated oncogenes, like MYC or RAS, can identify novel mechanisms of oncogenesis and point to actionable pathways with potential for anticancer therapy. To understand the molecular mechanisms underlying this cellular response, we used high-content microscopy to monitor the dynamic behavior of >60 GFP-tagged proteins linked to replication stress or DNA damage in primary human cells. Iterations of this screen identified a proteomic signature specific to MYC- and RAS-driven replication stress that was significantly different from responses activated by other sources of replication stress (i.e. DNA damaging drugs). These findings indicated that the replication stress response to oncogenes is somewhat unique. MYC-dependent replication stress responses typically activated the INO80 chromatin-remodeling complex — a highly conserved complex necessary for maintenance of genome stability in yeast. Core components of the INO80 complex assembled onto chromatin at MYC-bound sites and were quantitatively enriched at active replication forks. These molecular events were independent of transcription, and their disruption by pharmacological or genetic means specifically altered replication dynamics and cellular growth in cells overexpressing MYC. Notably, we identified recurrent somatic mutations in several genes encoding for INO80 complex components across multiple cancer types, with an abundance of nonsense and splice-site mutations consistent with loss of function. Most of these mutant variants behaved as hypomorphs when tested in cell-based assays: they were insensitive to MYC deregulation and failed to rescue cell proliferation in knock-out cells with MYC overexpression. Computational predictions also suggested that many of these mutations had been selected as potential cancer drivers. Consistent with this notion, we found that in vivo, INO80 complex mutations shortened tumor latency in a mouse model of MYC-dependent lymphomagenesis. Collectively, these findings indicate that the replication stress response to oncogene deregulation is unique in its molecular architecture, and modification of this response by partial loss-of-function mutations in cancer facilitates tumor progression. Citation Format: Rosa Vinas-Castells, Alja Kozulic-Pirher, Tomás Aparicio, Sandra Casas Recasens, Mark P. Roberto, Rachel Sue, Francisco Martinez, Nuria López-Bigas, Jean Gautier, David Dominguez-Sola. Molecular architecture of replication stress response to oncogene deregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1517.

A hexasome is the preferred substrate for the INO80 chromatin remodeling complex, allowing versatility of function

The critical role of the INO80 chromatin remodeling complex in transcription is commonly attributed to its nucleosome sliding activity. Here, we have found that INO80 prefers to mobilize hexasomes over nucleosomes. INO80's preference for hexasomes reaches up to ∼60 fold when flanking DNA overhangs approach ∼18-bp linkers in yeast gene bodies. Correspondingly, deletion of INO80 significantly affects the positions of hexasome-sized particles within yeast genes invivo. Our results raise the possibility that INO80 promotes nucleosome sliding by dislodging an H2A-H2B dimer, thereby making a nucleosome transiently resemble a hexasome. We propose that this mechanism allows INO80 to rapidly mobilize nucleosomes at promoters and hexasomes within gene bodies. Rapid repositioning of hexasomes that are generated in the wake of transcription may mitigate spurious transcription. More generally, such versatility may explain how INO80 regulates chromatin architecture during the diverse processes of transcription, replication, and repair.

Abstract 144: Nuclear Smooth Muscle α-actin Is Critical For Smooth Muscle Cell Differentiation And To Prevent Cerebrovascular Disease

Missense pathogenic variants in ACTA2, encoding α-smooth muscle actin (SMA), predispose to thoracic aortic aneurysms. A subset of these ACTA2 pathogenic variants also predispose to childhood onset cerebrovascular disease characterized by occlusion of the distal internal carotid arteries and small vessel disease. In our studies to identify how specific ACTA2 pathogenic variants predispose to cerebrovascular disease, we confirmed that SMA is localized to the nucleus in wildtype (WT) smooth muscle cells (SMCs), is enriched in the nucleus over β-actin with differentiation of SMCs, associates with the INO80 chromatin remodeling complex, and selectively binds to the promoters of SMC contractile genes. Pluripotent stem cell-derived SMCs from patients with ACTA2 -associated cerebrovascular disease ( ACTA2 R179C) as well as SMCs explanted from a knockin mouse model ( Acta2 SMC-R179C/+ ) confirm that R179 variants disrupt SMA nuclear localization. Both mouse and human cells harboring the R179C pathogenic variant proliferate and migrate more than WT SMCs, are less differentiated than WT SMCs, and have increased expression of pluripotency-associated genes. Although the variant is heterozygous, these cells show a dominant negative impact on nuclear SMA function through dramatically reduced levels of SMA in the nucleus, in the INO80 chromatin remodeling complex, and on the promoters of SMC-specific genes. Finally, single cell RNA sequencing analysis of aortic tissue from an ACTA2 R179H patient confirms dedifferentiation of SMCs as a key phenotype associated with this mutation. Taken together, we have identified a novel role for α-SMA in driving differentiation of SMCs, and our data supports that defects in this nuclear role drive cellular phenotypes consistent with the cerebrovascular disease seen in patients with ACTA2 R179 pathogenic variants.

Actin-related protein 5 functions as a novel modulator of MyoD and MyoG in skeletal muscle and in rhabdomyosarcoma.

Myogenic regulatory factors (MRFs) are pivotal transcription factors in myogenic differentiation. MyoD commits cells to the skeletal muscle lineage by inducing myogenic genes through recruitment of chromatin remodelers to its target loci. This study showed that actin-related protein 5 (Arp5) acts as an inhibitory regulator of MyoD and MyoG by binding to their cysteine-rich (CR) region, which overlaps with the region essential for their epigenetic functions. Arp5 expression was faint in skeletal muscle tissues. Excessive Arp5 in mouse hind limbs caused skeletal muscle fiber atrophy. Further, Arp5 overexpression in myoblasts inhibited myotube formation by diminishing myogenic gene expression, whereas Arp5 depletion augmented myogenic gene expression. Arp5 disturbed MyoD-mediated chromatin remodeling through competition with the three-amino-acid-loop-extension-class homeodomain transcription factors the Pbx1-Meis1 heterodimer for binding to the CR region. This antimyogenic function was independent of the INO80 chromatin remodeling complex, although Arp5 is an important component of that. In rhabdomyosarcoma (RMS) cells, Arp5 expression was significantly higher than in normal myoblasts and skeletal muscle tissue, probably contributing to MyoD and MyoG activity dysregulation. Arp5 depletion in RMS partially restored myogenic properties while inhibiting tumorigenic properties. Thus, Arp5 is a novel modulator of MRFs in skeletal muscle differentiation.

A hexasome is the preferred substrate of the INO80 chromatin remodeling complex

The critical role of the INO80 chromatin-remodeling complex in transcription is commonly attributed to its nucleosome sliding activity. We’ve found that INO80 prefers to mobilize hexasomes over nucleosomes by ∼60-fold. INO80’s preference for hexasomes is highest when linker DNA lengths approach the ∼18 bp linkers in yeast gene bodies. Correspondingly, deletion of INO80 significantly affects the positions of hexasome-sized particles within yeast genes in vivo. Our results suggest a model in which the Arp5 and Ies6 subunits of INO80 promote nucleosome sliding by dislodging an H2A/H2B dimer, thereby making a nucleosome transiently resemble a hexasome.

INO80 requires a polycomb subunit to regulate the establishment of poised chromatin in murine spermatocytes.

INO80 is the catalytic subunit of the INO80-chromatin remodeling complex that is involved in DNA replication, repair and transcription regulation. Ino80 deficiency in murine spermatocytes (Ino80cKO) results in pachytene arrest of spermatocytes due to incomplete synapsis and aberrant DNA double-strand break repair, which leads to apoptosis. RNA-seq on Ino80cKO spermatocytes revealed major changes in transcription, indicating that an aberrant transcription program arises upon INO80 depletion. In Ino80WT spermatocytes, genome-wide analysis showed that INO80-binding sites were mostly promoter proximal and necessary for the regulation of spermatogenic gene expression, primarily of premeiotic and meiotic genes. Furthermore, most of the genes poised for activity, as well as those genes that are active, shared INO80 binding. In Ino80cKO spermatocytes, most poised genes demonstrated de-repression due to reduced H3K27me3 enrichment and, in turn, showed increased expression levels. INO80 interacts with the core PRC2 complex member SUZ12 and promotes its recruitment. Furthermore, INO80 mediates H2A.Z incorporation at the poised promoters, which was reduced in Ino80cKO spermatocytes. Taken together, INO80 is emerging as a major regulator of the meiotic transcription program by mediating poised chromatin establishment through SUZ12 binding.

Abstract P174: Nuclear Smooth Muscle α-actin Is Critical For Smooth Muscle Cell Differentiation And To Prevent Cerebrovascular Disease

Missense mutations in ACTA2, encoding α-smooth muscle actin (α-SMA), predispose to thoracic aortic aneurysms. A subset of these ACTA2 mutations also predispose to childhood onset cerebrovascular disease characterized by occlusion of the distal internal carotid arteries and small vessel disease. In our studies to identify how specific ACTA2 mutations predispose to cerebrovascular disease, we confirmed that α-SMA translocates to the nucleus in wildtype (WT) smooth muscle cells (SMCs), is enriched in the nucleus over β-actin with differentiation of SMCs, associates with the INO80 chromatin remodeling complex, and selectively binds to the promoters of SMC contractile genes. Expression of individual mutant α-SMAs in 293 cells determined that missense mutations associated with cerebrovascular disease inhibit this nuclear translocation. To further examine the effects of reduced nuclear α-SMA with ACTA2 mutations, we used two model systems: pluripotent stem cell-derived SMCs from patients with ACTA2-associated cerebrovascular disease (ACTA2 R179C) as well as SMCs explanted from a knockin mouse model (Acta2SMC-R179C/+). Both mouse and human cells harboring the R179C mutation proliferate and migrate more than WT SMCs, are less differentiated than WT SMCs, and have increased expression of pluripotency-associated genes. Although the mutation is heterozygous, these cells show a dominant negative impact on nuclear α-SMA function through dramatically reduced levels of α-SMA in the nucleus, in the INO80 chromatin remodeling complex, and on the promoters of SMC-specific genes. Finally, forced nuclear localization of α-SMA in WT SMCs increases levels of SMC contractile proteins. Taken together, we have identified a novel role for α-SMA in driving differentiation of SMCs, and our data supports that defects in this nuclear role drive cellular phenotypes consistent with the cerebrovascular disease seen in patients with ACTA2 R179 mutations.

COMPASS functions as a module of the INO80 chromatin remodeling complex to mediate histone H3K4 methylation in Arabidopsis.

In the INO80 chromatin remodeling complex, all of the accessory subunits are assembled on the following three domains of INO80: N-terminal domain (NTD), HSA domain, and ATPase domain. Although the ATPase and HSA domains and their interacting accessory subunits are known to be responsible for chromatin remodeling, it is largely unknown how the accessory subunits that interact with the INO80 NTD regulate chromatin status. Here, we identify both conserved and nonconserved accessory subunits that interact with the three domains in the INO80 complex in Arabidopsis thaliana. While the accessory subunits that interact with all the three INO80 domains can mediate transcriptional repression, the INO80 NTD and the accessory subunits interact with it can contribute to transcriptional activation even when the ATPase domain is absent, suggesting that INO80 has an ATPase-independent role. A subclass of the COMPASS histone H3K4 methyltransferase complexes interact with the INO80 NTD in the INO80 complex and function together with the other accessory subunits that interact with the INO80 NTD, thereby facilitating H3K4 trimethylation and transcriptional activation. This study suggests that the opposite effects of the INO80 complex on transcription are required for the balance between vegetative growth and flowering under diverse environmental conditions.

The INO80 chromatin remodeling complex promotes thermomorphogenesis by connecting H2A.Z eviction and active transcription in Arabidopsis

Global warming poses a major threat to plant growth and crop production. In some plants, including Arabidopsis thaliana, elevated temperatures induce a series of morphological and developmental adjustments termed thermomorphogenesis, which facilitates plant cooling under high-temperature conditions. Plant thermal response is suppressed by histone variant H2A.Z. At warm temperatures, H2A.Z is evicted from nucleosomes at thermoresponsive genes, resulting in changes in their expression. However, the mechanisms that regulate H2A.Z eviction and subsequent transcriptional changes are largely unknown. Here, we show that the INO80 chromatin remodeling complex (INO80-C) promotes thermomorphogenesis and activates the expression of thermoresponsive and auxin-related genes. INO80-C associates with PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), a potent regulator of thermomorphogenesis, and mediates temperature-induced H2A.Z eviction at PIF4 targets. Moreover, INO80-C directly interacts with COMPASS-like and transcription elongation factors to promote active histone modification, histone H3 lysine 4 trimethylation, and RNA polymerase II elongation, leading to the thermal induction of transcription. Notably, the transcription elongation factors SPT4 and SPT5 are required for H2A.Z eviction at PIF4 targets, suggesting the cooperation of INO80-C and transcription elongation in H2A.Z removal. Taken together, these results suggest that the (PIF4)-(INO80-C)-(COMPASS-like)-(transcription elongator) module controls plant thermal response, thereby establishing a link between H2A.Z eviction and active transcription.

PHYTOCHROME-INTERACTING FACTORs trigger environmentally responsive chromatin dynamics in plants.

The interplay between light receptors and PHYTOCHROME-INTERACTING FACTORs (PIFs) serves as a regulatory hub that perceives and integrates environmental cues into transcriptional networks of plants1,2. Although occupancy of the histone variant H2A.Z and acetylation of histone H3 have emerged as regulators of environmentally responsive gene networks, how these epigenomic features interface with PIF activity is poorly understood3-7. By taking advantage of rapid and reversible light-mediated manipulation of PIF7 subnuclear localization and phosphorylation, we simultaneously assayed the DNA-binding properties of PIF7, as well as its impact on chromatin dynamics genome wide. We found that PIFs act rapidly to reshape the H2A.Z and H3K9ac epigenetic landscape in response to a change in light quality. Furthermore, we discovered that PIFs achieve H2A.Z removal through direct interaction with EIN6 ENHANCER (EEN), the Arabidopsis thaliana homolog of the chromatin remodeling complex subunit INO80 Subunit 6 (Ies6). Thus, we describe a PIF-INO80 regulatory module that is an intermediate step for allowing plants to change their growth trajectory in response to environmental changes.

CHIP and BAP1 Act in Concert to Regulate INO80 Ubiquitination and Stability for DNA Replication.

The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its half-life. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.