INO80 Complex Research Articles

The role of different subunits of INO80 remodeling complex in repair chromatin assembly in yeast <i>Saccharomyces cerevisiae</i>

Reparative chromatin assembly is an important step in maintaining genome stability. The correct assembly of chromatin is provided by histone chaperones, whose dysfunction can lead to the development of various forms of cancer and a number of hereditary diseases in humans. The effect of remodeling factors completes chromatin repair. The yeast chromatin remodeling complex INO80 plays an important role in chromatin architecture. We used induced mutagenesis and real-time PCR to study the role of INO80 in chromatin repair assembly. In double mutants ies5Δ hsm3Δ(hif1Δ), defects in the structure of nucleosomes caused by mutations hsm3Δ and hif1Δ lead to hypersensitivity of cells to UV radiation and the disappearance of hsm3- and hif1-specific mutagenesis. Double mutants carrying the nhp10Δ mutation and hsm3Δ or hif1Δ mutations were indistinguishable from a single mutant in terms of the lethal effect of UV irradiation, however, the high UV-induced mutagenesis characteristic of all mutations disappeared. Thus, we found that mutations in the genes controlling the subunits of the INO80 complex can exhibit strong interactions with mutations in histone chaperone genes. We have confirmed the hypothesis that the Him1 protein performs a chaperone function in the process of reparative chromatin assembly.

Truncating the spliceosomal ‘rope protein’ Prp45 results in Htz1 dependent phenotypes

ABSTRACT Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1–169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1–169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1–330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1–330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1–169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1–169), to the chromatin environment.

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.

Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma [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 138.

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.

Hexasome-INO80 complex reveals structural basis of noncanonical nucleosome remodeling.

Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5'-triphosphate-dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A-H2B. A large structural rearrangement switches the catalytic core of INO80 into a distinct, spin-rotated mode of remodeling while its nuclear actin module remains tethered to long stretches of unwrapped linker DNA. Direct sensing of an exposed H3-H4 histone interface activates INO80, independently of the H2A-H2B acidic patch. Our findings reveal how the loss of H2A-H2B grants remodelers access to a different, yet unexplored layer of energy-driven chromatin regulation.

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.

Resistance to Chemotherapeutic 5-Fluorouracil Conferred by Modulation of Heterochromatic Integrity through Ino80 Function in Fission Yeast

5-Fluorouracil (5-FU) is a conventional chemotherapeutic drug widely used in clinics worldwide, but development of resistance that compromises responsiveness remains a major hurdle to its efficacy. The mechanism underlying 5-FU resistance is conventionally attributed to the disruption of nucleotide synthesis, even though research has implicated other pathways such as RNA processing and chromatin dysregulation. Aiming to clarify resistance mechanisms of 5-FU, we tested the response of a collection of fission yeast (Schizosaccharomyces pombe) null mutants, which confer multiple environmental factor responsiveness (MER). Our screen identified disruption of membrane transport, chromosome segregation and mitochondrial oxidative phosphorylation to increase cellular susceptibility towards 5-FU. Conversely, we revealed several null mutants of Ino80 complex factors exhibited resistance to 5-FU. Furthermore, attenuation of Ino80 function via deleting several subunit genes reversed loss of chromosome-segregation fidelity in 5-FU in the loss-of-function mutant of the Argonaute protein, which regulates RNA interference (RNAi)-dependent maintenance of pericentromeric heterochromatin. Our study thus uncovered a critical role played by chromatin remodeling Ino80 complex factors in 5-FU resistance, which may constitute a possible target to modulate in reversing 5-FU resistance.

Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.

Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

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.

Structural mechanism of extranucleosomal DNA readout by the INO80 complex

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo-electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes.

Ino80 is required for H2A.Z eviction from hypha-specific promoters and hyphal development of Candida albicans.

ATP‐dependent chromatin remodeling complexes play important roles in many essential cellular processes, including transcription regulation, DNA replication, and repair. Evicting H2A.Z, a variant of histone H2A, from the promoter of hypha‐specific genes is required for hyphal formation in Candida albicans. However, the mechanism that regulates H2A.Z removal during hyphal formation remains unknown. In this study, we demonstrated that Ino80, the core catalytic subunit of the INO80 complex, was recruited to hypha‐specific promoters during hyphal induction in Arp8 dependent manner and facilitated the removal of H2A.Z. Deleting INO80 or mutating the ATPase site of Ino80 impairs the expression of hypha‐specific genes (HSGs) and hyphal development. In addition, we showed that Ino80 was essential for the virulence of C. albicans during systemic infections in mice. Interestingly, Arp5, an INO80 complex‐specific component, acts in concert with Ino80 during DNA damage responses but is dispensable for hyphal induction. Our findings clarified that Ino80 was critical for hyphal development, DNA damage response, and pathogenesis in C. albicans.

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.

Candidate therapeutic agents in a newly established triple wild-type mucosal melanoma cell line.

BackgroundMucosal melanoma has characteristically distinct genetic features and typically poor prognosis. The lack of representative mucosal melanoma models, especially cell lines, has hindered translational research on this melanoma subtype. In this study, we aimed to establish and provide the biological properties, genomic features and the pharmacological profiles of a mucosal melanoma cell line that would contribute to the understanding and treatment optimization of molecularly‐defined mucosal melanoma subtype.MethodsThe sample was collected from a 67‐year‐old mucosal melanoma patient and processed into pieces for the establishment of cell line and patient‐derived xenograft (PDX) model. The proliferation and tumorigenic property of cancer cells from different passages were evaluated, and whole‐genome sequencing (WGS) was performed on the original tumor, PDX, established cell line, and the matched blood to confirm the establishment and define the genomic features of this cell line. AmpliconArchitect was conducted to depict the architecture of amplified regions detected by WGS. High‐throughput drug screening (HTDS) assay including a total of 103 therapeutic agents was implemented on the established cell line, and selected candidate agents were validated in the corresponding PDX model.ResultsA mucosal melanoma cell line, MM9H‐1, was established which exhibited robust proliferation and tumorigenicity after more than 100 serial passages. Genomic analysis of MM9H‐1, corresponding PDX, and the original tumor showed genetic fidelity across genomes, and MM9H‐1 was defined as a triple wild‐type (TWT) melanoma subtype lacking well‐characterized “driver mutations”. Instead, the amplification of several oncogenes, telomerase reverse transcriptase (TERT), v‐Raf murine sarcoma viral oncogene homolog B1 (BRAF), melanocyte Inducing transcription factor (MITF) and INO80 complex ATPase subunit (INO80), via large‐scale genomic rearrangement potentially contributed to oncogenesis of MM9H‐1. Moreover, HTDS identified proteasome inhibitors, especially bortezomib, as promising therapeutic candidates for MM9H‐1, which was verified in the corresponding PDX model in vivo.ConclusionsWe established and characterized a new mucosal melanoma cell line, MM9H‐1, and defined this cell line as a TWT melanoma subtype lacking well‐characterized “driver mutations”. The MM9H‐1 cell line could be adopted as a unique model for the preclinical investigation of mucosal melanoma.

Nuclear Factor Related to KappaB Binding Protein (NFRKB) Is a Telomere-Associated Protein and Involved in Liver Cancer Development

Alternative lengthening of telomeres (ALT) is a homologous recombination-based telomere maintenance mechanism activated in 10-15% of human cancers. Although significant progress has been made, the key regulators of the ALT pathway and its role in cancer development remain elusive. Bioinformatics methods were used to predict novel telomere-associated proteins (TAPs) by analysis of large-scale ChIP-Seq data. Immunostaining and fluorescence in situ hybridization experiments were applied to detect the subcellular location of target genes and telomeres. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were used to examine the expression of targeting genes. Overall survival (OS) analyses were used to evaluate the relationship between gene expression and survival time; immunohistochemistry was used to detect the distribution of target genes in liver cancer tissues. We found that nuclear factor related to kappaB binding protein (NFRKB), a metazoan-specific subunit of the INO80 complex, can associate with telomeres in human ALT cells. Loss of NFRKB induces dysfunction of telomeres and less PML bodies in U2OS cells. In addition, NFRKB is low/moderately expressed in cytoplasm of normal hepatocytes but heavily accumulating in the nucleus of liver cancer cells. Finally, the high expression of NFRKB is associated with short OS time and poor prognosis. NFRKB is a TAP and protects telomeres from DNA damage in ALT cells. It is highly expressed in hepatocellular carcinoma (HCC) cells and predicts a poor prognosis. NFRKB may be a promising prognostic biomarker for the treatment of HCC in the future.

Complexes and complexities: INO80 takes center stage

Chromatin remodeling and transcriptional reprogramming are key mechanisms through which plants respond to environmental challenges. Nucleosomal dynamics associated with the presence/absence (incorporation/eviction) of the histone variant H2A.Z is one of the key chromatin remodeling mechanisms associated with environmental response (Coleman-Derr and Zilberman, 2012). Very recently, some excellent papers (Shang et al., 2021; Willige et al., 2021; Xue et al., 2021) have addressed the role of a less-explored component of this chromatin remodeling—the INO80 complex.

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.

Actin-Related Protein 6 (Arp6) Influences Double-Strand Break Repair in Yeast

DNA double-strand breaks (DSBs) are the most deleterious form of DNA damage and are repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Repair initiation, regulation and communication with signaling pathways require several histone-modifying and chromatin-remodeling complexes. In budding yeast, this involves three primary complexes: INO80-C, which is primarily associated with HR, SWR1-C, which promotes NHEJ, and RSC-C, which is involved in both pathways as well as the general DNA damage response. Here we identify ARP6 as a factor involved in DSB repair through an RSC-C-related pathway. The loss of ARP6 significantly reduces the NHEJ repair efficiency of linearized plasmids with cohesive ends, impairs the repair of chromosomal breaks, and sensitizes cells to DNA-damaging agents. Genetic interaction analysis indicates that ARP6, MRE11 and RSC-C function within the same pathway, and the overexpression of ARP6 rescues rsc2∆ and mre11∆ sensitivity to DNA-damaging agents. Double mutants of ARP6, and members of the INO80 and SWR1 complexes, cause a significant reduction in repair efficiency, suggesting that ARP6 functions independently of SWR1-C and INO80-C. These findings support a novel role for ARP6 in DSB repair that is independent of the SWR1 chromatin remodeling complex, through an apparent RSC-C and MRE11-associated DNA repair pathway.

RNAi and Ino80 complex control rate limiting translocation step that moves rDNA to eroding telomeres.

The discovery of HAATIrDNA, a telomerase-negative survival mode in which canonical telomeres are replaced with ribosomal DNA (rDNA) repeats that acquire chromosome end-protection capability, raised crucial questions as to how rDNA tracts ‘jump’ to eroding chromosome ends. Here, we show that HAATIrDNA formation is initiated and limited by a single translocation that juxtaposes rDNA from Chromosome (Chr) III onto subtelomeric elements (STE) on Chr I or II; this rare reaction requires RNAi and the Ino80 nucleosome remodeling complex (Ino80C), thus defining an unforeseen relationship between these two machineries. The unique STE-rDNA junction created by this initial translocation is efficiently copied to the remaining STE chromosome ends, independently of RNAi or Ino80C. Intriguingly, both RNAi and Ino80C machineries contain a component that plays dual roles in HAATI subtype choice. Dcr1 of the RNAi pathway and Iec1 of Ino80C both promote HAATIrDNA formation as part of their respective canonical machineries, but both also inhibit formation of the exceedingly rare HAATISTE (where STE sequences mobilize throughout the genome and assume chromosome end protection capacity) in non-canonical, pathway-independent manners. This work provides a glimpse into a previously unrecognized crosstalk between RNAi and Ino80C in controlling unusual translocation reactions that establish telomere-free linear chromosome ends.