The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80\'s ATPase activity to nucleosome movement. Our investigation reveals that the grappler domain of Arp5 interacts with the acidic pocket of nucleosomes through two distinct mechanisms: an arginine anchor or a hydrophobic/acidic patch. These two modes of binding serve distinct functions within INO80 as shown in vivo by mutations in these regions resulting in varying phenotypes and in vitro by diverse effects on nucleosome mobilization. Our findings suggest that the hydrophobic/acidic patch of Arp5 is likely important for dimer exchange by INO80, while the arginine anchor is crucial for mobilizing nucleosomes.

The aberrant function of ATP-dependent chromatin remodeler INO80 has been implicated in multiple types of cancers by altering chromatin architecture and gene expression; however, the underlying mechanism of the functional involvement of INO80 mutation in cancer etiology, especially in breast cancer, remains unclear. In the present study, we have performed a weighted gene co-expression network analysis (WCGNA) to investigate links between INO80 expression and breast cancer sub-classification and progression. Our analysis revealed that INO80 repression is associated with differential responsiveness of estrogen receptors (ERs) depending upon breast cancer subtype, ER networks, and increased risk of breast carcinogenesis. To determine whether INO80 loss induces breast tumors, a conditional INO80-knockout (INO80 cKO) mouse model was generated using the Cre-loxP system. Phenotypic characterization revealed that INO80 cKO led to reduced branching and length of the mammary ducts at all stages. However, the INO80 cKO mouse model had unaltered lumen morphology and failed to spontaneously induce tumorigenesis in mammary gland tissue. Therefore, our study suggests that the aberrant function of INO80 is potentially associated with breast cancer by modulating gene expression. INO80 mutation alone is insufficient for breast tumorigenesis.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells activated the DNA damage checkpoint due to persistent double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and non-homologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. In order to understand how Hhp1 and Hhp2 promote DSB repair, we identified new substrates using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 that is important for DSB repair. Our data suggest that Hhp1 and Hhp2 facilitate DSB repair by phosphorylating multiple substrates, including Arp8.

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.

Autophagy is a catabolic process to maintain homeostasis, and involved in cell differentiation and development. Autophagy is tightly regulated in response to nutrient availability but the underlying mechanism is not completely understood. Recently, we identified the chromatin remodeling complex INO80 (inositol-requiring mutant 80) and histone variant H2A.Z as new autophagy regulators and uncover how histone deacetylase Rpd3L (reduced potassium dependency 3 large) complex represses autophagy by deacetylating Ino80 and H2A.Z. In particular, Rpd3L complex deacetylates Ino80 at lysine 929, which protects Ino80 from being degraded by autophagy. The stabilized Ino80 then evicts H2A.Z from autophagy-related (ATG) genes, leading to their transcriptional repression. In parallel, Rpd3L complex also deacetylates H2A.Z, which further reduces its association with ATG gene promoters and repress ATG gene transcription. Under nutrient-rich conditions, Rpd3L-mediated deacetylation of Ino80 K929 and H2A.Z is enhanced by the TORC1 complex (target of rapamycin complex 1). Under nitrogen-starvation condition, TORC1 is inactivated, leading to reduced activity of Rpd3L complex and increased acetylation of Ino80 and H2A.Z, which in turn induce the transcription of ATG genes. These results reveal a critical role of chromatin remodelers and histone variants in regulating autophagy in response to nutrient availability.Abbreviations: INO80: inositol-requiring mutant 80; Rpd3: reduced potassium dependency 3; H2A.Z: histone H2A variant; Rpd3L complex: Rpd3 large complex; H4K16: H4 lysine 16; H3R17: H3 arginine 17; H3T11: H3 threonine 11; TORC1 complex: target of rapamycin complex 1; ATG: autophagy-related; SWI/SNF: switch/sucrose non-fermentable; SWR1: Swi2/Snf2-related ATPase complex; RSC: remodel the structure of chromatin; ISWI: imitation switch; CHD1: chromodomain helicase DNA binding protein 1; Arp8: actin-related protein 8; Sds3: suppressor of defective silencing 3; Ume6: unscheduled meiotic gene expression 6.

Gene transcription is a highly regulated process in all animals. In Drosophila, two major transcriptional programs, housekeeping and developmental, have promoters with distinct regulatory compatibilities and nucleosome organization. However, it remains unclear how the differences in chromatin structure relate to the distinct regulatory properties and which chromatin remodelers are required for these programs. Using rapid degradation of core remodeler subunits in Drosophila melanogaster S2 cells, we demonstrate that developmental gene transcription requires SWI/SNF-type complexes, primarily to maintain distal enhancer accessibility. In contrast, wild-type-level housekeeping gene transcription requires the Iswi and Ino80 remodelers to maintain nucleosome positioning and phasing at promoters. These differential remodeler dependencies relate to different DNA-sequence-intrinsic nucleosome affinities, which favor a default ON state for housekeeping but a default OFF state for developmental gene transcription. Overall, our results demonstrate how different transcription-regulatory strategies are implemented by DNA sequence, chromatin structure, and remodeler activity.

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 in vivo. 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.

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.

The Drosophila proteins Pleiohomeotic (Pho) and its paralog Pho-like (Phol) are the homologs of the mammalian transcription factor YY1. Pho and Phol are subunits of the Polycomb group protein complex PhoRC and they are also stably associated with the INO80 nucleosome remodeling complex. Drosophila lacking both Pho and Phol arrest development as larvae with small misshaped imaginal discs. The basis of this phenotype is poorly understood. We find that in pho phol mutant animals cells retain the capacity to proliferate but show a high incidence of apoptotic cell death that results in tissue hypoplasia. Clonal analyses establish that cells stringently require Pho and Phol to survive. In contrast, the PhoRC subunit Sfmbt and the ATP-dependent nucleosome remodeling factor Ino80 are not essential for cell viability. Pho and Phol, therefore, execute their critical role for cell survival through mechanisms that do not involve Sfmbt function or INO80 nucleosome remodeling.

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.
INO80蛋白是一种染色质重塑因子，参与调控胚胎干细胞多能性的维持以及体细胞诱导为多能干细胞的重编程过程。然而，INO80在猪着床前胚胎发育中的作用及机制尚不清楚。该研究中，我们证实INO80调节滋养层细胞的渗透能力以促进猪囊胚发育。在桑椹胚向囊胚转变过程，INO80蛋白在胚胎细胞核中呈高表达。功能研究证明RNA干扰介导的INO80敲低导致囊胚形成失败，并破坏囊胚中内细胞团和滋养层细胞谱系的分布。单胚胎RNA测序分析揭示INO80调控许多与囊胚细胞谱系分化、紧密连接组装和囊胚腔液体积累等基因的表达。细胞渗透性分析证实INO80敲低囊胚滋养层细胞之间的细胞封闭发生缺陷。对照组和INO80敲低组8-细胞胚胎的聚合试验表明，通过补偿缺陷型滋养层细胞可以诱导INO80敲低胚胎发育成囊胚并恢复正常的细胞谱系分布。因此，以上研究结果证明染色质重塑因子INO80通过调控细胞谱系特化、紧密连接组装和液体聚集等关键基因的表达以促进囊胚发育。.