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Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells’ fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER–mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory–like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. Significance: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells’ fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER–mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory–like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control.Significance:Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

Supplementary Figure from Carbon Monoxide Activates PERK-Regulated Autophagy to Induce Immunometabolic Reprogramming and Boost Antitumor T-cell Function

Adoptive transfer of tumor epitope–reactive T cells has emerged as a promising strategy to control tumor growth. However, chronically-stimulated T cells expanded for adoptive cell transfer are susceptible to cell death in an oxidative tumor microenvironment. Because oxidation of cell-surface thiols also alters protein functionality, we hypothesized that increasing the levels of thioredoxin (Trx), an antioxidant molecule facilitating reduction of proteins through cysteine thiol-disulfide exchange, in T cells will promote their sustained antitumor function. Using pre-melanosome protein (Pmel)–Trx1 transgenic mouse-derived splenic T cells, flow cytometry, and gene expression analysis, we observed here that higher Trx expression inversely correlated with reactive oxygen species and susceptibility to T-cell receptor restimulation or oxidation-mediated cell death. These Trx1-overexpressing T cells exhibited a cluster of differentiation 62L(hi) (CD62L(hi)) central memory-like phenotype with reduced glucose uptake (2-NBDG(lo)) and decreased effector function (interferon γ(lo)). Furthermore, culturing tumor-reactive T cells in the presence of recombinant Trx increased the dependence of T cells on mitochondrial metabolism and improved tumor control. We conclude that strategies for increasing the antioxidant capacity of antitumor T cells modulate their immunometabolic phenotype leading to improved immunotherapeutic control of established tumors.

Mitochondria and Endoplasmic reticulum (ER) share structural and functional networks to maintain cellular homeostasis and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS condition drives mitochondrial biogenesis and function. So far, various strategies have been tested to enhance the mitochondrial function in T cells to improve their anti-tumor potential. However, strategies targeting ER-Mitochondria crosstalk (by employing moderate ERS condition) to boost the anti-tumor T cell function have not been exploited yet. Thus, in the present study, we used carbon monoxide (CO), a short-lived gaseous molecule, to test if engaging moderate ERS conditions can improve T cells mitochondrial biogenesis, function, and anti-tumor therapeutic efficacy. Using melanoma antigen gp100 reactive T cells, we identify that CO induced transient activation of ERS sensor ‘protein kinase R-like endoplasmic reticulum kinase (PERK)’ dramatically increase anti-tumor T cell function. Furthermore, mechanistically we found that CO-induced PERK activation temporarily halts protein translation and induces protective autophagy (that includes mitophagy). To get further insight into how CO treatment kinetically regulates autophagic activity in T cells, we used LC3-GFP to differentiate between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter into the process (LC3-GFPneg). Using Ag-specific LC3-GFP expressing T cells, we demonstrate that LC3-GFPpos T cells show robust anti-tumor potential. Moreover, LC3-GFPneg shows a T regulatory-like phenotype, harbors dysfunctional mitochondria, and accumulates abnormal metabolite content. These anomalous ratios of metabolites render the cells with a hypermethylated state and distinct epigenetic profile limiting their anti-tumor activity. Overall, this preclinical data highlights that ERS-mediated activation of autophagy pathways modifies the mitochondrial function and epigenetically reprogram the T cells towards a superior anti-tumor phenotype and can be therapeutically translated to achieve robust tumor control in adoptive transfer therapies. Citation Format: Paramita Chakraborty, Rasesh Y. Parikh, Seungho Choi, Meenal Mehrotra, Eduardo N. Maldonado, Hongjun Wang, J. Alan Diehl, Vamsi K. Gangaraju, Shikhar Mehrotra. Carbon monoxide induced PERK regulated autophagy reprograms anti-tumor T cells with robust immunometabolic phenotype [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 LB014.

Over 1.2 million daily total tests are currently being performed for SARS-CoV-2, in the United States. The most common SARS-CoV-2 tests require RNA extraction and purification. Extraction of RNA is a time-consuming and costly step that requires a constant supply of reagents and accessories. With the current testing demand, the supply chain remains the bottleneck for RNA extraction. Here, we report Direct NP- a cost-effective extraction-free RT-qPCR based dualplex test for SARS-CoV-2 from NP swab specimens. Direct NP detects SARS-CoV-2 viral RNA from heat-denatured patient specimens using a dualplex RT-qPCR assay. Direct NP showed 92.5% positive percentage agreement (PPA) (95% Confidence Interval (CI) = 79.61% to 98.43%) and 97% negative percent agreement (NPA) (95% CI = 89.11 to 100%) with the CDC assay. Direct NP reduces the cost per test to $2, making it suitable for broad-scale testing while lowering the cost burden on the healthcare system. Funding: This work was funded by the Enterprise Strategic Funding for Pandemic Response from the Medical University of South Carolina. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: IRB review was waived by the Institutional Review Board for Human Research (IRB), a part of the Office of Research Integrity at the Medical University of South Carolina as this study is a process development and samples were deidentified and blinded.

Mitochondria and Endoplasmic reticulum (ER) share structural and functional networks and activates well-orchestrated signaling processes to shape a cell’s fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS condition promotes mitochondrial function. Strategies to boost anti-tumor T cell function by targeting ER-Mitochondria cross talk have not been exploited yet. Thus, we used carbon monoxide (CO), a short-lived gaseous molecule, to test if engaging moderate ERS conditions can improve T cells mitochondrial function and anti-tumor function. Using melanoma antigen specific T cells, we identify that CO induced transient activation of ERS sensor ‘protein kinase R-like endoplasmic reticulum kinase (PERK)’ dramatically increase anti-tumor T cell function. Furthermore, CO-induced PERK activation temporarily halts protein translation and induces protective autophagy (that includes mitophagy). To get further insight, we used LC3-GFP to differentiate between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter into the process (LC3-GFPneg). We found that LC3-GFPpos T cells show robust anti-tumor potential whereas, LC3-GFPneg cells show T regulatory-like phenotype, harbor dysfunctional mitochondria, and accumulate abnormal metabolite content. These anomalous ratios of metabolites render the cells with a hypermethylated state and distinct epigenetic profile limiting their anti-tumor activity. Overall, the study highlights that ERS activated autophagy pathways modify mitochondrial function and epigenetically reprogram the T cells towards a superior anti-tumor phenotype to achieve robust tumor control.

Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)–dependent ping-pong cycle generates most germline piRNAs. Loading of antisense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.

Piwi-interacting RNAs (piRNAs) are 26–30-nucleotide germ line-specific small non-coding RNAs that have evolutionarily conserved function in mobile genetic element (transposons) silencing and maintenance of genome integrity. Drosophila Hsp70/90-organizing protein homolog (Hop), a co-chaperone, interacts with piRNA-binding protein Piwi and mediates silencing of phenotypic variations. However, it is not known whether Hop has a direct role in piRNA biogenesis and transposon silencing. Here, we show that knockdown of Hop in the germ line nurse cells (GLKD) of Drosophila ovaries leads to activation of transposons. Hop GLKD females can lay eggs at the same rate as wild-type counterparts, but the eggs do not hatch into larvae. Hop GLKD leads to the accumulation of γ-H2Av foci in the germ line, indicating increased DNA damage in the ovary. We also show that Hop GLKD-induced transposon up-regulation is due to inefficient piRNA biogenesis. Based on these results, we conclude that Hop is a critical component of the piRNA pathway and that it maintains genome integrity by silencing transposons.

The SARS-CoV-2 viral pandemic has induced a global health crisis, which requires more in-depth investigation into immunological responses to develop effective treatments and vaccines. To understand protective immunity against COVID-19, we screened over 60,000 asymptomatic individuals in the Southeastern United States for IgG antibody positivity against the viral spike protein, and approximately three percent were positive. Of these three percent, individuals with the highest anti-S or anti-RBD IgG level showed a strong correlation with inhibition of ACE2 binding and cross-reactivity against non-SARS-CoV-2 coronavirus S-proteins. We also analyzed samples from 94 SARS-CoV-2 patients and compared them to asymptomatic individuals. SARS-CoV-2 symptomatic patients had decreased antibody responses, ACE2 binding inhibition, and antibody cross-reactivity. Our study shows that healthy individuals can mount robust immune responses against SARS-CoV-2 without symptoms. Further, IgG antibody responses against S and RBD may correlate with high inhibition of ACE2 binding in individuals tested for SARS-CoV-2 infection or vaccination., Graphical Abstract

A well-studied RNA-binding protein Hu Antigen-R (HuR), controls post-transcriptional gene regulation and undergoes stress-activated caspase-3 dependent cleavage in cancer cells. The cleavage products of HuR are known to promote cell death; however, the underlying molecular mechanisms facilitating caspase-3 activation and HuR cleavage remains unknown. Here, we show that HuR cleavage associated with active caspase-3 in oral cancer cells treated with ionizing radiation and chemotherapeutic drug, paclitaxel. We determined that oral cancer cells overexpressing cyclooxygenase-2 (COX-2) limited the cleavage of caspase-3 and HuR, which reduced the rate of cell death in paclitaxel resistant oral cancer cells. Specific inhibition of COX-2 by celecoxib, promoted apoptosis through activation of caspase-3 and cleavage of HuR in paclitaxel-resistant oral cancer cells, both in vitro and in vivo. In addition, oral cancer cells overexpressing cellular HuR increased the half-life of COX-2 mRNA, promoted COX-2 protein expression and exhibited enhanced tumor growth in vivo in comparison with cells expressing a cleavable form of HuR. Finally, our ribonucleoprotein immunoprecipitation and sequencing (RIP-seq) analyses of HuR in oral cancer cells treated with ionizing radiation (IR), determined that HuR cleavage product-1 (HuR-CP1) bound and promoted the expression of mRNAs encoding proteins involved in apoptosis. Our results indicated that, cellular non-cleavable HuR controls COX-2 mRNA expression and enzymatic activity. In addition, overexpressed COX-2 protein repressed the cleavage of caspase-3 and HuR to promote drug resistance and tumor growth. Altogether, our observations support the use of the COX-2 inhibitor celecoxib, in combination with paclitaxel, for the management of paclitaxel resistant oral cancer cells.

Formation of membrane pores/channels regulates various cellular processes, such as necroptosis or stem cell niche signaling. However, the roles of membrane lipids in the formation of pores and their biological functions are largely unknown. Here, using the cellular stress model evoked by the sphingolipid analog drug FTY720, we show that formation of ceramide-enriched membrane pores, referred to here as ceramidosomes, is initiated by a receptor-interacting Ser/Thr kinase 1 (RIPK1)–ceramide complex transported to the plasma membrane by nonmuscle myosin IIA–dependent trafficking in human lung cancer cells. Molecular modeling/simulation coupled with site-directed mutagenesis revealed that Asp(147) or Asn(169) of RIPK1 are key for ceramide binding and that Arg(258) or Leu(293) residues are involved in the myosin IIA interaction, leading to ceramidosome formation and necroptosis. Moreover, generation of ceramidosomes independently of any external drug/stress stimuli was also detected in the plasma membrane of germ line stem cells in ovaries during the early stages of oogenesis in Drosophila melanogaster. Inhibition of ceramidosome formation via myosin IIA silencing limited germ line stem cell signaling and abrogated oogenesis. In conclusion, our findings indicate that the RIPK1–ceramide complex forms large membrane pores we named ceramidosomes. They further suggest that, in addition to their roles in stress-mediated necroptosis, these ceramide-enriched pores also regulate membrane integrity and signaling and might also play a role in D. melanogaster ovary development.

Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)–dependent ping-pong cycle generates most germline piRNAs. Loading of antisense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.

The contribution of lncRNAs to tumour progression and the regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleocytoplasmic translocation or in response to TGFβ, allows alternative splicing and generates a non-coding isoform of PNUTS. Functionally the lncRNA-PNUTS serves as a competitive sponge for miR-205 during epithelial-mesenchymal transition (EMT). In mesenchymal breast tumour cells and in breast tumour samples, the expression of lncRNA-PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS mRNA and lncRNA-PNUTS, each eliciting distinct biological functions. While PNUTS mRNA is ubiquitously expressed, lncRNA-PNUTS appears to be tightly regulated dependent on the status of hnRNP E1 and tumour context.

Haifan Lin and colleagues report the identification in Drosophila of a protein complex composed of Hsp90 Piwi, and the Hsp70/Hsp90 Organizing Protein Homolog (Hop) and demonstrate the function of this complex in buffering against morphological changes in a sensitized background.

The hallmark of a stem cell is its ability to self-renew and to produce numerous differentiated cells. This unique property is controlled by dynamic interplays between extrinsic signalling, epigenetic, transcriptional and post-transcriptional regulations. Recent research indicates that microRNAs (miRNAs) have an important role in regulating stem cell self-renewal and differentiation by repressing the translation of selected mRNAs in stem cells and differentiating daughter cells. Such a role has been shown in embryonic stem cells, germline stem cells and various somatic tissue stem cells. These findings reveal a new dimension of gene regulation in controlling stem cell fate and behaviour.

Piwi proteins associate with piRNAs and functions in epigenetic programming, post-transcriptional regulation, transposon silencing, and germline development. However, it is not known whether the diverse functions of these proteins are molecularly separable. Here we report that Piwi interacts with Tudor-SN (Tudor staphylococcal nuclease, TSN) antagonistically in regulating spermatogenesis but synergistically in silencing transposons. However, it is not required for piRNA biogenesis. TSN is known to participate in diverse molecular functions such as RNAi, degradation of hyper-edited miRNAs, and spliceosome assembly. We show that TSN colocalizes with Piwi in primordial germ cells (PGCs) and embryonic somatic cells. In adult ovaries and testes, TSN is ubiquitously expressed and enriched in the cytoplasm of both germline and somatic cells. The tsn mutants display a higher mitotic index of spermatogonia, accumulation of spermatocytes, defects in meiotic cytokinesis, a decreased number of spermatids, and eventually reduced male fertility. Germline-specific TSN-expression analysis demonstrates that this function is germline-dependent. Different from other known Piwi interters, TSN represses Piwi expression at both protein and mRNA levels. Furthermore, reducing piwi expression in the germline rescues tsn mutant phenotype in a dosage-dependent manner, demonstrating that Piwi and TSN interact antagonistically in germ cells to regulate spermatogenesis. However, the tsn deficiency has little, if any, impact on piRNA biogenesis but displays a synergistic effect with piwi mutants in transposon de-silencing. Our results reveal the biological function of TSN and its contrasting modes of interaction with Piwi in spermatogenesis, transposon silencing, and piRNA biogenesis., Author Summary Piwi proteins bind to a large class of small noncoding RNAs called Piwi-interacting RNAs (piRNAs). These proteins have emerged as major players in germline development, stem cell self-renewal, transposon silencing, and gene regulation. However, it is not known whether these functions of Piwi proteins represent separate molecular mechanisms. Furthermore, although multiple Piwi interactors have been identified, including Tudor-domain-containing proteins, none of them regulates Piwi expression or interacts with Piwi antagonistically, or only impact on a subset of Piwi functions. Here we show that Drosophila Piwi interacts with a special Tudor-domain-containing protein called Tudor-SN (Tudor staphylococcal nuclease, TSN). TSN is drastically different from the known Piwi interactors because it represses Piwi mRNA and protein expression and interacts with Piwi antagonistically in spermatogenesis but synergistically in transposon silencing. However, this interaction is not required for piRNA biogenesis. Our study represents the first demonstration that different functions of Piwi are mediated by different molecular mechanisms. In addition, this is the first in vivo study that reveals the biological function of TSN protein in an organism.

Bisulfite genomic sequencing provides a single-molecule view of cytosine methylation states. After deamination, each cloned molecule contains a record of methylation within its sequence. The full power of this technique is harnessed by treating nuclei with an exogenous DNMT prior to DNA extraction. This exogenous methylation marks regions of accessibility and footprints nucleosomes, as well as other DNA-binding proteins. Thus, each cloned molecule records not only the endogenous methylation present (at CG sites, in mammals), but also the exogenous (GC, when using the Chlorella virus protein M.CviPI). We term this technique MAPit, methylation accessibility protocol for individual templates.

Bisulfite genomic sequencing provides a single-molecule view of cytosine methylation states. After deamination, each cloned molecule contains a record of methylation within its sequence. The full power of this technique is harnessed by treating nuclei with an exogenous DNMT prior to DNA extraction. This exogenous methylation marks regions of accessibility and footprints nucleosomes, as well as other DNA-binding proteins. Thus, each cloned molecule records not only the endogenous methylation present (at CG sites, in mammals), but also the exogenous (GC, when using the Chlorella virus protein M.CviPI). We term this technique MAPit, methylation accessibility protocol for individual templates.

Distinct stages in ATP-dependent chromatin remodeling are found as ISW2, an ISWI-type complex, forms a stable and processive complex with nucleosomes upon hydrolysis of ATP. There are two conformational changes of the ISW2-nucleosome complex associated with binding and hydrolysis of ATP. The initial binding of ISW2 to extranucleosomal DNA, to the entry site, and near the dyad axis of the nucleosome is enhanced by ATP binding, whereas subsequent ATP hydrolysis is required for template commitment and causes ISW2 to expand its interactions with nucleosomal DNA to an entire gyre of the nucleosome and a short approximately 3-4 bp site on the other gyre. The histone-fold-like subunit Dpb4 associates with nucleosomal DNA approximately 15 bp from the ATPase domain as part of this change and may help to disrupt histone-DNA interactions. These additional contacts are independent of the ATPase domain tracking along nucleosomal DNA and are maintained as ISW2 moves nucleosomes on DNA.

Chromatin remodeling complexes make chromatin more accessible for various cellular processes inside the cell by either covalently modifying histones (34) or mobilizing the histone octamer along DNA in cis or trans (7). A link between these two distinct chromatin remodeling activities has been shown, and in some instances, they even reside together in the same complex (20, 44). ATP-dependent chromatin remodeling causes changes in the nucleosome translational position (12, 18, 21, 42), the exchange of histone variants (3, 9, 15, 19, 26, 29), and even the removal of nucleosomes (31). ISWI, a class of ATP-dependent chromatin remodeling complexes (24), forms a number of distinct complexes in Saccharomyces cerevisiae (36), Drosophila melanogaster (37), and vertebrates (22). These remodeling complexes have an ATPase subunit that belongs to the SWI2/SNF2 subfamily of DEAD/H helicases (8). The ISWI family of ATPases is characterized by SANT and SANT-like domains in the catalytic subunit (11) that have been proposed to interact with histone tails (1, 2). ISWI requires histone H4 tail for stimulation of the ATPase activity, and several of the ISWI class remodelers generate regularly spaced nucleosomal arrays (13, 36, 39) and facilitate the deposition of histones onto DNA (14). In S. cerevisiae, there are two distinct ISWI genes, ISW1 and ISW2 (36). Isw1 is in two different complexes, ISW1a (Isw1 and Ioc3) and ISW1b (Isw1, Ioc2, and Ioc4), in addition to being present as a monomer (41). While no known protein motifs are evident in Ioc3, Ioc2 has a PHD finger, and Ioc4 has a PWWP motif, a putative DNA-binding (30) and chromatin-targeting domain (10). ISW2 is a four-subunit complex composed of Isw2, Itc1, Dpb4, and Dls1 (25). ISW1a and ISW1b share the same catalytic subunit but exhibit distinct remodeling properties (41). The differences in the biochemical activities of these two complexes are probably connected to their different functional roles inside the cell and are not yet well understood. ISW1a was shown to regularly space nucleosomes in vitro every 175 bp, but not ISW1b, as suggested earlier, due to an inability to mobilize nucleosomes (41). The inability of ISW1b to mobilize nucleosomes has been attributed to its low affinity for nucleosomes. Recently, Stockdale et al. have shown that ISW1a behaves like ISW2 in mobilizing nucleosomes to their thermodynamically preferred positions along DNA (33). There are, however, conflicting data in regard to ISW2 and ISW1a about whether their direction of mobilization is dictated by the thermodynamically preferred positioning of nucleosomes. Recently, in vivo analysis of ISW2 sliding has shown that ISW2 slides nucleosomes onto unfavorable positions on DNA (43). Other studies have also shown that ISW2 and ISW1a can efficiently move nucleosomes off of a DNA sequence to which nucleosomes are bound with an affinity higher than any known natural sequence (46). Furthermore, it seems unlikely that the nucleosome-spacing activity of ISW1a can be explained as moving nucleosomes to their thermodynamically preferred positions on DNA. The reason for ISW1a uniformly spacing nucleosomes every 175 bp is not evident, since many other complexes can mobilize nucleosomes without spacing nucleosomes. Some of the molecular details that influence nucleosome spacing were recently shown for ISW2, which spaces nucleosomes approximately every 200 bp (16). ISW2 has a built-in molecular spacer with its noncatalytic subunit, Itc1, interacting with ∼60 bp of extranucleosomal DNA to sterically block nucleosomes from moving too close together. As nucleosomes are moved together, ISW2 has a reduced affinity for nucleosomes and is more likely to dissociate so that it can search for nucleosomes that are farther apart to remodel. This study reveals that ISW1a remodeling activity is regulated by whether it is bound to both or just one of the extranucleosomal DNAs.

The inter-relationship between DNA repair and ATP dependent chromatin remodeling has begun to become very apparent with recent discoveries. ATP dependent remodeling complexes mobilize nucleosomes along DNA, promote the exchange of histones, or completely displace nucleosomes from DNA. These remodeling complexes are often categorized based on the domain organization of their catalytic subunit. The biochemical properties and structural information of several of these remodeling complexes are reviewed. The different models for how these complexes are able to mobilize nucleosomes and alter nucleosome structure are presented incorporating several recent findings. Finally the role of histone tails and their respective modifications in ATP-dependent remodeling are discussed.