Investigating the Role of the Nucleosome Remodeling Factor NURF as a Regulator of Gene Expression

The nucleosome remodeling factor (NURF) is an evolutionary conserved ATP-dependent chromatin remodeling factor. It was first isolated from Drosophila as a complex with enzymatic activity that once recruited to nucleosome, it slides the nucleosome to provide accessibility for transcription factors. Since then, numerous works from animal models and cell lines show the role of NURF as a regulator of gene expression. NURF interacts with H3K4me3 and sequence specific transcription factors that recruit the complex to promoter regions. Whether this is the only mechanism by which NURF regulates gene expression is not known. However, other ATP-dependent chromatin remodeling complexes are known to regulate gene expression independent from transcription initiation. In order to explore the role of NURF in regulating gene expression, we utilized two genome wide approaches to map NURF binding and NURF dependent changes in chromatin structure using ChIP-Seq and FAIRE-Seq, respectively. From these analyses, we discovered that NURF broadly localizes in the genome with preferences to gene bodies and 3’ends of genes. Also, we found that NURF maintains open chromatin regions at upstream, intron and downstream of genes. These novel findings shed light on new roles for NURF complex within genes, in addition to its classical role at promoter regions. Furthermore, we discovered the function of a previously uncharacterized domain in the NURF specific subunit BPTF. We show that the N-terminal the plant homeodomain (PHD) of BPTF directly interacts with THOC4, a protein associated with RNA-pol 2. Also, we show using ChIP analyses that this interaction recruits BPTF to gene bodies. Next, we investigated functional consequences for NURF recruitment to gene bodies using Cyclin D1 (Ccnd1) gene as a model. These analyses revealed that NURF is required for normal mRNA processing and loss of NURF induces intron retention, which results in unstable transcripts. Finally, we show that the defect in mRNA processing is not specific to the Ccnd1 gene, as we observe similar defects in four other BPTF dependent genes. Together, our work uncovered new role of mammalian NURF complex in regulating gene expression through mRNA processing

ATP-Dependent Histone Octamer Sliding Mediated by the Chromatin Remodeling Complex NURF

AbstractDrosophila NURF is an ATP-dependent chromatin remodeling complex that contains ISWI, a member of the SWI2/SNF2 family of ATPases. We demonstrate that NURF catalyzes the bidirectional redistribution of mononucleosomes reconstituted on hsp70 promoter DNA. In the presence of NURF, nucleosomes adopt one predominant position from an ensemble of possible locations within minutes. Movements occur in cis, with no transfer to competing DNA. Migrating intermediates trapped by Exo III digestion reveal progressive nucleosome motion in increments of several base pairs. All four core histones are retained quantitatively during this process, indicating that the general integrity of the histone octamer is maintained. We suggest that NURF remodels nucleosomes by transiently decreasing the activation energy for short-range sliding of the histone octamer

A Novel Pzg-NURF Complex Regulates Notch Target Gene Activity

The Putzig (Pzg) protein is associated with the NURF nucleosome remodeling complex, thereby promoting Notch target gene expression. Our findings suggest a novel Pzg-NURF complex that is responsible for the epigenetic regulation of Notch target genes

BPTF promotes tumor growth and predicts poor prognosis in lung adenocarcinomas.

BPTF, a subunit of NURF, is well known to be involved in the development of eukaryotic cell, but little is known about its roles in cancers, especially in non-small-cell lung cancer (NSCLC). Here we showed that BPTF was specifically overexpressed in NSCLC cell lines and lung adenocarcinoma tissues. Knockdown of BPTF by siRNA significantly inhibited cell proliferation, induced cell apoptosis and arrested cell cycle progress from G1 to S phase. We also found that BPTF knockdown downregulated the expression of the phosphorylated Erk1/2, PI3K and Akt proteins and induced the cleavage of caspase-8, caspase-7 and PARP proteins, thereby inhibiting the MAPK and PI3K/AKT signaling and activating apoptotic pathway. BPTF knockdown by siRNA also upregulated the cell cycle inhibitors such as p21 and p18 but inhibited the expression of cyclin D, phospho-Rb and phospho-cdc2 in lung cancer cells. Moreover, BPTF knockdown by its specific shRNA inhibited lung cancer growth in vivo in the xenografts of A549 cells accompanied by the suppression of VEGF, p-Erk and p-Akt expression. Immunohistochemical assay for tumor tissue microarrays of lung tumor tissues showed that BPTF overexpression predicted a poor prognosis in the patients with lung adenocarcinomas. Therefore, our data indicate that BPTF plays an essential role in cell growth and survival by targeting multiply signaling pathways in human lung cancers

Trithorax group proteins: switching genes on and keeping them active

Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology

BPTF Enhances Chemotherapy Induced Cytotoxicity

BPTF Enhances Chemotherapy Induced Cytotoxicity Valentina Posada, Depts. of Biology, Chemistry, & Religious Studies, with Dr. Joseph Landry, Dept. of Human Molecular Genetics New chemotherapies and immunotherapy treatments have greatly improved the outcomes of many cancers. However, for Triple Negative Breast Cancer (TNBC), existing therapies are not very effective long term as the disease becomes resistant and has low immunogenicity. Here we show the early development of a new way to treat the disease by combining existing chemotherapies with depletion of the Nucleosome Remodeling Factor (NURF). NURF is an ATP-dependent chromatin remodeling complex that is over-expressed in cancers and has shown to inhibit the anti-tumor immune response. The largest and essential subunit of the complex, BPTF is required for function. BPTF shRNA-mediated knockdown (KD) was done as a way to deplete cells of NURF. Our first aim was to determine if BPTF-KD cells showed enhanced sensitization to chemotherapies most prominently Doxorubicin. The results from completing this aim showed sensitization to several chemotherapies which correlated with enhanced therapy-induced autophagy. Our second aim was then to investigate the role of autophagy in the sensitization of BPTF-KD cells to chemotherapies. Autophagy is a process by which cells undergoing stress consume their cellular components. This process is mediated in part by the ATG5 protein. ATG5 KD was done through lentivirus transfection, and in turn, functional blockade of autophagy was achieved as confirmed by Western blotting. Results showed that BPTF-KD cells did not have enhanced sensitivity to Doxorubicin through the blockade of autophagy, which suggested a non-protective role in autophagy, while the BPTF-WT cells that had autophagy blocked did show an enhanced sensitization, suggesting a cytoprotective role. Aims were then tested in vivo to determine the role of autophagy in BPTF-KD cells in vivo. BPTF-KD and ATG5-KD 4T1 cells were transplanted into mice and tumor volume over time was measured. Syngeneic mouse models showed that the BPTF-KD tumors had significantly smaller tumor volumes than the control when treated with Doxorubicin, and therefore showed sensitization to Doxorubicin. Results for the ATG5 KD mice show tumors growing better in the WT while growing worse in the KD1/ KD2 mice suggesting that autophagy is required for sensitization of BPTF-KD tumors to Doxorubicin in vivo. The third aim of the project was to determine the possible immune-modulatory consequences of treating BPTF KD cells with chemotherapies. Natural Killer (NK) cells were depleted in mice to see if there would be a change in the sensitization to therapies. Results showed that once we depleted NK cells in mice with a mAb-depletion strategy, the sensitization to Doxorubicin was lost. Furthermore, a metabolomics screening was conducted and reductions in prostaglandin E2 (PGE2) were discovered in the therapy treated BPTF-KD cells. PGE2 is a well know immune suppressive metabolite produced by tumor cells to suppress the anti-tumor immune response. Further results showed PGE2 reductions when autophagy was blocked by ATG5 KD in the BPTF-KD cells. This result could explain the improvements in tumor growth within the mice since PGE2 is a known NK cell inhibitor. Together, these results suggest that NURF could be a therapeutic target for enhancing clinical outcomes in Triple Negative Breast Cancer Patients.https://scholarscompass.vcu.edu/uresposters/1328/thumbnail.jp

The Role of the Nucleosome Remodeling Factor NURF in Inhibiting T and Natural Killer Cell Mediated Antitumor Immunity by Suppressing Tumor Antigenicity and Natural Cytotoxicity Receptor Co-ligands

Tumor immunoediting is a dynamic process in which the immune response attacks tumor cells by detecting danger signals and tumor antigens. In order to survive, tumor cells develop mechanisms to avoid detection or destruction by the immune system. To counteract this, several strategies are being developed to enhance the antitumor immune response, including the depletion of immunosuppressive cells, enhancing the activation of antitumor immune cells and increasing tumor cell immunogenicity. These therapies have seen limited success individually, however, and it is likely that combination therapy with novel targets will be necessary to see reproducible beneficial responses. Epigenetic modifications are attractive therapeutic targets because they are reversible and affect gene expression in cancer cells. Within this framework, this study aimed to elucidate the role of the chromatin remodeling complex nucleosome remodeling factor (NURF) in cancer immunoediting by silencing of bromodomain PHD-finger containing transcription factor (BPTF), the largest and essential subunit of NURF. Using two syngeneic mouse models of cancer, BPTF was found to suppress T cell antitumor activity in the tumor microenvironment. In vitro, enhanced cytolytic activity was observed for individual CD8 T cell clones only from mice bearing BPTF-silenced tumors, implicating the involvement of novel antigens. Mechanistic investigations revealed that NURF directly suppresses the expression of genes encoding immunoproteasome subunits Psmb8 and Psmb9 and the antigen transporter genes Tap1 and Tap2. PSMB8 inhibition reversed the effects of BPTF ablation, consistent with a critical role for the immunoproteasome in improving tumor immunogenicity. Thus, NURF normally suppresses tumor cell antigenicity and its depletion improves CD8 T cell antitumor immunity. In a concurrent study using different tumor lines, BPTF was also found to suppress natural killer (NK) cell antitumor immunity in vivo. Enhanced NK cell cytolytic activity toward BPTF-depleted targets in vitro was dependent on the natural cytotoxicity receptors (NCR). Molecular studies revealed that BPTF directly activates heparanase (Hpse) expression, resulting in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans. Thus, NURF represses NCR co-ligand abundance and its depletion enhances NK cell cytotoxicity. Therefore, NURF emerges as a candidate therapeutic target to enhance CD8 T or NK cell antitumor immunity

ISWI, a member of the SWl2/SNF2 ATPase family, encodes the 140 kDa subunit of the nucleosome remodeling factor

AbstractThe generation of an accessible heat shock promoter in chromatin in vitro requires the concerted action of the GAGA transcription factor and NURF, an ATP-dependent nucleosome remodeling factor. NURF is composed of four subunits and is biochemically distinct from the SWI2/SNF2 multiprotein complex, a transcriptional activator that also appears to alter nucleosome structure. We have obtained protein microsequence and immunological evidence identifying the 140 kDa subunit of NURF as ISWI, previously of unknown function but highly related to SWI2/SNF2 only in the ATPase domain. The ISWI protein is localized to the cell nucleus and is expressed throughout Drosophila development at levels as high as 100,000 molecules/cell. The convergence of biochemical and genetic studies on ISWI and SWI2/SNF2 underscores these ATPases and their close relatives as key components of independent systems for chromatin remodeling

Evolution of Yin and Yang isoforms of a chromatin remodeling subunit precedes the creation of two genes

Genes can encode multiple isoforms, broadening their functions and providing a molecular substrate to evolve phenotypic diversity. Evolution of isoform function is a potential route to adapt to new environments. Here we show that de novo, beneficial alleles in the nurf-1 gene became fixed in two laboratory lineages of C. elegans after isolation from the wild in 1951, before methods of cryopreservation were developed. nurf-1 encodes an ortholog of BPTF, a large (>300 kD) multidomain subunit of the NURF chromatin remodeling complex. Using CRISPR-Cas9 genome editing and transgenic rescue, we demonstrate that in C. elegans, nurf-1 has split into two, largely non-overlapping isoforms (NURF-1.D and NURF-1.B, which we call Yin and Yang, respectively) that share only two of 26 exons. Both isoforms are essential for normal gametogenesis but have opposite effects on male/female gamete differentiation. Reproduction in hermaphrodites, which involves production of both sperm and oocytes, requires a balance of these opposing Yin and Yang isoforms. Transgenic rescue and genetic position of the fixed mutations suggest that different isoforms are modified in each laboratory strain. In a related clade of Caenorhabditis nematodes, the shared exons have duplicated, resulting in the split of the Yin and Yang isoforms into separate genes, each containing approximately 200 amino acids of duplicated sequence that has undergone accelerated protein evolution following the duplication. Associated with this duplication event is the loss of two additional nurf-1 transcripts, including the long-form transcript and a newly identified, highly expressed transcript encoded by the duplicated exons. We propose these lost transcripts are non-functional side products necessary to transcribe the Yin and Yang transcripts in the same cells. Our work demonstrates how gene sharing, through the production of multiple isoforms, can precede the creation of new, independent genes.National Institute of General Medical Sciences R01GM114170 Patrick T McGrat National Institute of General Medical Sciences R01GM121688 Ronald E Ellis.The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S