Genome-wide Location Analysis Research Articles

Functional Portrait of Irf1 (Orf19.217), a Regulator of Morphogenesis and Iron Homeostasis in Candida albicans.

The alternate growth of Candida albicans between a unicellular yeast form and a multicellular hyphal form is crucial for its ability to cause disease. Interestingly, both morphological forms support distinct functions during proliferation in the human host. We previously identified ORF19.217 (C2_08890W_A), encoding a zinc-finger transcription factor of the C2H2 family, in a systematic screen of genes whose overexpression contributes to C. albicans’ morphological changes. Conditional overexpression of ORF19.217 with the strong tetracycline-inducible promoter (P TET ) resulted in a hyperfilamentous phenotype. We examined growth of the orf19.217 knockout-mutant in different hypha-inducing conditions and found that the mutant still formed hyphae under standard hypha-inducing conditions. To further investigate the function of Orf19.217 in C. albicans, we combined genome-wide expression (RNA-Seq) and location (ChIP-Seq) analyses. We found that Orf19.217 is involved in regulatory processes comprising hyphal morphogenesis and iron acquisition. Comparative analysis with existing C. albicans hyphal transcriptomes indicates that Orf19.217-mediated filamentation is distinct from a true hyphal program. Further, the orf19.217 knockout-mutant did not show increased sensitivity to iron deprivation, but ORF19.217 overexpression was able to rescue the growth of a hap5-mutant, defective in a subunit of the CCAAT-complex, which is essential for iron acquisition. This suggested that Orf19.217 is involved in regulation of iron acquisition genes during iron deprivation and acts in a parallel pathway to the established CCAAT-complex. Interestingly, the orf19.217-mutant turned out to be defective in its ability to form filaments under iron-deficiency. Taken together our findings propose that the transcription factor Orf19.217 stimulates expression of the hyphal regulators EFG1 and BRG1 to promote filamentous growth under iron deprivation conditions, allowing the fungus to escape these iron-depleted conditions. The transcription factor therefore appears to be particularly important for adaptation of C. albicans to diverse environmental conditions in the human host. In regard to the newly identified functions, we have given the regulator the name Irf1, Iron-dependent Regulator of Filamentation.

A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency.

SUMMARYTET1 maintains hypomethylation at bivalent promoters through its catalytic activity in embryonic stem cells (ESCs). However, TET1 catalytic activity-independent function in regulating bivalent genes is not well understood. Using a proteomics approach, we map the TET1 interactome in ESCs and identify PSPC1 as a TET1 partner. Genome-wide location analysis reveals that PSPC1 functionally associates with TET1 and Polycomb repressive complex-2 (PRC2). We establish that PSPC1 and TET1 repress, and the lncRNA Neat1 activates, bivalent gene expression. In ESCs, Neat1 is preferentially bound to PSPC1 alongside its PRC2 association at bivalent promoters. During the ESC-to-epiblast-like stem cell (EpiLC) transition, PSPC1 and TET1 maintain PRC2 chromatin occupancy at bivalent gene promoters, while Neat1 facilitates the activation of certain bivalent genes by promoting PRC2 binding to their mRNAs. Our study demonstrates a TET1-PSPC1-Neat1 molecular axis that modulates PRC2-binding affinity to chromatin and bivalent gene transcripts in controlling stem cell bivalency

Six1 promotes skeletal muscle thyroid hormone response through regulation of the MCT10 transporter

BackgroundThe Six1 transcription factor is implicated in controlling the development of several tissue types, notably skeletal muscle. Six1 also contributes to muscle metabolism and its activity is associated with the fast-twitch, glycolytic phenotype. Six1 regulates the expression of certain genes of the fast muscle program by directly stimulating their transcription or indirectly acting through a long non-coding RNA. We hypothesized that additional mechanisms of action of Six1 might be at play.MethodsA combined analysis of gene expression profiling and genome-wide location analysis data was performed. Results were validated using in vivo RNA interference loss-of-function assays followed by measurement of gene expression by RT-PCR and transcriptional reporter assays.ResultsThe Slc16a10 gene, encoding the thyroid hormone transmembrane transporter MCT10, was identified as a gene with a transcriptional enhancer directly bound by Six1 and requiring Six1 activity for full expression in adult mouse tibialis anterior, a predominantly fast-twitch muscle. Of the various thyroid hormone transporters, MCT10 mRNA was found to be the most abundant in skeletal muscle, and to have a stronger expression in fast-twitch compared to slow-twitch muscle groups. Loss-of-function of MCT10 in the tibialis anterior recapitulated the effect of Six1 on the expression of fast-twitch muscle genes and led to lower activity of a thyroid hormone receptor-dependent reporter gene.ConclusionsThese results shed light on the molecular mechanisms controlling the tissue expression profile of MCT10 and identify modulation of the thyroid hormone signaling pathway as an additional mechanism by which Six1 influences skeletal muscle metabolism.

OCT4 cooperates with distinct ATP-dependent chromatin remodelers in na\xefve and primed pluripotent states in human

Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes. In naïve hESCs, OCT4 is associated with both BRG1 and BRM, the two paralog ATPases of the BAF complex. Genome-wide location analyses and genetic studies reveal that these two enzymes cooperate in a functionally redundant manner in the transcriptional regulation of blastocyst-specific genes. In contrast, in primed hESCs, OCT4 cooperates with BRG1 and SOX2 to promote chromatin accessibility at ectodermal genes. This work reveals how a common transcription factor utilizes differential BAF complexes to control distinct transcriptional programs in naïve and primed hESCs.

Pioneer factor Foxa2 enables ligand-dependent activation of type II nuclear receptors FXR and LXRα

ObjectiveType II nuclear hormone receptors, including farnesoid X receptors (FXR), liver X receptors (LXR), and peroxisome proliferator-activated receptors (PPAR), which serve as drug targets for metabolic diseases, are permanently positioned in the nucleus and thought to be bound to DNA regardless of the ligand status. However, recent genome-wide location analysis showed that LXRα and PPARα binding in the liver is largely ligand-dependent. We hypothesized that pioneer factor Foxa2 evicts nucleosomes to enable ligand-dependent binding of type II nuclear receptors and performed genome-wide studies to test this hypothesis. MethodsATAC-Seq was used to profile chromatin accessibility; ChIP-Seq was performed to assess transcription factors (Foxa2, FXR, LXRα, and PPARα) binding; and RNA-Seq analysis determined differentially expressed genes in wildtype and Foxa2 mutants treated with a ligand (GW4064 for FXR, GW3965, and T09 for LXRα). ResultsWe reveal that chromatin accessibility, FXR binding, LXRα occupancy, and ligand-responsive activation of gene expression by FXR and LXRα require Foxa2. Unexpectedly, Foxa2 occupancy is drastically increased when either receptor, FXR or LXRα, is bound by an agonist. In addition, co-immunoprecipitation experiments demonstrate that Foxa2 interacts with either receptor in a ligand-dependent manner, suggesting that Foxa2 and the receptor, bind DNA as an interdependent complex during ligand activation. Furthermore, PPARα binding is induced in Foxa2 mutants treated with FXR and LXR ligands, leading to the activation of PPARα targets. ConclusionsOur model requires pioneering activity for ligand activation that challenges the existing ligand-independent binding mechanism. We also demonstrate that Foxa2 is required to achieve activation of the proper receptor – one that binds the added ligand – by repressing the activity of a competing receptor.

SOX4-mediated repression of specific tRNAs inhibits proliferation of human glioblastoma cells

Transfer RNAs (tRNAs) are products of RNA polymerase III (Pol III) and essential for mRNA translation and ultimately cell growth and proliferation. Whether and how individual tRNA genes are specifically regulated is not clear. Here, we report that SOX4, a well-known Pol II-dependent transcription factor that is critical for neurogenesis and reprogramming of somatic cells, also directly controls, unexpectedly, the expression of a subset of tRNA genes and therefore protein synthesis and proliferation of human glioblastoma cells. Genome-wide location analysis through chromatin immunoprecipitation-sequencing uncovers specific targeting of SOX4 to a subset of tRNA genes, including those for tRNAiMet Mechanistically, sequence-specific SOX4-binding impedes the recruitment of TATA box binding protein and Pol III to tRNA genes and thereby represses their expression. CRISPR/Cas9-mediated down-regulation of tRNAiMet greatly inhibits growth and proliferation of human glioblastoma cells. Conversely, ectopic tRNAiMet partially rescues SOX4-mediated repression of cell proliferation. Together, these results uncover a regulatory mode of individual tRNA genes to control cell behavior. Such regulation may coordinate codon usage and translation efficiency to meet the demands of diverse tissues and cell types, including cancer cells.

DYRK1A interacts with histone acetyl transferase p300 and CBP and localizes to enhancers

DYRK1A, dual-specificity tyrosine phosphorylation-regulated kinase 1A, which is linked to mental retardation and microcephaly, is a member of the CMGC group of kinases. It has both cytoplasmic and nuclear functions, however, molecular mechanisms of how DYRK1A regulates gene expression is not well understood. Here, we identify two histone acetyltransferases, p300 and CBP, as interaction partners of DYRK1A through a proteomics study. We show that overexpression of DYKR1A causes hyperphosphorylation of p300 and CBP. Using genome-wide location (ChIP-sequencing) analysis of DYRK1A, we show that most of the DYRK1A peaks co-localize with p300 and CBP, at enhancers or near the transcription start sites (TSS). Modulation of DYRK1A, by shRNA mediated reduction or transfection mediated overexpression, leads to alteration of expression of downstream located genes. We show that the knockdown of DYRK1A results in a significant loss of H3K27acetylation at these enhancers, suggesting that DYRK1A modulates the activity of p300/CBP at these enhancers. We propose that DYRK1A functions in enhancer regulation by interacting with p300/CBP and modulating their activity. Overall, DYRK1A function in the regulation of enhancer activity provides a new mechanistic understanding of DYRK1A mediated regulation of gene expression, which may help in better understanding of the roles of DYRK1A in human pathologies.

Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut.

Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature‐tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome‐wide expression and gene‐set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome‐wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase‐ and cell‐wall protein‐encoding genes, suggesting a link with cell‐wall function. We show that CRZ2 overexpression alters cell‐wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

Native Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) from Low Cell Numbers.

ChIP-seq is the current method of choice for genome-wide protein location analysis. Here, we present a native (non-cross-linked) ChIP procedure suitable for histone proteins, coupled with an efficient library preparation technique for subsequent next-generation sequencing. The method enables ChIP-seq starting with 50,000 or more cells.

Abstract 382: Genome-wide location analysis of DNA adducts in vivo

Abstract DNA adducts are the biochemical consequences of exposure to UV light, carcinogens or DNA-reactive drugs. They play a central role in malignant transformation and the selective killing of cancer cells by chemotherapeutic agents. The functional consequences of DNA adducts is likely to depend on their exact location in the genome with respect to histones and sites where histones and DNA are modified. However, tools that permit precise genome-wide mapping of adduct locations have poor resolution or are too specific to the type of DNA-adduct. Here we present the development of a molecular assay (Ad-Seq) capable of identifying the genomic location of damaged DNA bases in whole cells. Using naked DNA treated with cisplatin (cDDP) or ultra-violet (UV) light as the damaging agents, the strategy relies on differential exonuclease digestion to enrich genome-wide libraries for fragments containing DNA adducts, followed by high-throughput sequencing. Consistent with the expected adduct chemistry, the 5’ end of the reads were enriched in TT, TC or CT di-nucleotides in the UV treated DNA (>4x) and in AG or GG in the cDDP treated DNA (1.5x), suggesting a positive predictive value of 76% and 44% and UV (TT) and cDDP (GG) adduct detection, respectively. Applied in vivo to IMR90 cells, Ad-Seq revealed that the fraction of AG/GG loci was higher in DNA from the cDDP-treated cells at all coverage depths, reaching 1.5x enrichment for the highest depth quartile. This indicates that the method is selective and that even low covered loci show enrichment of AG/GG sequences. The enrichment in purines was limited to 3 nucleotides downstream of the read start site, confirming the high resolution of the exonuclease digestion. A total of 11 x 103 AG/GG loci were identified in replicate in cDDP-treated samples and were absent from untreated controls. With about 60 x103 expected adducts per genome, the current assay therefore captured up to 18% of cDDP adducts. The most covered adduct sites were significantly enriched in the quiescent chromatin domains (depleted of histone modifications, p=6.7x10-31), and depleted in the transcription start sites (p=4.9x10-12), as defined by the ENCODE consortium. This may be reflective of the differences in DNA accessibility or the mechanisms of repair in these distinct regions of the genome. The analysis was performed using the PipeDuct computational package, which was developed to align the sequencing reads and provide general statistics and quality control information of Ad-Seq data. Ad-Seq is therefore a novel, unbiased method to map DNA-adducts and study in vivo the genome-wide specificity of their formation and repair. Citation Format: Brian Woo, Christopher Fang, Paolo Abada, Stephen B. Howell, Olivier Harismendy. Genome-wide location analysis of DNA adducts in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 382. doi:10.1158/1538-7445.AM2017-382

Human NOTCH4 Is a Key Target of RUNX1 in Megakaryocytic Differentiation

Megakaryocytes (MK), which produce platelets, play important roles in blood coagulation and hemostasis. The master transcription factor RUNX1 regulates lineage-specific transcriptional targets and key signaling pathways, and is known to be essential for megakaryopoiesis. Mono-allelic RUNX1 mutations lead to familial platelet disorder (FPD), which is characterized by thrombocytopenia and abnormal platelet functions. A high percentage (~50%) of these FPD patients later develop myelodysplastic syndromes and acute myeloid leukemia. The exact mechanisms underlying deregulated megakaryopoiesis in FPD remain unclear, partially due to the lack of an adequate experimental model mimicking the human disease. For example, engineered laboratory mice and zebrafish with only one copy of the Runx1 gene do not develop bleeding disorders or leukemia. Using an in vitro hematopoietic differentiation system, we found that megakaryocytic differentiation from FPD-derived induced pluripotent stem cells (iPSCs) were defective (Connelly et al., 2014). Targeted correction of the mutated RUNX1 allele by genome editing restored the MK production and functions, validating the central role of RUNX1 in megakaryopoiesis (Connelly et al., 2014). In this new study, we pursued the hypothesis that direct target genes regulated by RUNX1 play important roles in human megakaryopoiesis.We first performed RNA-Seq analysis on differentiated hematopoietic cells from FPD-iPSCs (harboring a mono-allelic RUNX1 mutation) and RUNX1-corrected isogenic iPSCs. Seventy-nine genes were expressed at a significantly higher level (p<0.01, FDR<0.05) while 93 genes were expressed at a significantly lower level (p<0.01, FDR<0.05) in the RUNX1-corrected cells as compared to the FPD-iPSCs. To determine whether these differentially expressed genes (DEGs) are the direct targets of RUNX1, we additionally performed genome-wide location analysis of RUNX1 by ChIP-Seq using the same hematopoietic cell population differentiated from the RUNX1-corrected isogenic iPSCs. We detected 5266 (FDR<0.05) binding sites in 4526 gene loci. Combined with the DEG data from RNA-Seq analyses, we further identified 37 up-regulated genes (such as ITGB3 and PF4) and 27 down-regulated genes with RUNX1 binding to the gene’s proximity. Among the 64 differentially expressed genes with RUNX1 binding, Gene Ontology (GO) analysis revealed that only 13 genes including PF4 have been reported to be relevant to megakaryopoiesis. In order to verify the roles of these RUNX1 target genes in hematopoiesis and megakaryopoiesis, we carried out gene knockout (KO) experiments by CRISPR-Cas9 in normal human iPSCs followed by in vitro hematopoietic differentiation assays. We first focused on the "down-regulated" genes by RUNX1 binding, with the hypothesis that their KO may enhance hematopoiesis and/or megakaryopoiesis from normal iPSCs. One of such genes is NOTCH4, a member of NOTCH receptor family that plays important roles in development and cell fate determination. A previous study showed that NOTCH signaling specifies MK development from mouse hematopoietic progenitor cells (Mercher et al., 2008), while we have not seen publications on the NOTCH4 in human MK development.Using the improved CRISPR technology, we successfully achieved KO of one copy of NOTCH4 in the wildtype iPSCs. We found that heterozygous KO of NOTCH4 increased MK (progenitor) production by 95% (p<0.05), while the production of CD34+ multipotent hematopoietic progenitor cells were not affected. To further verify its function, we inhibited NOTCH4 signaling with a gamma-secretase inhibitor. Notably, inhibition of NOTCH4 signal starting at day 2 of hematopoietic differentiation improved the efficiency of MK progenitor production by 50% (p<0.05) and more mature MK production by 70% (p<0.05). Taken together, we conclude that NOTCH4, a newly discovered RUNX1 target gene, negatively regulates megakaryopoiesis in a developmental-stage specific manner. Unlocking this inhibitory effect by small molecule inhibitors can promote MK production ex vivo. The described approach will enable us to discover additional novel genes that influence human hematopoiesis and megakaryopoiesis, which in turn will help to promote ex vivo generation of MKs from human iPSCs or postnatal hematopoietic stem/progenitor cells. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3752: A cell cycle-related kinase (CCRK) self-regulatory circuitry in NAFLD-associated hepatocarcinogenesis

Abstract Background and aim: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome including obesity and diabetes which elevate the risk of hepatocellular carcinoma (HCC). Accumulating evidence has unmasked the molecular linkage between androgen receptor (AR) signaling and gender disparity in NAFLD and HCC incidence. Our previous genome-wide location and functional analysis has pinpointed cell cycle-related kinase (CCRK) as a critical mediator of AR oncogenic activity in hepatitis B virus-associated HCC through glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling (1-3). Here we investigated whether CCRK plays an oncogenic role in NAFLD-related HCC. Methods: We investigated the functional role of CCRK in a high-fat high carbohydrate diet-induced obesity model exposed to low-dose diethylnitrosamine using lentiviral-mediated gene knockdown, followed by measurement of glucose/lipid metabolism via intraperitoneal glucose tolerance test and triglyceride/non-esterified fatty acids levels, and HCC incidence. Gene regulation was assessed by chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, co-immunoprecipitation and expression analyses. Results: Lentiviral-mediated Ccrk silencing in male mice significantly reduced obesity-promoted tumor multiplicity and size by &amp;gt;70%. The reduced tumorigenicity was also associated with significant restoration of circulating metabolic profiles and reduction in hepatocellular lipid level. Mechanistic studies demonstrated that the pro-inflammatory cytokine interleukin-6 (IL-6) triggered signal transducer and activator of transcription 3 (STAT3) signaling to up-regulate CCRK expression in an AR-dependent manner. Simultaneously, the phosphorylation of STAT3 by CCRK facilitated the co-occupancy of CCRK promoter by STAT3-AR complex and its subsequent transcriptional activation, thus forming a self-reinforcing circuitry. In addition, both in vitro and in vivo data suggested that CCRK phosphorylated GSK-3β to activate not only β-catenin but also mechanistic target of rapamycin complex 1 (mTORC1) signaling pathways that are crucial for cell survival, proliferation and lipogenesis. Furthermore, transgenic over-expression of CCRK in mouse liver led to concordant activation of STAT3, β-catenin and mTORC1 signaling pathways. Conclusion: Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates NAFLD-associated hepatocarcinogenesis and offer novel therapeutic strategies to combat this dreadful disease in an era of global obesity. Acknowledgement: This project was supported by the Collaborative Research Fund C4017-14G and General Research Fund 14102914.

FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function.

Forkhead box A1 (FOXA1) is an FKHD family protein that plays pioneering roles in lineage-specific enhancer activation and gene transcription. Through genome-wide location analyses, here we show that FOXA1 expression and occupancy are, in turn, required for the maintenance of these epigenetic signatures, namely DNA hypomethylation and histone 3 lysine 4 methylation. Mechanistically, this involves TET1, a 5-methylcytosine dioxygenase. We found that FOXA1 induces TET1 expression via direct binding to its cis-regulatory elements. Further, FOXA1 physically interacts with the TET1 protein through its CXXC domain. TET1 thus co-occupies FOXA1-dependent enhancers and mediates local DNA demethylation and concomitant histone 3 lysine 4 methylation, further potentiating FOXA1 recruitment. Consequently, FOXA1 binding events are markedly reduced following TET1 depletion. Together, our results suggest that FOXA1 is not only able to recognize but also remodel the epigenetic signatures at lineage-specific enhancers, which is mediated, at least in part, by a feed-forward regulatory loop between FOXA1 and TET1.

Widespread colocalization of the Drosophila histone acetyltransferase homolog MYST5 with DREF and insulator proteins at active genes.

MYST family histone acetyltransferases play important roles in gene regulation. Here, we have characterized the Drosophila MYST histone acetyltransferase (HAT) encoded by cg1894, whose closest homolog is Drosophila MOF, and which we have termed MYST5. We found it localized to a large number of interbands as well as to the telomeres of polytene chromosomes, and it showed strong colocalization with the interband protein Z4/Putzig and RNA polymerase II. Accordingly, genome-wide location analysis by ChIP-seq showed co-occurrence of MYST5 with the Z4-interacting partner Chriz/Chromator. Interestingly, MYST5 bound to the promoter of actively transcribed genes, and about half of MYST5 sites colocalized with the transcription factor DNA replication-related element-binding factor (DREF), indicating a role for MYST5 in gene expression. Moreover, we observed substantial overlap of MYST5 binding with that of the insulator proteins CP190, dCTCF, and BEAF-32, which mediate the organization of the genome into functionally distinct topological domains. Altogether, our data suggest a broad role for MYST5 both in gene-specific transcriptional regulation and in the organization of the genome into chromatin domains, with the two roles possibly being functionally interconnected.

Abstract 4063: The oncogenic cell cycle-related kinase promotes tumor immune escape via modulation of myeloid-derived suppressor cells in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a sexually dimorphic cancer significantly associated with elevated levels of male sex hormone androgen and pro-inflammatory cytokine interleukin-6 (IL-6) (ref 1). Our previous genome-wide location and functional analysis has pinpointed cell cycle-related kinase (CCRK) as a critical mediator of androgen receptor (AR) oncogenic activity in HCC through a self-reinforcing circuitry involving β-catenin and the Polycomb repressor enhancer of zeste 2 (EZH2) (ref 2-4). As these key circuit players have also been shown to trigger inflammatory response and aggressiveness of HCC cells (ref 5,6), we aimed to investigate whether CCRK modulates tumor microenvironment during hepatocarcinogenesis. Expression profiling of 16 pro-inflammatory cytokines and chemokines revealed that ectopic expression of wild-type but not kinase-defective CCRK significantly induced the production of IL-6, IL-1β, tumor necrosis factor-α and cyclooxygenase 2 in immortal hepatic and HCC cells. Rescue experiments showed that CCRK-induced IL-6 production was dependent on EZH2 up-regulation and subsequent nuclear factor-κB activation. Based on the causal link between IL-6 and myeloid-derived suppressor cells (MDSCs), we next examined the relationship between CCRK and MDSCs in vitro and in vivo. By co-culturing CCRK-expressed or silenced hepatic and HCC cell lines with peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, we found that CCRK stimulated the expansion of Lin-CD33+CD11b+HLA-DR-MDSCs from PBMCs by flow cytometry analysis. The induced MDSCs also showed higher suppressive activity against CD3+ T cell proliferation. In a high-fat diet-fed mouse model exposed to low-dose carcinogen diethylnitrosamine, administration of lentivirus expressing short-hairpin (sh)RNA against Ccrk significantly reduced &amp;gt;70% obesity-promoted tumor multiplicity and size compared to mice treated with control shRNA (p&amp;lt;0.01). Notably, Ccrk down-regulation also significantly reduced the population of circulating CD11b+Gr-1intMDSCs in the mice (p&amp;lt;0.05). In summary, our findings demonstrate that the oncogenic CCRK in hepatic cells can promote MDSC expansion and suppressive functions possibly via IL-6 production. Such tumor-stromal cell interaction provides survival advantage of cancer cells via immune escape. In conclusion, CCRK not only functions as a signaling hub but also an immunoregulator, thus representing a new cancer immunotherapy target.

An epigenomic role of Fe65 in the cellular response to DNA damage

Previous findings describe Fe65 as a key protein in the cellular response to genotoxic stress. However, the precise molecular mechanism by which Fe65 contributes to DNA damage signaling remains unclear. In this study, we hypothesized that the transcriptional activity of Fe65 may contribute to DNA damage pathways by regulating gene expression patterns activated in response to genotoxic stress. To address this hypothesis, we mapped the global binding profile of Fe65 by chromatin immunoprecipitation (ChIP)-sequencing in the SK-N-SH cells exposed to genotoxic stress. Unexpectedly, the genome-wide location analysis showed a substantial enrichment of Fe65 in the promoter regions of coding genes linked to DNA damage signaling pathways. To further investigate the role of Fe65 in the transcriptional regulation of putative coding target genes identified by ChIP-seq, we performed microarray assays using wild-type (WT) or Fe65 deficient mouse embryonic fibroblasts (MEFs) exposed to oxidative stress with multiple recovery times. Gene ontology analysis of the Fe65-depedent transcriptome suggested that Fe65 modulates the expression of genes critical for DNA damage response. Motif enrichment analysis of regulatory regions occupied by Fe65 revealed a strong correlation with key transcription factors involved in DNA damage signaling pathways, including E2F1, p53, and Jun. Comparison of ChIP-sequencing results with microarray results ultimately identified 248 Fe65-depedent target genes, the majority of which were known regulators of cell cycle, cell death, and DNA replication and repair pathways. We validated the target genes identified by in silico analysis by qPCR experiments. Collectively, our results provide strong evidence that Fe65 plays a role in DNA damage response and cell viability by epigenomic regulation of specific transcriptional programs activated upon genotoxic stress.

A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea.

Networks of interacting transcription factors are central to the regulation of cellular responses to abiotic stress. Although the architecture of many such networks has been mapped, their dynamic function remains unclear. Here we address this challenge in archaea, microorganisms possessing transcription factors that resemble those of both eukaryotes and bacteria. Using genome-wide DNA binding location analysis integrated with gene expression and cell physiological data, we demonstrate that a bacterial-type transcription factor (TF), called RosR, and five TFIIB proteins, homologs of eukaryotic TFs, combinatorially regulate over 100 target genes important for the response to extremely high levels of peroxide. These genes include 20 other transcription factors and oxidative damage repair genes. RosR promoter occupancy is surprisingly dynamic, with the pattern of target gene expression during the transition from rapid growth to stress correlating strongly with the pattern of dynamic binding. We conclude that a hierarchical regulatory network orchestrated by TFs of hybrid lineage enables dynamic response and survival under extreme stress in archaea. This raises questions regarding the evolutionary trajectory of gene networks in response to stress.

Abstract 4500: An AR-regulated kinase CCRK activates a tumor-initiating circuitry through dual regulation of EZH2

Abstract Hepatocellular carcinoma (HCC) is one of the commonest and deadliest cancers worldwide with a striking gender prevalence observed in men. Androgen receptor (AR) is a ligand-activated nuclear receptor that regulates the development of male sexual phenotype. Aberrant AR signaling, however, has detrimental consequences in the development of male-predominant cancers. Using genome-wide location analysis, we have recently uncovered cell cycle-related kinase (CCRK) as an AR direct transcriptional target that drives aberrant hepatocellular proliferation and tumorigenicity through β-catenin/T cell factor signaling (Feng et al. 2011). We also found that the Polycomb protein EZH2 promotes β-catenin-dependent hepatocarcinogenesis through concordant repression of Wnt antagonists via histone H3 lysine 27 trimethylation (Cheng et al. 2011). Recent studies have revealed the Polycomb-independent role of EZH2 in gene activation via AKT-mediated phosphorylation, however, the regulation and signaling network of EZH2 in HCC are poorly-understood. Here we show that the AR-regulated kinase CCRK activates a tumor-initiating circuitry by dual regulation of EZH2. Activation of CCRK phosphorylates glycogen synthase kinase 3β (GSK-3β) and AKT leading to transcriptional up-regulation and phosphorylation of EZH2 at Ser21 (pEZH2Ser9), respectively. pEZH2Ser9 in turn physically interacts with AR and cooperatively activate CCRK transcription via direct binding to the CCRK promoter, thus forming a feedback circuitry. In a chemical-induced murine HCC model, activation of the AR/CCRK/EZH2 regulatory loop precedes HCC formation, which is significantly reduced (&amp;gt;80%) by lentiviral-mediated knockdown of CCRK. In human HCC tissues, these proteins are highly up-regulated, starting from the adjacent precancerous liver tissues. Most importantly, hyper-activation of this loop is significantly associated with shorter overall (p&amp;lt;0.0001) and disease-free survival rates (p&amp;lt;0.01) of the HCC patients. This study not only advances our fundamental understanding of the gender disparity in HCC, but also provides a new paradigm for nuclear receptor regulation of cancer epigenome. Elucidating the detailed signaling network of AR/CCRK/EZH2 will lead to the discovery and development of targeted therapies tailored to inhibit cancer-specific pathways. This study was supported by a CUHK Focused Investments Scheme - Scheme B (fis1112/0674/12yc), a National Natural Science Foundation of China (373492), a Health and Medical Research Fund (12110532) and a General Research Fund (462710).

Eaf5/7/3 form a functionally independent NuA4 submodule linked to RNA polymerase II-coupled nucleosome recycling.

The NuA4 histone acetyltransferase complex is required for gene regulation, cell cycle progression, and DNA repair. Dissection of the 13-subunit complex reveals that the Eaf7 subunit bridges Eaf5 with Eaf3, a H3K36me3-binding chromodomain protein, and this Eaf5/7/3 trimer is anchored to NuA4 through Eaf5. This trimeric subcomplex represents a functional module, and a large portion exists in a native form outside the NuA4 complex. Gene-specific and genome-wide location analyses indicate that Eaf5/7/3 correlates with transcription activity and is enriched over the coding region. In agreement with a role in transcription elongation, the Eaf5/7/3 trimer interacts with phosphorylated RNA polymerase II and helps its progression. Loss of Eaf5/7/3 partially suppresses intragenic cryptic transcription arising in set2 mutants, supporting a role in nucleosome destabilization. On the other hand, loss of the trimer leads to an increase of replication-independent histone exchange over the coding region of transcribed genes. Taken together, these results lead to a model where Eaf5/7/3 associates with elongating polymerase to promote the disruption of nucleosomes in its path, but also their refolding in its wake.

Early B-cell Factor-1 (EBF1) Is a Key Regulator of Metabolic and Inflammatory Signaling Pathways in Mature Adipocytes

EBF1 plays a crucial role in early adipogenesis; however, despite high expression in mature adipocytes, its function in these cells is currently unknown. To identify direct and indirect EBF1 targets in fat, we undertook a combination of transcriptional profiling of EBF1-deficient adipocytes and genome-wide EBF1 location analysis. Our results indicate that many components of metabolic and inflammatory pathways are positively and directly regulated by EBF1, including PI3K/AKT, MAPK, and STAT1 signaling. Accordingly, we observed significant reduction of multiple signaling events in EBF1 knockdown cells as well as a reduction in insulin-stimulated glucose uptake and lipogenesis. Inflammatory signaling, gene expression, and secretion of inflammatory cytokines were also significantly affected by loss of EBF1 in adipocytes, although ChIP-sequencing results suggest that these actions are indirect. We also found that EBF1 occupies some 35,000 sites in adipocytes, most of which occur in enhancers. Significantly, comparison with three other published EBF1 ChIP-sequencing data sets in B-cells reveals both gene- and cell type-specific patterns of EBF1 binding. These results advance our understanding of the transcriptional mechanisms regulating signaling pathways in mature fat cells and indicate that EBF1 functions as a key integrator of signal transduction, inflammation, and metabolism.