POU2F2-mediated upregulation of lncRNA PTPRG-AS1 inhibits ferroptosis in breast cancer via miR-376c-3p/SLC7A11 axis.

Background: Triple-negative breast cancer (TNBC) is a subtype of BC with high rates of mortality. The mechanism of PTPRG-AS1 in ferroptosis of TNBC was investigated. Methods: Chromatin immunoprecipitation and dual-luciferase reporter assays were used to measure intermolecular relationships. MTT and colony formation assays detected cell viability and proliferation. Kits detected Fe2+ and reactive oxygen species levels. The role of PTPRG-AS1 in tumor growth was analyzed in vivo. Results: PTPRG-AS1 was increased in TNBC tissues and cells. PTPRG-AS1 silencing increased the reduction of glutathione and GPX4, increased Fe2+ and reactive oxygen species in erastin-treated cells and inhibited proliferation. POU2F2 transcriptionally upregulated PTPRG-AS1. PTPRG-AS1 targeted miR-376c-3p to upregulate SLC7A11. PTPRG-AS1 knockdown suppressed tumor growth in vivo. Conclusion: POU2F2 transcriptionally activates PTPRG-AS1 to modulate ferroptosis and proliferation by miR-376c-3p/SLC7A11, promoting TNBC.

Triple-negative breast cancer (TNBC) is a kind of breast cancer with high recurrence and low survival rates. Activation of the ferroptosis pathway can inhibit BC proliferation and distant metastasis. Therefore, identifying effective biomarkers and molecular mechanisms of ferroptosis in TNBC is important for its earlier detection and therapy. PTPRG-AS1 is a new type of lncRNA discovered in recent years that is increased in various diseases and is related to prognosis. In the present study, the authors found that POU2F2 promoted PTPRG-AS1 transcription. PTPRG-AS1 knockdown activated ferroptosis in TNBC and inhibited proliferation. Mechanistically, PTPRG-AS1 targeted miR-376c-3p to upregulate SLC7A11, thereby inhibiting ferroptosis and promoting TNBC development. These results indicate that PTPRG-AS1 is a possible therapeutic target in TNBC.

Transcriptional Coactivator BOB1 (OBF1, OCA-B) Modulates the Specificity of DNA Recognition by the POU-Domain Factors OCT1 and OCT2 in a Monomeric Configuration.

BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA-SELEX-Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1-an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies.

CEBPB/POU2F2 modulates endothelin 1 expression in prehypertensive SHR vascular smooth muscle cells.

The pathogenesis of hypertension is not fully understood; endothelin 1 (EDN1) is involved in developing essential hypertension. EDN1 can promote vascular smooth muscle cell (VSMC) proliferation or hypertrophy through autocrine and paracrine effects. Proliferating smooth muscle cells in the aorta are 'dedifferentiated' cells that cause increased arterial stiffness and remodeling. Male SHRs had higher aortic stiffness than normal control male WKY rats. Male SHR VSMCs expressed high levels of the EDN1 gene, but endothelial cells did not. Therefore, it is necessary to understand the molecular mechanism of enhanced EDN1 expression in SHR VSMCs. We identified POU2F2 and CEBPB as the main molecules that enhance EDN1 expression in male SHR VSMCs. A promoter activity analysis confirmed that the enhancer region of the Edn1 promoter in male SHR VSMCs was from -1309 to -1279 bp. POU2F2 and CEBPB exhibited an additive role in the enhancer region of the EdnET1 promoter. POU2F2 or CEBPB overexpression sufficiently increased EDN1 expression, and co-transfection with the CEBPB and POU2F2 expression plasmids had additive effects on the activity of the Edn1 promoter and EDN1 secretion level of male WKY VSMCs. In addition, the knockdown of POU2F2 also revealed that POU2F2 is necessary to enhance EDN1 expression in SHR VSMCs. The enhancer region of the Edn1 promoter is highly conserved in rats, mice, and humans. POU2F2 and CEBPB mRNA levels were significantly increased in remodeled human VMSCs. In conclusion, the novel regulation of POU2F2 and CEBPB in VSMCs will help us understand the pathogenesis of hypertension and support the development of future treatments for hypertension.

POU Domain Class 2 Transcription Factor 2 Inhibits Ferroptosis in Cerebral Ischemia Reperfusion Injury by Activating Sestrin2.

Cerebral ischemia reperfusion injury (CIRI) is the commonest cause of brain dysfunction. Up-regulation of POU domain class 2 transcription factor 2 (POU2F2) has been reported in patients with cerebral ischemia, while the role of POU2F2 in CIRI remains elusive. Middle cerebral artery occlusion/reperfusion (MCAO/R) in mice and oxygen and glucose deprivation/reperfusion (OGD/R) in mouse primary cortical neurons were used as models of CIRI injury in vivo and in vitro. Lentivirus-mediated POU2F2 knockdown further impaired CIRI induced by MCAO/R in mice, which was accompanied by increased-neurological deficits, cerebral infarct volume and neuronal loss. Our evidence suggested that POU2F2 deficiency deteriorated oxidative stress and ferroptosis according to the phenomenon such as the abatement of SOD, GSH, glutathione peroxidase 4 (GPX4) activity and accumulation of ROS, lipid ROS, 4-hydroxynonenal (4-HNE) and MDA. In vivo, primary cortical neurons with POU2F2 knockdown also showed worse neuronal damage, oxidative stress and ferroptosis. Sestrin2 (Sesn2) was reported as a neuroprotection gene and involved in ferroptosis mechanism. Up-regulation of Sesn2 was observed in the ischemic penumbra and OGD/R-induced neuronal cells. Further, we proved that POU2F2, as a transcription factor, could bind to Sesn2 promoter and positively regulate its expression. Sesn2 overexpression relieved oxidative stress and ferroptosis induced by POU2F2 knockdown in OGD/R-treated neurons. This research demonstrated that CIRI induced a compensatory increase of POU2F2 and Sesn2. Down-regulated POU2F2 exacerbated CIRI through the acceleration of oxidative stress and ferroptosis possibly by decreasing Sesn2 expression, which offers new sights into therapeutic mechanisms for CIRI.

CBX7 represses the POU2F2 to inhibit the PD-L1 expression and regulate the immune response in bladder cancer.

Bladder cancer (BC) is one of the most common malignant tumors of the urinary system worldwide. To date, immune checkpoint inhibitors (including PD-1/PD-L1) have been applied to treat patients with bladder cancer in the clinic and achieved the promising outcome. Further improvement of the anticancer efficiency of these immune therapies is crucial for bladder cancer. Our previous RNA-seq data on CBX7 (GSE185630) suggested that CBX7 might repress PD-L1 expression and PD-1 checkpoint pathway in cancer. In this study, we revealed that CBX7 downregulated the expression of POU2F2 that indirectly repressed the PD-L1 in BC cells. Depletion of CBX7 resulted in resistance to PD-1 blockade in bladder cancer. Collectively, our results suggested that the CBX7/POU2F2/PD-L1 axis plays an important role in determining the antitumor effect of PD-1 blockade in bladder cancer.

BOB.1/OBF.1 is required during B-cell ontogeny for B-cell differentiation and germinal center function.

BOB.1/OBF.1 is a lymphocyte-specific transcriptional co-activator of octamer-dependent transcription. It regulates the expression of genes important for lymphocyte physiology together with the Oct-1 and Oct-2 transcription factors. So far, BOB.1/OBF.1 has been studied in conventional knockout mice, whereby a function of BOB.1/OBF.1 in B but also in T cells was described. The main characteristic of BOB.1/OBF.1-deficient mice is the complete absence of germinal centers. However, it is entirely unsolved at which stage of B-cell development BOB.1/OBF.1 expression is essential for germinal center formation. Still, it is not known whether defects observed late in B-cell development of BOB.1/OBF.1-deficient mice are merely a consequence of defective early B-cell development. To answer the question, whether BOB.1/OBF.1 expression is required before or during the process of germinal center formation, we established a mouse system, which allows the conditional deletion of BOB.1/OBF.1 at different stages of B-cell development. Our data reveal a requirement for BOB.1/OBF.1 during both early antigen-independent and late antigen-dependent B-cell development, and further a requirement for efficient germinal center reaction during complete B-cell ontogeny. By specifically deleting BOB.1/OBF.1 in germinal center B cells, we provide evidence that the failure to form germinal centers is a germinal center B-cell intrinsic defect and not exclusively a consequence of defective early B-cell maturation.

Long Non-Coding RNA ARAP1-AS1 Facilitates the Progression of Cervical Cancer by Regulating miR-149-3p and POU2F2.

BACKGROUND: Emerging research has demonstrated that long non-coding RNAs (lncRNAs) attach great importance to the progression of cervical cancer (CC). LncRNA ARAP1-AS1 was involved in the development of several cancers; however, its role in CC is far from being elucidated. METHODS: Real-time PCR (RT-PCR) was employed to detect ARAP1-AS1 and miR-149-3p expression in CC samples. CC cell lines (HeLa and C33A cells) were regarded as the cell models. The biological effect of ARAP1-AS1 on cancer cells was measured using CCK-8 assay, colony formation assay, flow cytometry, Transwell assay and wound healing assay in vitro, and subcutaneous xenotransplanted tumor model and tail vein injection model in vivo. Furthermore, interactions between ARAP1-AS1 and miR-149-3p, miR-149-3p and POU class 2 homeobox 2 (POU2F2) were determined by bioinformatics analysis, qRT-PCR, Western blot, luciferase reporter and RNA immunoprecipitation assay, respectively. RESULTS: The expression of ARAP1-AS1 was enhanced in CC samples, while miR-149-3p was markedly suppressed. Additionally, ARAP1-AS1 overexpression enhanced the viability, migration, and invasion of CC cells. ARAP1-AS1 downregulated miR-149-3p via sponging it. ARAP1-AS1 and miR-149-3p exhibited a negative correlation in CC samples. On the other hand, ARAP1-AS1 enhanced the expression of POU2F2, which was validated as a target gene of miR-149-3p. CONCLUSION: ARAP1-AS1 was abnormally upregulated in CC tissues and indirectly modulated the POU2F2 expression via reducing miR-149-3p expression. Our study identified a novel axis, ARAP1-AS1/miR-149-3p/POU2F2, in CC tumorigenesis.

POU2F2 regulates glycolytic reprogramming and glioblastoma progression via PDPK1-dependent activation of PI3K/AKT/mTOR pathway.

The POU Class Homeobox 2 (POU2F2) is a member of POU transcription factors family, which involves in cell immune response by regulating B cell proliferation and differentiation genes. Recent studies have shown that POU2F2 acts as tumor-promoting roles in some cancers, but the underlying mechanism remains little known. Here, we identified that the highly expressed POU2F2 significantly correlated with poor prognosis of glioblastoma (GBM) patients. POU2F2 promoted cell proliferation and regulated glycolytic reprogramming. Mechanistically, the AKT/mTOR signaling pathway played important roles in the regulation of POU2F2-mediated aerobic glycolysis and cell growth. Furthermore, we demonstrated that POU2F2 activated the transcription of PDPK1 by directly binding to its promoter. Reconstituted the expression of PDPK1 in POU2F2-knockdown GBM cells reactivated AKT/mTOR pathway and recovered cell glycolysis and proliferation, whereas this effect was abolished by the PDPK1/AKT interaction inhibitor. In addition, we showed that POU2F2-PDPK1 axis promoted tumorigenesis by regulating glycolysis in vivo. In conclusion, our findings indicate that POU2F2 leads a metabolic shift towards aerobic glycolysis and promotes GBM progression in PDPK1-dependent activation of PI3K/AKT/mTOR pathway.

POU2F2 promotes the proliferation and motility of lung cancer cells by activating AGO1.

BACKGROUND: To detect and investigate the expression of POU domain class 2 transcription factor 2 (POU2F2) in human lung cancer tissues, its role in lung cancer progression, and the potential mechanisms. METHODS: Immunohistochemical (IHC) assays were conducted to assess the expression of POU2F2 in human lung cancer tissues. Immunoblot assays were performed to assess the expression levels of POU2F2 in human lung cancer tissues and cell lines. CCK-8, colony formation, and transwell-migration/invasion assays were conducted to detect the effects of POU2F2 and AGO1 on the proliferaion and motility of A549 and H1299 cells in vitro. CHIP and luciferase assays were performed for the mechanism study. A tumor xenotransplantation model was used to detect the effects of POU2F2 on tumor growth in vivo. RESULTS: We found POU2F2 was highly expressed in human lung cancer tissues and cell lines, and associated with the lung cancer patients' prognosis and clinical features. POU2F2 promoted the proliferation, and motility of lung cancer cells via targeting AGO1 in vitro. Additionally, POU2F2 promoted tumor growth of lung cancer cells via AGO1 in vivo. CONCLUSION: We found POU2F2 was highly expressed in lung cancer cells and confirmed the involvement of POU2F2 in lung cancer progression, and thought POU2F2 could act as a potential therapeutic target for lung cancer.

OBF1 and Oct factors control the germinal center transcriptional program.

OBF1 is a specific coactivator of the POU family transcription factors OCT1 and OCT2. OBF1 and OCT2 are B cell-specific and indispensable for germinal center (GC) formation, but their mechanism of action is unclear. Here, we show by chromatin immunoprecipitation-sequencing that OBF1 extensively colocalizes with OCT1 and OCT2. We found that these factors also often colocalize with transcription factors of the ETS family. Furthermore, we showed that OBF1, OCT2, and OCT1 bind widely to the promoters or enhancers of genes involved in GC formation in mouse and human GC B cells. Short hairpin RNA knockdown experiments demonstrated that OCT1, OCT2, and OBF1 regulate each other and are essential for proliferation of GC-derived lymphoma cell lines. OBF1 downregulation disrupts the GC transcriptional program: genes involved in GC maintenance, such as BCL6, are downregulated, whereas genes related to exit from the GC program, such as IRF4, are upregulated. Ectopic expression of BCL6 does not restore the proliferation of GC-derived lymphoma cells depleted of OBF1 unless IRF4 is also depleted, indicating that OBF1 controls an essential regulatory node in GC differentiation.

The people behind the papers - Awais Javed and Michel Cayouette.

The mammalian retina contains a variety of functionally distinct cell types that are generated by progenitor cells in a specific chronological order. A new paper in Development probes the role of the POU-homeodomain factors Pou2f1 and Pou2f2 in the timely generation of cone photoreceptors in mice. We caught up with first author and PhD student Awais Javed and his supervisor Michel Cayouette (Director of the Cellular Neurobiology Research Unit at the Montreal Clinical Research Institute, Professor at the Université de Montréal and Adjunct Professor at McGill University) to hear more about their work.

Pou2f1 and Pou2f2 cooperate to control the timing of cone photoreceptor production in the developing mouse retina.

Multipotent retinal progenitor cells (RPCs) generate various cell types in a precise chronological order, but how exactly cone photoreceptor production is restricted to early stages remains unclear. Here, we show that the POU-homeodomain factors Pou2f1/Pou2f2, the homologs of Drosophila temporal identity factors nub/pdm2, regulate the timely production of cones in mice. Forcing sustained expression of Pou2f1 or Pou2f2 in RPCs expands the period of cone production, whereas misexpression in late-stage RPCs triggers ectopic cone production at the expense of late-born fates. Mechanistically, we report that Pou2f1 induces Pou2f2 expression, which binds to a POU motif in the promoter of the rod-inducing factor Nrl to repress its expression. Conversely, conditional inactivation of Pou2f2 in RPCs increases Nrl expression and reduces cone production. Finally, we provide evidence that Pou2f1 is part of a cross-regulatory cascade with the other temporal identity factors Ikzf1 and Casz1. These results uncover Pou2f1/2 as regulators of the temporal window for cone genesis and, given their widespread expression in the nervous system, raise the possibility of a general role in temporal patterning.This article has an associated 'The people behind the papers' interview.

Overexpressing of POU2F2 accelerates fracture healing via regulating HMGA1/Wnt/ß-catenin signaling pathway.

To elucidate the role of POU2F2 (POU class 2 homeobox 2) in fracture healing, 30 rats with femoral fracture were randomly grouped into three groups: FF group, LV-POU2F2 group and LV-scramble group. Rats were injected with PBS, lentivirus expressing POU2F2 or scramble lentivirus once a week for 4 weeks. Results showed that overexpressing of POU2F2 promoted fracture healing and callus growth. Besides, overexpressing of POU2F2 promoted protein and mRNA expression of Col10a1, Runx2, Osterix, and Osteocalcin. High Mobility Group AT-hook 1 (HMGA1) is a non-histone protein participating in chromatin remodeling of cells. Western blotting manifested HMGA1/Wnt/ß-catenin pathway was activated in POU2F2 group. Moreover, in-vitro study of hMSCs cells supported the above data. In conclusion, POU2F2 promotes fracture healing via activating the HMGA1/Wnt/ß-catenin signaling pathway.

Network analyses-based identification of circular ribonucleic acid-related pathways in intrahepatic cholangiocarcinoma.

Circular ribonucleic acids are non-coding ribonucleic acids that can be identified from genome sequencing studies. Although they can be readily detected, their regulation and functional role in human diseases such as cancer are unknown. Using a systematic approach, we analyzed ribonucleic acid-sequencing data from a well-characterized cohort of intrahepatic cholangiocarcinoma to identify genetic pathways related to circular ribonucleic acids. Although the expression of most circular ribonucleic acids was similar in both the cancer and non-cancer tissues, expression of circ2174 was significantly increased in cancer tissues. Network analysis of co-related genes identified several pathways associated with circ2174, and common regulatory mediators between genes in these pathways and circ2174. Among these, alterations in several genes involved in interleukin-16 signaling responses such Lck, interleukin-16, and macrophage inflammatory protein-1-beta were the most prominent. Octamer transcription factor (Oct)-2 was identified as a signal transducer that was common to both circ2174 and interleukin-16. Circ2174 has sequence complementarity to miR149 which can target Oct-2. These data suggest a mechanism whereby circ2174 can act as a sponge to regulate the expression of miR149, and thereby modulate Oct-2 and interleukin-16 signaling pathways in cholangiocarcinoma.

A physiologically based pharmacokinetic (PBPK) parent-metabolite model of the chemotherapeutic zoptarelin doxorubicin-integration of in vitro results, Phase I and Phase II data and model application for drug-drug interaction potential analysis.

PURPOSE: Zoptarelin doxorubicin is a fusion molecule of the chemotherapeutic doxorubicin and a luteinizing hormone-releasing hormone receptor (LHRHR) agonist, designed for drug targeting to LHRHR positive tumors. The aim of this study was to establish a physiologically based pharmacokinetic (PBPK) parent-metabolite model of zoptarelin doxorubicin and to apply it for drug-drug interaction (DDI) potential analysis. METHODS: The PBPK model was built in a two-step procedure. First, a model for doxorubicin was developed, using clinical data of a doxorubicin study arm. Second, a parent-metabolite model for zoptarelin doxorubicin was built, using clinical data of three different zoptarelin doxorubicin studies with a dosing range of 10-267 mg/m2, integrating the established doxorubicin model. DDI parameters determined in vitro were implemented to predict the impact of zoptarelin doxorubicin on possible victim drugs. RESULTS: In vitro, zoptarelin doxorubicin inhibits the drug transporters organic anion-transporting polypeptide 1B3 (OATP1B3) and organic cation transporter 2 (OCT2). The model was applied to evaluate the in vivo inhibition of these transporters in a generic manner, predicting worst-case scenario decreases of 0.5% for OATP1B3 and of 2.5% for OCT2 transport rates. Specific DDI simulations using PBPK models of simvastatin (OATP1B3 substrate) and metformin (OCT2 substrate) predict no significant changes of the plasma concentrations of these two victim drugs during co-administration. CONCLUSIONS: The first whole-body PBPK model of zoptarelin doxorubicin and its active metabolite doxorubicin has been successfully established. Zoptarelin doxorubicin shows no potential for DDIs via OATP1B3 and OCT2.

CoQ10 increases mitochondrial mass and polarization, ATP and Oct4 potency levels, and bovine oocyte MII during IVM while decreasing AMPK activity and oocyte death.

PURPOSE: We tested whether mitochondrial electron transport chain electron carrier coenzyme Q10 (CoQ10) increases ATP during bovine IVM and increases %M2 oocytes, mitochondrial polarization/mass, and Oct4, and decreases pAMPK and oocyte death. METHODS: Bovine oocytes were aspirated from ovaries and cultured in IVM media for 24 h with 0, 20, 40, or 60 µM CoQ10. Oocytes were assayed for ATP by luciferase-based luminescence. Oocyte micrographs were quantitated for Oct4, pAMPK (i.e., activity), polarization by JC1 staining, and mitochondrial mass by MitoTracker Green staining. RESULTS: CoQ10 at 40 µM was optimal. Oocytes at 40 µM enabled 1.9-fold more ATP than 0 µM CoQ10. There was 4.3-fold less oocyte death, 1.7-fold more mitochondrial charge polarization, and 3.1-fold more mitochondrial mass at 40 µM than at 0 µM CoQ10. Increased ATP was associated with 2.2-fold lower AMPK thr172P activation and 2.1-fold higher nuclear Oct4 stemness/potency protein at 40 µM than at 0 µM CoQ10. CoQ10 is hydrophobic, and at all doses, 50% was lost from media into oil by ~ 12 h. Replenishing CoQ10 at 12 h did not significantly diminish dead oocytes. CONCLUSIONS: The data suggest that CoQ10 improves mitochondrial function in IVM where unwanted stress, higher AMPK activity, and Oct4 potency loss are induced.

J chain and myocyte enhancer factor 2B are useful in differentiating classical Hodgkin lymphoma from nodular lymphocyte predominant Hodgkin lymphoma and primary mediastinal large B-cell lymphoma.

Although most classical Hodgkin lymphomas (CHLs) are easily distinguished from nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and primary mediastinal large B-cell lymphoma (PMBL), cases with significant CD20 expression cause diagnostic confusion. Although the absence of OCT-2 and BOB.1 are useful in these circumstances, a variable proportion of CHLs are positive for these antigens. We investigated the utility of J chain and myocyte enhancer factor 2B (MEF2B) in the diagnosis of CHL; NLPHL; PMBL; T-cell/histiocyte-rich large B-cell lymphoma (TCRLBL); and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL, compared with OCT-2 and BOB.1. J chain and MEF2B highlighted lymphocyte predominant (LP) cells in 20/20 (100%) NLPHLs and were negative in 43/43 (100%) CHLs. Fourteen of 15 (93%) PMBLs and 4/4 (100%) TCRLBLs were MEF2B positive, whereas 67% of PMBLs and 50% of TCRLBLs were J chain positive. Three of 3 B-cell lymphomas, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and CHL, were negative for J chain and MEF2B. J chain and MEF2B were 100% sensitive and specific for NLPHL versus CHL. MEF2B was 100% sensitive and 98% specific for PMBL versus CHL. Whereas loss of OCT-2 and/or BOB.1 expression had a sensitivity of only 86% and specificity of 100% for CHL versus NLPHL, PMBL, and TCRLBL, lack of both J chain and MEF2B expression was 100% sensitive and 97% specific. J chain and MEF2B are highly sensitive and specific markers of NLPHL versus CHL; are particularly useful in highlighting LP cells; and, with rare exception, are of greater utility than OCT-2 and BOB.1 in differentiating CHL from NLPHL and other large B-cell lymphomas.

Identification of novel prostate cancer drivers using RegNetDriver: a framework for integration of genetic and epigenetic alterations with tissue-specific regulatory network.

We report a novel computational method, RegNetDriver, to identify tumorigenic drivers using the combined effects of coding and non-coding single nucleotide variants, structural variants, and DNA methylation changes in the DNase I hypersensitivity based regulatory network. Integration of multi-omics data from 521 prostate tumor samples indicated a stronger regulatory impact of structural variants, as they affect more transcription factor hubs in the tissue-specific network. Moreover, crosstalk between transcription factor hub expression modulated by structural variants and methylation levels likely leads to the differential expression of target genes. We report known prostate tumor regulatory drivers and nominate novel transcription factors (ERF, CREB3L1, and POU2F2), which are supported by functional validation.

Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation.

The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.

Flexible, symmetry-directed approach to assembling protein cages.

The assembly of individual protein subunits into large-scale symmetrical structures is widespread in nature and confers new biological properties. Engineered protein assemblies have potential applications in nanotechnology and medicine; however, a major challenge in engineering assemblies de novo has been to design interactions between the protein subunits so that they specifically assemble into the desired structure. Here we demonstrate a simple, generalizable approach to assemble proteins into cage-like structures that uses short de novo designed coiled-coil domains to mediate assembly. We assembled eight copies of a C3-symmetric trimeric esterase into a well-defined octahedral protein cage by appending a C4-symmetric coiled-coil domain to the protein through a short, flexible linker sequence, with the approximate length of the linker sequence determined by computational modeling. The structure of the cage was verified using a combination of analytical ultracentrifugation, native electrospray mass spectrometry, and negative stain and cryoelectron microscopy. For the protein cage to assemble correctly, it was necessary to optimize the length of the linker sequence. This observation suggests that flexibility between the two protein domains is important to allow the protein subunits sufficient freedom to assemble into the geometry specified by the combination of C4 and C3 symmetry elements. Because this approach is inherently modular and places minimal requirements on the structural features of the protein building blocks, it could be extended to assemble a wide variety of proteins into structures with different symmetries.