Interaction of human CRX and NRL in live HEK293T cells measured using fluorescence resonance energy transfer (FRET).

CRX and NRL are retina-specific transcription factors that control rod photoreceptor differentiation and synergistically activate rod phototransduction gene expression. Previous experiments showed they interact in vitro and in yeast two-hybrid assays. Here, we examined CRX-NRL interaction in live HEK293T cells using two fluorescence resonance energy transfer (FRET) approaches: confocal microscopy and flow cytometry (FC-FRET). FC-FRET can provide measurements from many cells having wide donor-acceptor expression ranges. FRET efficiencies were calibrated with a series of donor (EGFP)-acceptor (mCherry) fusion proteins separated with linkers between 6-45 amino acids. CRX and NRL were fused at either terminus with EGFP or mCherry to create fluorescent proteins, and all combinations were tested in transiently transfected cells. FRET signals between CRX or NRL homo-pairs were highest with both fluorophores fused to the DNA binding domains (DBD), lower with both fused to the activation domains (AD), and not significant when fused on opposite termini. NRL had stronger FRET signals than CRX. A significant FRET signal between CRX and NRL hetero-pairs was detected when donor was fused to the CRX DNA binding domain and the acceptor fused to the NRL activation domain. FRET signals increased with CRX or NRL expression levels at a rate much higher than expected for collisional FRET alone. Together, our results show the formation of CRX-NRL complexes in live HEK293T cells that are close enough for FRET.

HLF regulates ferroptosis, development and chemoresistance of triple-negative breast cancer by activating tumor cell-macrophage crosstalk.

Tumor-associated macrophages (TAMs) are major components of the tumor microenvironment (TME) which are closely associated with the tumor malignant progression. However, the regulatory mechanisms by which TAMs influence the progression of triple-negative breast cancer (TNBC) remain unclear. Here, we report that hepatic leukemia factor (HLF) acts as a novel oncoprotein in TNBC. We found that HLF was regulated by transforming growth factor-beta1 (TGF-ß1) that is secreted by TAMs. Then, HLF transactivated gamma-glutamyltransferase 1 (GGT1) to promote the ferroptosis resistance, thus driving TNBC cell proliferation, metastasis and cisplatin resistance. Reciprocally, IL-6 produced by TNBC cells activated the JAK2/STAT3 axis to induce TGF-ß1 secretion by TAMs, thus constituted a feed-forward circuit. The accuracy of TNBC patient prognosis could be improved by employing a combination of HLF and GGT1 values. Thus, our findings document that the interactive dialogue between TNBC cells and TAMs promotes sustained activation of HLF in tumor cells through the IL-6-TGF-ß1 axis. Subsequently, HLF promotes the ferroptosis resistance in TNBC cells via GGT1 and ultimately facilitates the malignant tumor progression. Our study provides a potential target for the treatment of TNBC.

Nuclear factor erythroid 2-related factors 1 and 2 are able to define the worst prognosis group among high-risk diffuse large B cell lymphomas treated with R-CHOEP.

AIMS: Oxidative stress markers and antioxidant enzymes have previously been shown to have prognostic value and associate with adverse outcome in patients with diffuse large B cell lymphoma (DLBCL). Nuclear factor erythroid 2-related factor 1 (Nrf1) and factor 2 (Nrf2) are among the principal inducers of antioxidant enzyme production. Kelch ECH associating protein 1 (Keap1) is a negative regulator of Nrf2, and BTB (BR-C, ttk and bab) domain and CNC homolog 1 (Bach1) represses the function of both factors. Their significance in DLBCL prognosis is unknown. METHODS: Diagnostic biopsy samples of 76 patients with high-risk DLBCL were retrospectively stained with immunohistochemistry for Nrf1, Nrf2, Keap1 and Bach1, and correlated with clinical data and outcome. RESULTS: Nuclear Nrf2 and nuclear Bach1 expression were associated with adverse clinical features (anaemia, advanced stage, high IPI, high risk of neutropaenic infections), whereas cytoplasmic Nrf1 and Nrf2 were associated with favourable clinical presentation (normal haemoglobin level, no B symptoms, limited stage). None of the evaluated factors could predict survival alone. However, when two of the following parameters were combined: high nuclear score of Nrf2, low nuclear score of Nrf1, high cytoplasmic score of Nrf1 and low cytoplasmic score of Keap1 were associated with significantly worse overall survival. CONCLUSIONS: Nrf1 and Nrf2 are relevant in disease presentation and overall survival in high-risk DLBCL. Low nuclear expression of Nrf1, high cytoplasmic expression of Nrf1, high nuclear expression of Nrf2 and low cytoplasmic expression of Keap1 are associated with adverse outcome in this patient group.

Batf3-Dependent Dendritic Cells Promote Optimal CD8 T Cell Responses against Respiratory Poxvirus Infection.

Respiratory infection with vaccinia virus (VacV) elicits robust CD8+ T cell responses that play an important role in host resistance. In the lung, VacV encounters multiple tissue-resident antigen-presenting cell (APC) populations, but which cell plays a dominant role in priming of virus-specific CD8+ effector T cell responses remains poorly defined. We used Batf3-/- mice to investigate the impact of CD103+ and CD8α+ dendritic cell (DC) deficiency on anti-VacV CD8+ T cell responses. We found that Batf3-/- mice were more susceptible to VacV infection, exhibiting profound weight loss, which correlated with impaired accumulation of gamma interferon (IFN-γ)-producing CD8+ T cells in the lungs. This was largely due to defective priming since early in the response, antigen-specific CD8+ T cells in the draining lymph nodes of Batf3-/- mice expressed significantly reduced levels of Ki67, CD25, and T-bet. These results underscore a specific role for Batf3-dependent DCs in regulating priming and expansion of effector CD8+ T cells necessary for host resistance against acute respiratory VacV infection.IMPORTANCE During respiratory infection with vaccinia virus (VacV), a member of Poxviridae family, CD8+ T cells play important role in resolving the primary infection. Effector CD8+ T cells clear the virus by accumulating in the infected lungs in large numbers and secreting molecules such as IFN-γ that kill virally infected cells. However, precise cell types that regulate the generation of effector CD8+ T cells in the lungs are not well defined. Dendritic cells (DCs) are a heterogeneous population of immune cells that are recognized as key initiators and regulators of T-cell-mediated immunity. In this study, we reveal that a specific subset of DCs that are dependent on the transcription factor Batf3 for their development regulate the magnitude of CD8+ T cell effector responses in the lungs, thereby providing protection during pulmonary VacV infection.

The expression change of RORγt, BATF, and IL-17 in Chinese vitiligo patients with 308 nanometers excimer laser treatment.

This study aims to explore the expression of RORγt, BATF, and IL-17 in Chinese vitiligo patients with 308 nm excimer laser treatment. One hundred and sixty-four vitiligo patients treated with 308 nm excimer laser were enrolled as Case group and 137 health examiners as Control group. Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expressions of RORγt, BATF, and IL-17. Expression of RORγt, BATF, IL-17A, and IL-17F were higher in Case group than Control group, with the diagnostic accuracy of 88.04, 87.38, 97.34, and 89.04%, respectively. Pearson correlation analysis showed a positive correlation in RORγt, BATF, IL-17A, and IL-17F mRNAs in vitiligo patients. Moreover, their expressions were higher in active vitiligo patients than stable ones. Besides, the expressions of RORγt, BATF, IL-17A, and IL-17F in vitiligo skin were significantly higher than those in non lesional skin and normal controls. After treatment, their expressions were significantly decreased. Active vitiligo and the high expressions of RORγt, BATF, and IL-17F were the independent risk factors for the ineffectiveness of 308 nm excimer laser treatment. The expressions of RORγt, BATF, IL-17 were significantly enhanced in vitiligo patients, which were correlated with the activity of vitiligo and 308 nm excimer laser therapeutic effects.

Generation of a rod-specific NRL reporter line in human pluripotent stem cells.

Reporter lines generated in human pluripotent stem cells can be highly useful for the analysis of specific cell types and lineages in live cultures. We created the first human rod reporter line using CRISPR/Cas9 genome editing to replace one allele of the Neural Retina Leucine zipper (NRL) gene with an eGFP transgene in the WA09 human embryonic stem cell (hESC) line. After confirming successful targeting, three-dimensional optic vesicle structures were produced to examine reporter specificity and to track rod differentiation in culture. The NRL+/eGFP hESC line robustly and exclusively labeled the entirety of rods throughout differentiation, eventually revealing highly mature structural features. This line provides a valuable tool for studying human rod development and disease and testing therapeutic strategies for retinitis pigmentosa.

Bifurcated BACH2 control coordinates mantle cell lymphoma survival and dispersal during hypoxia.

BACH2, a B-cell-specific transcription factor, plays a critical role in oxidative stress-mediated drug resistance in mantle cell lymphoma (MCL); however, the biological functions of BACH2 and its regulation of B-cell malignancies in chronic hypoxic microenvironment have not been studied. Here, we found that silencing BACH2 led to not only increased tumor formation and colony formation but also increased tumor dispersal to spleen and bone marrow. Decreased BACH2 levels in patients were also correlated with bone marrow and gastrointestinal dispersal of MCL and blastoid subtypes of MCL. Unexpectedly, decreased BACH2 levels in dispersed MCL cells were due to direct transcriptional repression by hypoxia-induced factor 1α (HIF-1α) and increased heme-mediated protein degradation. In normoxic conditions, BACH2 was able to modulate HIF-1α degradation by suppressing prolyl hydroxylase 3 expression. Bifurcated BACH2 controls during hypoxia and normoxia coordinate not only MCL tumor dispersal but also drug resistance, including bortezomib resistance, via plasmacytic differentiation. Our data highlight an interactive relationship between tumor cells and local microenvironment and the mechanisms of B-cell transcription factor in the regulation of MCL dispersal.

Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8.

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

Unique expression pattern of viral proteins in human herpesvirus 8-positive plasmablastic lymphoma: a case report.

BACKGROUND: Human herpesvirus 8 (HHV8)-positive plasmablastic lymphoma is a disease which correlates with acquired immunodeficiency syndrome (AIDS). Little is known about the pathogenesis of the disease due to its rarity. We report an autopsy case about AIDS related HHV-8-positive plasmablastic lymphoma and presents an examination about HHV8 related proteins for the disease by using immunohistochemical techniques. CASE PRESENTATION: Two kinds of tumors complicated the male AIDS patient: one was HHV-8-positive plasmablastic lymphoma and the other was Kaposi's sarcoma (KS). Immunohistochemically, the lymphoma cells were positive for HHV8-associated lytic early proteins as well as HHV8 latency-associated nuclear antigen 1 (LANA-1), and, on the other hand, the lymphoma cells were negative for lytic immediately early proteins. KS was positive for only LANA-1. CONCLUSION: These findings indicate that the lymphoma cells acquired an ability to proliferate without de novo HHV8 replication. Moreover, the onset mechanisms of HHV-8-positive plasmablastic lymphoma may be different from those of KS.

Selective and efficient generation of functional Batf3-dependent CD103+ dendritic cells from mouse bone marrow.

Multiple subsets of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent dendritic cells (DCs) control T-cell tolerance and immunity. In mice, Batf3-dependent CD103(+) DCs efficiently enter lymph nodes and cross-present antigens, rendering this conserved DC subset a promising target for tolerance induction or vaccination. However, only limited numbers of CD103(+) DCs can be isolated with current methods. Established bone marrow culture protocols efficiently generate monocyte-derived DCs or produce a mixture of FLT3L-dependent DC subsets. We show that CD103(+) DC development requires prolonged culture time and continuous action of both FLT3L and granulocyte macrophage colony-stimulating factor (GM-CSF), explained by a dual effect of GM-CSF on DC precursors and differentiating CD103(+) DCs. Accordingly, we established a novel method to generate large numbers of CD103(+) DCs (iCD103-DCs) with limited presence of other DC subsets. iCD103-DCs develop in a Batf3- and Irf8-dependent fashion, express a CD8α/CD103 DC gene signature, cross-present cell-associated antigens, and respond to TLR3 stimulation. Thus, iCD103-DCs reflect key features of tissue CD103(+) DCs. Importantly, iCD103-DCs express high levels of CCR7 upon maturation and migrate to lymph nodes more efficiently than classical monocyte-derived DCs. Finally, iCD103-DCs induce T cell-mediated protective immunity in vivo. Our study provides insights into CD103(+) DC development and function.

Batf3-independent langerin- CX3CR1- CD8α+ splenic DCs represent a precursor for classical cross-presenting CD8α+ DCs.

This study tests the hypothesis that CD8α(+) DCs in the spleen of mice contain an immature precursor for functionally mature, "classical" cross-presenting CD8α(+) DCs. The lymphoid tissues contain a network of phenotypically distinct DCs with unique roles in surveillance and immunity. Splenic CD8α(+) DCs have been shown to exhibit a heightened capacity for phagocytosis of cellular material, secretion of IL-12, and cross-priming of CD8(+) T cells. However, this population can be subdivided further on the basis of expression of both langerin/CD207 and CX(3)CR1. We therefore evaluated the functional capacities of these different subsets. The CX(3)CR1(+) CD8α(+) DC subset does not express langerin and does not exhibit the classical features above. The CX(3)CR1(-) CD8α(+) DC can be divided into langerin-positive and negative populations, both of which express DEC205, Clec9A, and high basal levels of CD86. However, the langerin(+) CX(3)CR1(-) CD8α(+) subset has a superior capacity for acquiring cellular material and producing IL-12 and is more susceptible to activation-induced cell death. Significantly, following purification and adoptive transfer into new hosts, the langerin(-) CX(3)CR1(-) CD8α(+) subset survives longer, up-regulates expression of langerin, and becomes more susceptible to activation-induced cell death. Last, in contrast to langerin(+) CX(3)CR1(-) CD8α(+), the langerin(-) CX(3)CR1(-) CD8α(+) are still present in Batf3(-/-) mice. We conclude that the classical attributes of CD8α(+) DC are confined primarily to the langerin(+) CX(3)CR1(-) CD8α(+) DC population and that the langerin(-) CX(3)CR1(-) subset represents a Batf3-independent precursor to this mature population.

Long-term adaptation of Saccharomyces cerevisiae to the burden of recombinant insulin production.

High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30 g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation.

Duodenal follicular lymphoma lacks AID but expresses BACH2 and has memory B-cell characteristics.

We have reported previously that duodenal follicular lymphoma (FL) is distinct from nodal FL and showed more resemblance to mucosa-associated lymphoid tissue lymphoma, and that FL frequently involved the duodenal second portion. In the present study, we examined duodenal FLs and gastric/colonic FLs to clarify the clinicopathological and immunological differences between the tumor types. We analyzed 8 samples of gastric FL, 17 of duodenal ones, and 5 of colonic/rectal ones, and characterized them by immunohistochemistry, immunogenotyping, and histology. Gastric and colonic FLs presented in submucosal to subserosal areas, whereas duodenal ones presented in the mucosal to submucosal layers. Immunohistochemical analysis revealed that duodenal FLs exhibited the following phenotypes: CD10 (+), B-cell lymphoma 2 (BCL-2) (+), BCL-6 (+), activation-induced cytidine deaminase (AID) (-), BACH2 (+), CD27 (+), MUM-1 (-), Blimp-1 (-), and loose CD21 network (duodenal pattern). Gastric/colonic FLs exhibited the following phenotypes: CD10 (+), BCL-2 (+), BCL-6 (+), AID (+), BACH2 (+), CD27 (-), MUM-1 (-), Blimp-1 (-), and a dense CD21 network (nodal pattern). Expression of AID and CD27 in lymphoma cells and the CD21 network pattern were considerably different between duodenal FLs and gastric/colonic ones. Moreover, in situ hybridization revealed that, in the duodenal FLs, BACH2 was expressed at the periphery of the tumor follicle and tumor villi. The number of immunoglobulin heavy-chain variable domains VH4 and VH5 were higher in duodenal follicular lymphomoas than in gastric FLs. The lymphoma cells of duodenal FLs are different from those of gastric/colonic FLs, and duodenal FL is distinct even within the gastrointestinal tract. Somatic hypermutation in immunoglobulin genes and CD27 expression are hallmarks of memory B cells. We suggest that duodenal FL cells are in the memory B-cell stage, and require BACH2 instead of AID for ongoing mutation.

Multiparametric flow cytometry for identification and fluorescence activated cell sorting of five distinct B-cell subpopulations in normal tonsil tissue.

The purpose of this study was to establish a procedure capable of isolating distinct B-cell subpopulations from human tonsils as a basis for subsequent molecular analyses. Overall, 5 distinct B-cell subpopulations were purified from fresh tonsils based on their fluorescence surface marker expression: naive B cells, centroblasts, centrocytes, memory B cells, and plasmablasts. The immunophenotypic identity of the subpopulations was verified by quantitative real-time reverse transcriptase-polymerase chain reaction using the proliferation marker MKI-67 and 6 B-cell-associated differentiation markers (BACH2, BCL6, PAX5, IRF4, PRDM1, and XBP1). Furthermore, within the centroblast compartment, large and small centroblasts could be distinguished and large centroblasts were shown to proliferate with a morphologic appearance of a "centroblast"-like cell but with lower gene expression of the germinal center markers BCL6 and BACH2 vs small centroblasts. This study has established a detailed and fast procedure for simultaneous sorting of up to 5 distinct maturation-associated B-cell subpopulations from human tonsils.

Intrinsic and extrinsic effects of mafG deficiency on hematopoietic recovery following bone marrow transplant.

OBJECTIVE: MafG is the small subunit of the transcription factor NF-E2 that controls terminal megakaryocyte maturation and platelet release. Studies were conducted to evaluate the intrinsic and extrinsic effects of mafG deficiency on bone marrow engraftment kinetics. MATERIALS AND METHODS: We used mafG knockout mice either as donors or recipients in bone marrow transplantations with wild-type mice and compared the engraftment kinetics to transplantations using wild-type donors and recipients. We measured peripheral cell counts, the presence of circulating donor-derived cells by flow cytometry, changes in the cellularity of the bone marrow and splenic weight on day 5, 7, 14, and 1 month post-transplantation. RESULTS: Compared to wild-type recipients, mafG recipients had delayed platelet and leukocyte recovery and lower spleen weight at early time points after transplantation. Intrinsic effects: When mafG-deficient bone marrow served as donor source, we observed more rapid recovery of bone marrow cellularity and increased splenic hematopoiesis. The finding of increased short-term hematopoietic stem cells and progenitors in the mafG-deficient bone marrow could explain the accelerated hematopoietic recovery after transplantation. Furthermore, the expression of Bach 2, which can form a heterodimer with mafG protein, was found to be greatly reduced, while Notch 1 expression was increased in mafG-deficient mice. Extrinsic effects: When mafG-deficient mice were transplant recipients, there were delays in recovery of normal levels of marrow and splenic hematopoiesis as well as circulating leukocytes and platelets. CONCLUSIONS: Our study demonstrates that mafG expression has intrinsic and extrinsic effects on hematopoietic engraftment following bone marrow transplantation.

Significantly improved sensitivity of Q-band PELDOR/DEER experiments relative to X-band is observed in measuring the intercoil distance of a leucine zipper motif peptide (GCN4-LZ).

Pulsed electron double resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy is a very powerful structural biology tool in which the dipolar coupling between two unpaired electron spins (site-directed nitroxide spin-labels) is measured. These measurements are typically conducted at X-band (9.4 GHz) microwave excitation using the four-pulse DEER sequence and can often require up to 12 h of signal averaging for biological samples (depending on the spin-label concentration). In this work, we present for the first time a substantial increase in DEER sensitivity obtained by collecting DEER spectra at Q-band (34 GHz), when compared to X-band. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents a 169-fold decrease in data collection time, reveals a greatly improved frequency spectrum and higher-quality distance data, and significantly increases sample throughput. Thus, the availability of Q-band DEER spectroscopy should have a major impact on structural biology studies using site-directed spin labeling EPR techniques.

Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice.

OsbZIP23 is a member of the basic leucine zipper (bZIP) transcription factor family in rice (Oryza sativa). Expression of OsbZIP23 is strongly induced by a wide spectrum of stresses, including drought, salt, abscisic acid (ABA), and polyethylene glycol treatments, while other stress-responsive genes of this family are slightly induced only by one or two of the stresses. Transactivation assay in yeast demonstrated that OsbZIP23 functions as a transcriptional activator, and the sequences at the N terminus (amino acids 1-59) and a region close to the C terminus (amino acids 210-240) are required for the transactivation activity. Transient expression of OsbZIP23-green fluorescent protein in onion (Allium cepa) cells revealed a nuclear localization of the protein. Transgenic rice overexpressing OsbZIP23 showed significantly improved tolerance to drought and high-salinity stresses and sensitivity to ABA. On the other hand, a null mutant of this gene showed significantly decreased sensitivity to a high concentration of ABA and decreased tolerance to high-salinity and drought stress, and this phenotype can be complemented by transforming the OsbZIP23 back into the mutant. GeneChip and real-time polymerase chain reaction analyses revealed that hundreds of genes were up- or down-regulated in the rice plants overexpressing OsbZIP23. More than half of these genes have been annotated or evidenced for their diverse functions in stress response or tolerance. In addition, more than 30 genes that are possible OsbZIP23-specific target genes were identified based on the comparison of the expression profiles in the overexpressor and the mutant of OsbZIP23. Collectively, these results indicate that OsbZIP23 functions as a transcriptional regulator that can regulate the expression of a wide spectrum of stress-related genes in response to abiotic stresses through an ABA-dependent regulation pathway. We propose that OsbZIP23 is a major player of the bZIP family in rice for conferring ABA-dependent drought and salinity tolerance and has high potential usefulness in genetic improvement of stress tolerance.

The mobility of Bach2 nuclear foci is regulated by SUMO-1 modification.

The small ubiquitin-like modifier-1 (SUMO-1) modulates the functions of nuclear proteins by changing their structure and/or subnuclear localization. Several nuclear proteins form dynamic higher order nuclear structures, termed non-chromatin nuclear domains, which are involved in the regulation of nuclear function. However, the role that SUMO modification of the component proteins plays in the regulation of the activity and function of nuclear domains is unclear. Here we demonstrate that nuclear domains formed by Bach2, a transcription repressor, show restricted movement and undergo fusion events upon oxidative stress. Mutation of the SUMO-acceptor lysines in Bach2 alters the behavior of these nuclear foci and results in a decreased frequency of fusion events. We propose that SUMO modification is an important regulatory system for the mobility of the nuclear domains formed by Bach2.

Evaluation of AIbZIP and Cdc47 as markers for human prostatic diseases.

OBJECTIVES: Benign prostatic hyperplasia and prostate cancer are important public health issues. However, histologic markers for these diseases are limited. METHODS: Immunocytochemistry was used to analyze the cellular localization of AIbZIP, Cdc47, androgen receptor and estrogen receptor-beta markers. AIbZIP is a protein recently found to be more abundant in prostate cancer than in benign prostatic tissue, and Cdc47 is a cell proliferation-associated protein. The localization and modulation of androgen receptor and estrogen receptor-beta through the carcinogenesis process have been examined in several studies but controversial results were obtained. These four proteins were evaluated as potential markers of prostatic diseases in 210 needle core biopsies, including normal prostate, benign prostatic hyperplasia, low-grade and high-grade prostatic intraepithelial neoplasia, and different Gleason grades of prostatic adenocarcinoma. RESULTS: Androgen receptor and estrogen receptor-beta do not discriminate between benign and malignant specimens, while AIbZIP was able to distinguish between them. Cdc47, in contrast, discriminated not only between malignant and benign prostatic tissue, but also between benign prostatic hyperplasia and normal prostatic tissue. CONCLUSIONS: Cdc47 appears to be a sensitive marker of prostatic diseases since its expression gradually increased in parallel with the severity of the lesion. AIbZIP discriminated between benign tissue and cancer. AIbZIP and Cdc47 thus appear to be useful markers with diagnostic and prognostic values.

SCFCdc4-mediated degradation of the Hac1p transcription factor regulates the unfolded protein response in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae basic leucine zipper transcription factor Hac1p is synthesized in response to the accumulation of unfolded polypeptides in the lumen of the endoplasmic reticulum (ER), and it is responsible for up-regulation of approximately 5% of all yeast genes, including ER-resident chaperones and protein-folding catalysts. Hac1p is one of the most short-lived yeast proteins, having a half-life of approximately 1.5 min. Here, we have shown that Hac1p harbors a functional PEST degron and that degradation of Hac1p by the proteasome involves the E2 ubiquitin-conjugating enzyme Ubc3/Cdc34p and the SCF(Cdc4) E3 complex. Consistent with the known nuclear localization of Cdc4p, rapid degradation of Hac1p requires the presence of a functional nuclear localization sequence, which we demonstrated to involve basic residues in the sequence (29)RKRAKTK(35). Two-hybrid analysis demonstrated that the PEST-dependent interaction of Hac1p with Cdc4p requires Ser146 and Ser149. Turnover of Hac1p may be dependent on transcription because it is inhibited in cell mutants lacking Srb10 kinase, a component of the SRB/mediator module of the RNA polymerase II holoenzyme. Stabilization of Hac1p by point mutation or deletion, or as the consequence of defects in components of the degradation pathway, results in increased unfolded protein response element-dependent transcription and improved cell viability under ER stress conditions.