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Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most abundantly expressed RNA binding proteins in the cell and play major roles in all facets of RNA metabolism. hnRNPs are increasingly appreciated as essential for mammalian B cell development by regulating the carefully ordered expression of specific genes. Due to this tight regulation of the hnRNP-RNA network, it is no surprise that a growing number of genes encoding hnRNPs have been causally associated with the onset or progression of many cancers, including B cell neoplasms. Here we discuss our current understanding of hnRNP-driven regulation in normal, perturbed, and malignant B cells, and the most recent and emerging therapeutic innovations aimed at targeting the hnRNP–RNA network in lymphoma.

The basic leucine zipper transcription factor CREBZF (Zhangfei or ZF) was identified through its interaction with Herpes Simplex Virus-1 related cellular protein HCF-1. CREBZF has been implicated in cellular stress responses through its interaction with other proteins, such as CREB3/Luman and ATF4. Here we investigated the production of four CREBZF isoforms, which arise from translational initiation of a downstream AUG at codon 83, and mRNA alternative splicing that adds an IFFFR pentapeptidyl tail to the C-terminus. We found that in addition to transcriptional activation, the short-tailed CREBZF (stZF) isoform was specifically induced by prolonged ER stress treatment. This stZF isoform is a potent transcriptional activator of the pro-apoptotic protein CHOP. Overexpression of stZF activates transcription of CHOP through a CCAAT enhancer binding protein (C/EBP)-ATF site, and promotes apoptosis. We propose that 1) CREBZF is a key component of the Integrated Stress Response (ISR); 2) stZF is essential for the role of CREBZF in inducing CHOP and promoting cell death upon prolonged cellular stress.

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.

In response to environmental stress, human cells have been shown to form reversible amyloid aggregates within the nucleus, termed amyloid bodies (A-bodies). These protective physiological structures share many of the biophysical characteristics associated with the pathological amyloids found in Alzheimer's and Parkinson's disease. Here, we show that A-bodies are evolutionarily conserved across the eukaryotic domain, with their detection in Drosophila melanogaster and Saccharomyces cerevisiae marking the first examples of these functional amyloids being induced outside of a cultured cell setting. The conditions triggering amyloidogenesis varied significantly among the species tested, with results indicating that A-body formation is a severe, but sublethal, stress response pathway that is tailored to the environmental norms of an organism. RNA-sequencing analyses demonstrate that the regulatory low-complexity long non-coding RNAs that drive A-body aggregation are both conserved and essential in human, mouse and chicken cells. Thus, the identification of these natural and reversible functional amyloids in a variety of evolutionarily diverse species highlights the physiological significance of this protein conformation, and will be informative in advancing our understanding of both functional and pathological amyloid aggregation events. This article has an associated First Person interview with the first author of the paper.

Physiological amyloid aggregation occurs within the nuclei of stress-treated cells. These structures, termed Amyloid bodies (A-bodies), assemble through the rapid accumulation of proteins into dense membrane-less organelles, which possess the same biophysical properties as plaques observed in many amyloid-based diseases. Here, we demonstrate that A-body proteomic compositions vary significantly between stimuli, as constituent proteins can be sequestered by one or more stressors. Stimulus exposure alone was insufficient to induce aggregation, demonstrating that this pathway is not regulated solely by stress-induced conformational changes of the A-body targets. We propose that different environmental conditions induce the formation of A-body subtypes containing distinct protein residents. This selective immobilization of proteins may have evolved as a finely tuned mechanism for surviving divergent stressors.

Biomolecular condensates concentrate molecules to facilitate basic biochemical processes, including transcription and DNA replication. While liquid-like condensates have been ascribed various functions, solid-like condensates are generally thought of as amorphous sites of protein storage. Here, we show that solid-like amyloid bodies coordinate local nuclear protein synthesis (LNPS) during stress. On stimulus, translationally active ribosomes accumulate along fiber-like assemblies that characterize amyloid bodies. Mass spectrometry analysis identified regulatory ribosomal proteins and translation factors that relocalize from the cytoplasm to amyloid bodies to sustain LNPS. These amyloidogenic compartments are enriched in newly transcribed messenger RNA by Heat Shock Factor 1 (HSF1). Depletion of stress-induced ribosomal intergenic spacer noncoding RNA (rIGSRNA) that constructs amyloid bodies prevents recruitment of the nuclear protein synthesis machinery, abolishes LNPS, and impairs the nuclear HSF1 response. We propose that amyloid bodies support local nuclear translation during stress and that solid-like condensates can facilitate complex biochemical reactions as their liquid counterparts can.

Objectives Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features known to contribute to differences in clinical course remain limited. We previously discovered non-coding and silent mutations in HNRNPH1 that affect its splicing and contribute to poor outcomes for patients with MCL. We sought to extend our understanding of the mechanisms by which HNRNPH1 contributes to MCL pathology using a combination of in vitro models and integrative analysis of RNA sequencing from MCL tumors. Methods We previously sequenced ribosomal RNA-depleted RNA from 130 MCL tumors. Based on our earlier identification of mutations in HNRNPH1 and altered splicing of this gene, we performed differential splicing analyses using rMATS and leafcutter. We investigated the functional and phenotypic effect of deregulated hnRNP H1 protein through siRNA knockdown. Results Our previous work identified that splicing of HNRNPH1, and not total mRNA expression, correlated with protein abundance in MCL tumors. As a result, our analysis of alternative splicing focused on events associated with altered splicing of HNRNPH1. We identified 155 unique alternative splicing events (ΔPSI > 0.1, FDR < 0.1). Gene ontology analysis identified various aspects of RNA processing which are significantly enriched within this gene list, including mRNA splicing, transport, and metabolic process. This nominates HNRNPH1 as part of the complex network controlling alternative splicing within MCL. Available CLIP-seq in HeLa cells provides evidence for direct interactions between hnRNP H1 and transcripts identified by our analysis (e.g. RBM25, EIF4A1, HNRNPA2B1). Of the 155 events we identified, more than half involved retained introns. Generally, retained introns result in non-productive RNA species, which indicates that this program of intron retention in MCL is a mechanism by which protein abundance can be regulated by hnRNP H1. For all cases with available Mantle Cell Lymphoma International Prognostic Indicator (MIPI) classification, we determined the splicing ratio for HNRNPH1 and observed a general association between high MIPI scores and a lower ratio of non-productive HNRNPH1 transcripts. This suggested that the increased hnRNP H1 abundance we observed in HNRNPH1-mutant tumors contributes to increased proliferation of MCL cells. We verified this in vitro with siRNA knockdown of HNRNPH1 in HEK cells, which resulted in a significant decrease in cell proliferation. Conclusions We have described a pattern of alternative splicing in MCL that is associated with alterations in HNRNPH1 splicing and related protein abundance. The prevalence of retained introns suggests that hnRNP H1 regulates the abundance of protein-coding transcripts via alternative splicing coupled to nonsense-mediated decay. We continue to explore targets of hnRNP H1, a novel oncoprotein in MCL. Disclosures Morin: Celgene: Consultancy.

The amyloid state of protein organization is typically associated with debilitating human neuropathies and is seldom observed in physiology. Here, we uncover a systemic program that leverages the amyloidogenic propensity of proteins to regulate cell adaptation to stressors. On stimulus, cells assemble the amyloid bodies (A-bodies), nuclear foci containing heterogeneous proteins with amyloid-like biophysical properties. A discrete peptidic sequence, termed the amyloid-converting motif (ACM), is capable of targeting proteins to the A-bodies by interacting with ribosomal intergenic noncoding RNA (rIGSRNA). The pathological β-amyloid peptide, involved in Alzheimer's disease, displays ACM-like activity and undergoes stimuli-mediated amyloidogenesis in vivo. Upon signal termination, elements of the heat-shock chaperone pathway disaggregate the A-bodies. Physiological amyloidogenesis enables cells to store large quantities of proteins and enter a dormant state in response to stressors. We suggest that cells have evolved a post-translational pathway that rapidly and reversibly converts native-fold proteins to an amyloid-like solid phase.

SummaryProtein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, and not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4FH. O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, given that they occur during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus.

Historically, amyloids were perceived as toxic/irreversible protein aggregates associated with neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Recent papers are challenging this perception by uncovering widespread cellular roles for physiological amyloidogenesis. These findings suggest that the amyloid-fold should be considered, alongside the native-fold and unfolded configurations, as a physiological and reversible protein organization.

Non-Hodgkin lymphomas (NHL) are a collection of cancers with each malignancy having distinct clinical management and prognosis. Mantle cell lymphoma (MCL) is particularly genetically heterogeneous and is considered incurable. Through a combination of exome, genome, and targeted sequencing of MCL tumors, we identified recurrent mutations in HNRNPH1 (heterogeneous nuclear ribonucleoprotein H1). These mutations are largely intronic or silent and are associated with a putative cis regulatory region involving a single exon. In RNA-seq data from matched cases, we identified variable representation of two distinct HNRNPH1 isoforms. Based on the reading frame of the affected exons, canonical splicing is predicted to produce a functional protein, while alternative splicing introduces a premature termination codon leading to nonsense-mediated decay. We observed a significantly higher proportion of canonical transcripts in MCL tumors bearing HNRNPH1 mutations, leading us to conclude that mutations in HNRNPH1 significantly alter its splicing. Furthermore, increased canonical splicing results in higher HNRNPH1 protein abundance as determined by immunohistochemical analysis of an MCL tissue microarray. HNRNPH1 mutation status and splicing ratio are associated with shorter survival of MCL patients. We developed an in vitro reporter minigene and introduced three specific mutations corresponding to the patient-identified mutations in HNRNPH1. These mutations lead to an increase in canonical splicing and translation of the HNRNPH1 minigene-derived peptide. Additionally, when HNRNPH1 is overexpressed, we observe a decrease of this peptide, implicating HNRNPH1 in the regulation of its own splicing. This is supported by preliminary data indicating that HNRNPH1 binds its own RNA. Beyond its own splicing, HNRNPH1 is likely also involved in the splicing of additional splicing factors. Overexpression of HNRNPH1 affects the splicing of SRSF3 and HNRNPDL, and many other targets are likely to be identified. This work elucidates a functional role for recurrent noncoding HNRNPH1 mutations and implicates HNRNPH1 expression and splicing of its downstream targets in lymphomagenesis. We continue to explore trans regulatory targets of HNRNPH1 using in vitro and cell-based models. While splicing is a growing field of interest in lymphoma biology, the unique pattern and consequences of these largely silent mutations specifically implicate alternative splicing as an oncogenic mechanism in MCL. Citation Format: Krysta M. Coyle, Quratulain Qureshi, Prasath Pararajalingam, Nicole Thomas, Timothy E. Audas, Ryan D. Morin. Noncoding mutations in mantle cell lymphoma disrupt regulation of HNRNPH1 by alternative splicing [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-04.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3′ UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics., The driver mutations for the two main molecular subgroups of diffuse large B-cell lymphoma (DLBCL) are poorly defined. Here, an integrative genomics analysis identifies 3′ UTR NFKBIZ mutations within the activated B-cell DLBCL subgroup and small FCGR2B amplifications in the germinal centre B-cell DLBCL subgroup.

Epigenetic regulation of gene expression by DNA methylation plays a central role in the maintenance of cellular homeostasis. Here we present evidence implicating the DNA methylation program in the regulation of hypoxia-inducible factor (HIF) oxygen-sensing machinery and hypoxic cell metabolism. We show that DNA methyltransferase 3a (DNMT3a) methylates and silences the HIF-2α gene (EPAS1) in differentiated cells. Epigenetic silencing of EPAS1 prevents activation of the HIF-2α gene program associated with hypoxic cell growth, thereby limiting the proliferative capacity of adult cells under low oxygen tension. Naturally occurring defects in DNMT3a, observed in primary tumors and malignant cells, cause the unscheduled activation of EPAS1 in early dysplastic foci. This enables incipient cancer cells to exploit the HIF-2α pathway in the hypoxic tumor microenvironment necessary for the formation of cellular masses larger than the oxygen diffusion limit. Reintroduction of DNMT3a in DNMT3a-defective cells restores EPAS1 epigenetic silencing, prevents hypoxic cell growth, and suppresses tumorigenesis. These data support a tumor-suppressive role for DNMT3a as an epigenetic regulator of the HIF-2α oxygen-sensing pathway and the cellular response to hypoxia.

Mammalian hibernators survive low body temperatures, ischemia-reperfusion, and restricted nutritional resources via global reductions in energy-expensive cellular processes and selective increases in stress pathways. Consequently, studies that analyze hibernation uncover mechanisms which balance metabolism and support survival by enhancing stress tolerance. We hypothesized processing factors that influence messenger ribonucleic acid (mRNA) maturation and translation may play significant roles in hibernation. We characterized the amino acid sequences of three RNA processing proteins (T cell intracellular antigen 1 (TIA-1), TIA1-related (TIAR), and poly(A)-binding proteins (PABP-1)) from thirteen-lined ground squirrels (Ictidomys tridecemlineatus), which all displayed a high degree of sequence identity with other mammals. Alternate Tia-1 and TiaR gene variants were found in the liver with higher expression of isoform b versus a in both cases. The localization of RNA-binding proteins to subnuclear structures was assessed by immunohistochemistry and confirmed by subcellular fractionation; TIA-1 was identified as a major component of subnuclear structures with up to a sevenfold increase in relative protein levels in the nucleus during hibernation. By contrast, there was no significant difference in the relative protein levels of TIARa/TIARb in the nucleus, and a decrease was observed for TIAR isoforms in cytoplasmic fractions of torpid animals. Finally, we used solubility tests to analyze the formation of reversible aggregates that are associated with TIA-1/R function during stress; a shift towards the soluble fraction (TIA-1a, TIA-1b) was observed during hibernation suggesting enhanced protein aggregation was not present during torpor. The present study identifies novel posttranscriptional regulatory mechanisms that may play a role in reducing translational rates and/or mRNA processing under unfavorable environmental conditions.

The recently identified Luman/CREB3-binding partner LRF (Luman/CREB3 recruitment factor) was shown to localize to discrete sub-nuclear foci. Luman is implicated in herpes simplex virus-1 (HSV-1) latency/reactivation and the unfolded protein response (UPR) pathway; therefore, we sought to characterize the formation of the LRF nuclear foci in the context of cellular signaling and HSV-1 replication. Here, we mapped the nuclear foci-targeting sequence to the central region containing the first leucine zipper (a.a.415-519), and found that the integrity of the whole region appears essential for LRF foci formation. LRF foci integrity was unaffected by inhibition of cellular DNA replication and translation, however, disruption of transcription resulted in altered LRF localization. When compared to other cellular and viral foci LRF co-localized with the nuclear receptor co-activator GRIP1, while the HSV-1 gene products ICP4, ICP27 and VP13/14 disrupted foci formation to varying degrees. Interestingly, cells over-expressing LRF were resistant to productive HSV-1 infection and this resistance was dependent upon protein targeting and an N-terminal transactivation domain. When LRF knockdown cells were subjected to primary infection, HSV-1 gene expression and progeny virus yield were enhanced by ∼3 fold compared to wildtype cells. Taken together, these results indicate that LRF is a key regulator that may act direct or indirectly as a repressor of essential genes required for productive viral infection.

Accumulation of mis- and unfolded proteins during viral replication can cause stress in the endoplasmic reticulum (ER) and trigger the unfolded protein response (UPR). If unchecked, this process may induce cellular changes detrimental to viral replication. In the report, we investigated the impact of HSV-1 on the UPR during lytic replication. We found that HSV-1 effectively disarms the UPR in early stages of viral infection. Only ATF6 activation was detected during early infection, but with no upregulation of target chaperone proteins. Activity of the eIF2α/ATF4 signaling arm increased at the final stage of HSV-1 replication, which may indicate completion of virion assembly and egress, thus releasing suppression of the UPR. We also found that the promoter of viral ICP0 was responsive to ER stress, an apparent mimicry of cellular UPR genes. These results suggest that HSV-1 may use ICP0 as a sensor to modulate the cellular stress response.

Genomic studies have revealed that humans possess far fewer protein-encoding genes than originally predicted. These over-estimates were drawn from the inherent developmental and stimuli-responsive complexity found in humans and other mammals, when compared to lower eukaryotic organisms. This left a conceptual void in many cellular networks, as a new class of functional molecules was necessary for "fine-tuning" the basic proteomic machinery. Transcriptomics analyses have determined that the vast majority of the genetic material is transcribed as noncoding RNA, suggesting that these molecules could provide the functional diversity initially sought from proteins. Indeed, as discussed in this review, long noncoding RNAs (lncRNAs), the largest family of noncoding transcripts, have emerged as common regulators of many cellular stressors; including heat shock, metabolic deprivation and DNA damage. These stimuli, while divergent in nature, share some common stress-responsive pathways, notably inhibition of cell proliferation. This role intrinsically makes stress-responsive lncRNA regulators potential tumor suppressor or proto-oncogenic genes. As the list of functional RNA molecules continues to rapidly expand it is becoming increasingly clear that the significance and functionality of this family may someday rival that of proteins. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

Luman (or LZIP, CREB3) is a transcription factor with an endoplasmic reticulum (ER)-transmembrane domain. Due to its structural similarities with ATF6, it is thought that Luman might also be involved in cellular stress responses. Here we report that Luman can bind and activate transcription from the consensus unfolded protein response element (UPRE). Mutations that disrupted the binding of Luman to the UPREs impaired its ability to activate transcription from these sites. Overexpression of Luman stimulated transcription of EDEM, a downstream effector of the mammalian unfolded protein response involved in ER-associated degradation (ERAD). Unlike ATF6, however, Luman was not activated by proteolytic cleavage in response to endoplasmic reticulum stressors such as tunicamycin and thapsigargin. These results suggest that the activation of ERAD by Luman is likely through a pathway different from the common ER stress response, and that additional factor(s) are required for the activation of this Luman-mediated pathway.

Environmental signals, such heat shock and acidosis, induce a structural and functional remodeling of the nucleolus. This process, which depends on the expression of intergenic long noncoding RNA, reversibly converts the nucleolus from a transcriptionally active ribosome factory into a transcriptionally inert prison for proteins., The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Its structural plasticity has long been appreciated, particularly in response to transcriptional inhibition and other cellular stresses, although the mechanism and physiological relevance of these phenomena are unclear. Using MCF-7 and other mammalian cell lines, we describe a structural and functional adaptation of the nucleolus, triggered by heat shock or physiological acidosis, that depends on the expression of ribosomal intergenic spacer long noncoding RNA (IGS lncRNA). At the heart of this process is the de novo formation of a large subnucleolar structure, termed the detention center (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate–positive hydrophobic signature. Its formation is accompanied by redistribution of nucleolar factors and arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA by environmental signals operates as a molecular switch that regulates the structure and function of the nucleolus.

Molecular dynamics ensures that proteins and other factors reach their site of action in a timely and efficient manner. This is essential to the formation of molecular complexes, as they require an ever-changing framework of specific interactions to facilitate a model of self-assembly. Therefore, the absence or reduced availability of any key component would significantly impair complex formation and disrupt all downstream molecular networks. Recently, we identified a regulatory mechanism that modulates protein mobility through the inducible expression of a novel family of long noncoding RNA. In response to diverse environmental stimuli, the nucleolar detention pathway (NoDP) captures and immobilizes essential cellular factors within the nucleolus away from their effector molecules. The vast array of putative NoDP targets, including DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the delta catalytic subunit of DNA polymerase (POLD1), suggests that this may be a common and significant regulatory mechanism. Here, we discuss the implications of this new posttranslational strategy for regulating molecular networks.

Cellular pathways are established and maintained by stochastic interactions of highly mobile molecules. The nucleolus plays a central role in the regulation of these molecular networks by capturing and immobilizing proteins. Here, we report a function for noncoding RNA (ncRNA) in the regulation of protein dynamics of key cellular factors, including VHL, Hsp70 and MDM2/PML. Stimuli-specific loci of the nucleolar intergenic spacer produce ncRNA capable of capturing and immobilizing proteins that encode a discrete peptidic code referred to as the nucleolar detention sequence (NoDS). Disruption of the NoDS/intergenic RNA interaction enables proteins to evade nucleolar sequestration and retain their dynamic profiles. Mislocalization of intergenic ncRNA triggers protein immobilization outside of the nucleolus, demonstrating that these ncRNA species can operate independently from the nucleolar architecture. We propose a model whereby protein immobilization by ncRNA is a posttranslational regulatory mechanism.

Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER)-bound cellular transcription factor. It has been implicated in the mammalian unfolded protein response (UPR), as well as herpes simplex virus reactivation from latency in sensory neurons. Here, we report the identification of a novel Luman recruitment factor (LRF). Like Luman, LRF is a UPR-responsive basic-region leucine zipper protein that is prone to proteasomal degradation. Being a highly unstable protein, LRF interacts with Luman through the leucine zipper region and promotes Luman degradation. LRF was found to recruit the nuclear form of Luman to discrete nuclear foci, which overlap with the nuclear receptor coactivator GRIP1 bodies, and repress the transactivation activity of Luman. Compared to LRF+/+ mouse embryonic fibroblast (MEF) cells, the levels of CHOP, EDEM, and Herp were elevated in LRF-/- MEF cells. We propose that LRF is a negative regulator of the UPR. For Luman, it may represent another level of regulation following Luman proteolytic cleavage on the ER and nuclear translocation. In addition to inducing rapid Luman turnover, LRF may repress the transactivation potential of Luman by sequestering it in the LRF nuclear bodies away from key cofactors (such as HCF-1) that are required for transcriptional activation.

Luman/CREB3 (also called LZIP) is an endoplasmic reticulum (ER) membrane-bound transcription factor which is believed to undergo regulated intramembrane proteolysis in response to cellular cues. We previously found that Luman activates transcription from the unfolded protein response element. Here we report the identification of Herp, a gene involved in ER stress-associated protein degradation (ERAD), as a direct target of Luman. We found that Luman was transcriptionally induced and proteolytically activated by the ER stress inducer thaspsigargin. Overexpression of Luman activated transcription of cellular Herp via ER stress response element II (ERSE-II; ATTGG-N-CCACG) in the promoter region. Mutagenesis studies and chromatin immunoprecipitation assays showed that Luman physically associates with the Herp promoter, specifically the second half-site (CCACG) of ERSE-II. Luman was also necessary for the full activation of Herp during the ER stress response, since Luman small interfering RNA knockdown or functional repression by a dominant negative mutant attenuated Herp gene expression. Like Herp, overexpression of Luman protected cells against ER stress-induced apoptosis. With Luman structurally similar to ATF6 but resembling XBP1 in DNA-binding specificities, we propose that Luman is a novel factor that plays a role in ERAD and a converging point for various signaling pathways channeling through the ER.

The basic leucine zipper transcription factor CREBZF (Zhangfei or ZF) was identified through its interaction with Herpes Simplex Virus-1 related cellular protein HCF-1, and has been implicated in cellular stress responses through its interaction with other proteins, such as CREB3/Luman and ATF4. Here we investigated the production of four CREBZF isoforms, which arise from translational initiation of a downstream AUG at codon 83 and mRNA alternative splicing that adds an IFFFR pentapeptidyl tail to the C-terminus. We found that in addition to transcriptional induction, the short-tailed CREBZF (stZF) isoform was specifically induced by prolonged ER stress treatment and amino acid deprivation. This stZF isoform is a potent transcriptional activator of the pro-apoptotic protein CHOP. Overexpression of stZF activates transcription of CHOP through a CCAAT enhancer binding protein (C/EBP)-ATF site, and promotes apoptosis. We propose that 1) CREBZF is a key component of the integrated stress response; 2) stZF is tightly regulated and induced primarily under prolonged cellular stress, and is essential for the role of CREBZF in inducing CHOP and promoting cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-333.