Mammalian Transcription Factor Research Articles

Exploring the transcriptional cooperation between RUNX2 and its associated elncRNA RAIN

Recent insights into the mechanisms controlling gene expression identified enhancer-associated long non-coding RNAs (elncRNAs) as master players of transcription in cancers. RUNX2, a mammalian RUNT-related transcription factor, is increasingly recognized in cancer biology for its role in supporting survival and progression also in thyroid cancer (TC). We recently identified, within the RUNX2 locus, a novel elncRNA that we named RAIN (RUNX2 associated intergenic lncRNA). We showed that RAIN and RUNX2 expression correlate in TC, both in vitro and in vivo, and that RAIN promotes RUNX2 expression by interacting with and affecting the activity of the RUNX2 P2 promoter through two distinct mechanisms. Here, we took forward these observations to explore the genome-wide transcriptional function of RAIN and its contribution to the RUNX2-dependent gene expression program in TC. By combining multiple omics data, we demonstrated that RAIN functionally cooperates with RUNX2 to the regulation of a subset of functionally related genes involved in promoting matrix remodeling, migration, and loss of differentiation. We showed that RAIN interacts with RUNX2 and its expression is required for the efficient recruitment of this TF to its target regulatory regions. In addition, our data revealed that besides RUNX2, RAIN governs a hierarchically organized complex transcriptional program by controlling a core of cancer-associated TFs that, in turn, orchestrate the expression of downstream genes. This evidence indicates that the functional cooperation observed between RAIN and RUNX2 can be a diffuse work mechanism for this elncRNA.

Engineered Transcription Factor Binding Arrays for DNA-based Gene Expression Control in Mammalian Cells.

Manipulating gene expression in mammalian cells is critical for cell engineering applications. Here we explore the potential of transcription factor (TF) recognition element arrays as DNA tools for modifying free TF levels in cells and thereby controlling gene expression. We first demonstrate proof-of-concept, showing that Tet TF-binding recognition element (RE) arrays of different lengths can tune gene expression and alter gene circuit performance in a predictable manner. We then open-up the approach to interface with any TF with a known binding site by developing a new method called Cloning Troublesome Repeats in Loops (CTRL) that can assemble plasmids with up to 256 repeats of any RE sequence. Transfection of RE array plasmids assembled by CTRL into mammalian cells show potential to modify host cell gene regulation at longer array sizes by sequestration of the TF of interest. RE array plasmids built using CTRL were demonstrated to target both synthetic and native mammalian TFs, illustrating the ability to use these tools to modulate genetic circuits and instruct cell fate. Together this work advances our ability to assemble repetitive DNA arrays and showcases the use of TF-binding RE arrays as a method for manipulating mammalian gene expression, thus expanding the possibilities for mammalian cell engineering.

The SINA1-BSD1 Module Regulates Vegetative Growth Involving Gibberellin Biosynthesis in Tomato.

In plants, vegetative growth is controlled by synergistic and/or antagonistic effects of many regulatory factors. Here, the authors demonstrate that the ubiquitin ligase seven in absentia1 (SINA1) mammalian BTF2-like transcription factors, Drosophila synapse-associated proteins, and yeast DOS2-like proteins (BSD1) function as a regulatory module to control vegetative growth in tomato via regulation of the production of plant growth hormone gibberellin (GA). SINA1 negatively regulates the protein level of BSD1 through ubiquitin-proteasome-mediated degradation, and the transgenic tomato over-expressing SINA1 (SINA1-OX) resembles the dwarfism phenotype of the BSD1-knockout (BSD1-KO) tomato plant. BSD1 directly activates expression of the BSD1-regulated gene 1 (BRG1) via binding to a novel core BBS (standing for BSD1 binding site) binding motif in the BRG1 promoter. Knockout of BRG1 (BRG1-KO) in tomato also results in a dwarfism phenotype, suggesting BRG1 plays a positive role in vegetative growth as BSD1 does. Significantly, GA contents are attenuated in transgenic SINA1-OX, BSD1-KO, and BRG1-KO plants exhibiting dwarfism phenotype and exogenous application of bioactive GA3 restores their vegetative growth. Moreover, BRG1 is required for the expression of multiple GA biosynthesis genes and BSD1 activates three GA biosynthesis genes promoting GA production. Thus, this study suggests that the SINA1-BSD1 module controls vegetative growth via direct and indirect regulation of GA biosynthesis in tomato.

Expression profiling of BSD domain-containing genes in apricot during different developmental stages

Plant transcription factors are evolutionarily conserved proteins that play an important role in the transcriptional regulation of gene expression by binding to their specific DNA sequences. BSD (mammalian BTF2-like transcription factors, synapse-associated proteins, and DOS2-like proteins) transcription factors are conserved in various species, from protozoa to humans, and are characterized by a typical BSD domain. However, little information is available about their possible roles in plant growth and development, and to date, members of this transcription factor family have not been systematically identified and analyzed in apricot. In this study, two BSD domain-encoding genes were identified in the apricot genome. Expression profile analysis by RT-qPCR revealed that both genes participate in different developmental stages of three different organs in apricot. PaBSD1 was expressed higher than PaBSD2 only in the stamen. Moreover, PaBSD2 was higher expressed than PaBSD1 in four different fruit stages, young leaf, leaf bud, sepal and petal. This study reveals the critical roles of BSD transcription factors in apricot development, with PaBSD1 showing higher expression in stamen and PaBSD2 in various fruit stages and leaf tissues. These findings provide a foundation for future functional studies and apricot breeding programs.

Transcriptional upregulation of the myo-inositol biosynthesis pathway is enhanced by NFAT5 in hyperosmotically stressed tilapia cells.

Euryhaline fish experience variable osmotic environments requiring physiological adjustments to tolerate elevated salinity. Mozambique tilapia (Oreochromis mossambicus) possess one of the highest salinity tolerance limits of any fish. In tilapia and other euryhaline fish species, the myo-inositol biosynthesis (MIB) pathway enzymes, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1), are among the most upregulated mRNAs and proteins indicating the high importance of this pathway for hyperosmotic (HO) stress tolerance. These abundance changes must be precluded by HO perception and signaling mechanism activation to regulate the expression of MIPS and IMPA1.1 genes. In previous work using a O. mossambicus cell line (OmB), a reoccurring osmosensitive enhancer element (OSRE1) in both MIPS and IMPA1.1 was shown to transcriptionally upregulate these enzymes in response to HO stress. The OSRE1 core consensus (5'-GGAAA-3') matches the core binding sequence of the predominant mammalian HO response transcription factor, nuclear factor of activated T-cells (NFAT5). HO-challenged OmB cells showed an increase in NFAT5 mRNA suggesting NFAT5 may contribute to MIB pathway regulation in euryhaline fish. Ectopic expression of wild-type NFAT5 induced an IMPA1.1 promoter-driven reporter by 5.1-fold (P < 0.01). Moreover, expression of dominant negative NFAT5 in HO media resulted in a 47% suppression of the reporter signal (P < 0.005). Furthermore, reductions of IMPA1.1 (37-49%) and MIPS (6-37%) mRNA abundance were observed in HO-challenged NFAT5 knockout cells relative to control cells. Collectively, these multiple lines of experimental evidence establish NFAT5 as a tilapia transcription factor contributing to HO-induced activation of the MIB pathway.NEW & NOTEWORTHY In our study, we use a multi-pronged synthetic biology approach to demonstrate that the fish homolog of the predominant mammalian osmotic stress transcription factor nuclear factor of activated T-cells (NFAT5) also contributes to the activation of hyperosmolality inducible genes in cells of extremely euryhaline fish. However, in addition to NFAT5 the presence of other strong osmotically inducible signaling mechanisms is required for full activation of osmoregulated tilapia genes.

Transcription factor PBX4 regulates limb development and haematopoiesis in mice.

The mammalian Pre-B cell leukaemia transcription factors 1-4 (PBX1-4) constitutes the PBC class of the homeodomain (HD)-containing proteins, which play important roles in diverse developmental processes. The functions and the underlying molecular mechanisms of PBX1-3 but not PBX4 have been extensively studied, and they have been reported to direct essential morphogenetic processes and organogenesis. In the present study, we generated knockin mice of FLAG-tagged PBX4 and the Pbx4 knockout (KO) mice and carried out in-depth characterisation of PBX4 expression and function. PBX4 was initially detected only in the testis among several organs of the adult mice and was expressed in spermatocytes and spermatids. However, no abnormality in spermatogenesis, but growth retardation and premature death after birth were observed in most adult Pbx4 KO mice. These animals were inactive and had shorter hindlimbs and lower numbers of reticulocytes and lymphocytes, probably caused by abnormalities at earlier developmental stages. Pbx4 mRNAs were indeed detected in several embryonic cell types related to limb development by in situ hybridisation and single-cell RNA-sequencing analysis. Pbx4 protein was also detected in the bone marrow of adult mice with a lower level compared with that in the testis. PBX4 preferentially binds to the promoters of a large number of genes including those for other HD-containing proteins and ribosomal proteins whose mutations are related to anaemia. PBX4-binding sites are enriched in motifs similar to those of other HD-containing proteins such as PKNOX1 indicating that PBX4 may also act as a co-transcription factor like other PBC proteins. Together, these results show that PBX4 participates in limb development and haematopoiesis while its function in spermatogenesis has not been revealed by gene KO probably due to the complementary effects of other genes.

SKN-1 isoform-c is essential for C. elegans development.

The transcription factor SKN-1 in Caenorhabditis elegans is a critical regulator of various biological processes, impacting development, diet and immune responses, cellular detoxification, and lipid metabolism; thereby playing a pivotal role in regulating the health and lifespan of the organism. The primary isoforms of SKN-1 ( SKN-1 a, SKN-1 b, and SKN-1 c) exhibit distinct functions resembling mammalian Nrf transcription factors. This study investigates the specific role of the SKN-1 c isoform in development by generating mutants with targeted missense mutations in the skn-1 c and skn-1 a isoforms. The skn-1 c Met1Ala mutants, which replaces a start methionine with alanine, renders SKN-1 c non-functional while preserving other isoforms, produced inviable embryos, requiring a balancer chromosome for proper embryonic development. In contrast, skn-1 a Met1Ala mutants, which replaces the start methionine with alanine for this isoform, displayed normal embryonic development and hatching. Moreover, the data suggest that SKN-1 c plays a crucial role in embryonic development, as strains without maternally deposited SKN-1 c lead to embryos that are developmentally arrested. Together, these findings contribute to our understanding of SKN-1 c's specific role in influencing embryogenesis and development in C. elegans.

Increased expression of metabolism and lysosome-associated genes in a C. elegans dpy-7 cuticle furrow mutant.

The collagen-based epidermal 'cuticle' of Caenorhabditis elegans functions as an extracellular sensor for damage that regulates genes promoting osmotic balance, innate immunity, and detoxification. Prior studies demonstrate that SKN-1 , an ortholog of the mammalian Nrf transcription factors, activates core detoxification genes downstream from cuticle damage. Prior RNAseq data suggested that expression of five genes with functions in redox balance, ATP homeostasis, and lysosome function ( gst-15 , gst-24 , cyts-1 , argk-1 , and mfsd-8.4 ) were increased in a cuticle collagen mutant; this study employed RT-qPCR to verify this observation and to test the role of SKN-1 . Activation of all five genes was verified in dpy-7 mutants, but none were reduced by skn-1 (RNAi) suggesting parallel or distinct regulatory mechanisms.

Identification of mammalian transcription factors that bind to inaccessible chromatin.

Transcription factors (TFs) are proteins that affect gene expression by binding to regulatory regions of DNA in a sequence specific manner. The binding of TFs to DNA is controlled by many factors, including the DNA sequence, concentration of TF, chromatin accessibility and co-factors. Here, we systematically investigated the binding mechanism of hundreds of TFs by analysing ChIP-seq data with our explainable statistical model, ChIPanalyser. This tool uses as inputs the DNA sequence binding motif; the capacity to distinguish between strong and weak binding sites; the concentration of TF; and chromatin accessibility. We found that approximately one third of TFs are predicted to bind the genome in a DNA accessibility independent fashion, which includes TFs that can open the chromatin, their co-factors and TFs with similar motifs. Our model predicted this to be the case when the TF binds to its strongest binding regions in the genome, and only a small number of TFs have the capacity to bind dense chromatin at their weakest binding regions, such as CTCF, USF2 and CEBPB. Our study demonstrated that the binding of hundreds of human and mouse TFs is predicted by ChIPanalyser with high accuracy and showed that many TFs can bind dense chromatin.

Cognitive Impairment in Multiple Sclerosis.

Almost three million individuals suffer from multiple sclerosis (MS) throughout the world, a demyelinating disease in the nervous system with increased prevalence over the last five decades, and is now being recognized as one significant etiology of cognitive loss and dementia. Presently, disease modifying therapies can limit the rate of relapse and potentially reduce brain volume loss in patients with MS, but unfortunately cannot prevent disease progression or the onset of cognitive disability. Innovative strategies are therefore required to address areas of inflammation, immune cell activation, and cell survival that involve novel pathways of programmed cell death, mammalian forkhead transcription factors (FoxOs), the mechanistic target of rapamycin (mTOR), AMP activated protein kinase (AMPK), the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1), and associated pathways with the apolipoprotein E (APOE-ε4) gene and severe acute respiratory syndrome coronavirus (SARS-CoV-2). These pathways are intertwined at multiple levels and can involve metabolic oversight with cellular metabolism dependent upon nicotinamide adenine dinucleotide (NAD+). Insight into the mechanisms of these pathways can provide new avenues of discovery for the therapeutic treatment of dementia and loss in cognition that occurs during MS.

A parasite DNA binding protein with potential to influence disease susceptibility acts as an analogue of mammalian HMGA transcription factors.

Intracellular pathogens construct their environmental niche, and influence disease susceptibility, by deploying factors that manipulate infected host cell gene expression. Theileria annulata is an important tick-borne parasite of cattle that causes tropical theileriosis. Excellent candidates for modulating host cell gene expression are DNA binding proteins bearing AT-hook motifs encoded within the TashAT gene cluster of the parasite genome. In this study, TashAT2 was transfected into bovine BoMac cells to generate three expressing and three non-expressing (opposite orientation) cell lines. RNA-Seq was conducted and differentially expressed (DE) genes identified. The resulting dataset was compared with genes differentially expressed between infected cells and non-infected cells, and DE genes between infected cell lines from susceptible Holstein vs tolerant Sahiwal cattle. Over 800 bovine genes displayed differential expression associated with TashAT2, 209 of which were also modulated by parasite infection. Network analysis showed enrichment of DE genes in pathways associated with cellular adhesion, oncogenesis and developmental regulation by mammalian AT-hook bearing high mobility group A (HMGA) proteins. Overlap of TashAT2 DE genes with Sahiwal vs Holstein DE genes revealed that a significant number of shared genes were associated with disease susceptibility. Altered protein levels encoded by one of these genes (GULP1) was strongly linked to expression of TashAT2 in BoMac cells and was demonstrated to be higher in infected Holstein leucocytes compared to Sahiwal. We conclude that TashAT2 operates as an HMGA analogue to differentially mould the epigenome of the infected cell and influence disease susceptibility.

Construction and validation of customized genomes for human and mouse ribosomal DNA mapping

rRNAs are transcribed from ribosomal DNA (rDNA) repeats, the most intensively transcribed loci in the genome. Due to their repetitive nature, there is a lack of genome assemblies suitable for rDNA mapping, creating a vacuum in our understanding of how the most abundant RNA in the cell is regulated. Our recent work revealed binding of numerous mammalian transcription and chromatin factors to rDNA. Several of these factors were known to play critical roles in development, tissue function, and malignancy, but their potential roles in rDNA regulation remained unexplored. This demonstrated the blind spot into which rDNA has fallen in genetic and epigenetic studies and highlighted an unmet need for public rDNA-optimized genome assemblies. Here, we customized five human and mouse assemblies-hg19 (GRCh37), hg38 (GRCh38), hs1 (T2T-CHM13), mm10 (GRCm38), and mm39 (GRCm39)-to render them suitable for rDNA mapping. The standard builds of these genomes contain numerous fragmented or repetitive rDNA loci. We identified and masked all rDNA-like regions, added a single rDNA reference sequence of the appropriate species as a ∼45 kb chromosome designated "chromosome R," and created annotation files to aid visualization of rDNA features in browser tracks. We validated these customized genomes for mapping of known rDNA-binding proteins and present a simple workflow for mapping chromatin immunoprecipitation-sequencing datasets. Customized genome assemblies, annotation files, positive and negative control tracks, and Snapgene files of standard rDNA reference sequences have been deposited to GitHub. These resources make rDNA mapping and visualization more readily accessible to a broad audience.

Chronic exposure to di(2-ethylhexyl) phthalate (DEHP) weakens innate immunity and leads to immunosenescence in C. elegans

Di(2-ethylhexyl) phthalate (DEHP), a widespread contaminant, has numerous adverse impacts on human health and ecosystems. Chronic DEHP exposure has been found to accelerate aging; however, its potential threat to age-dependent innate immune decline remains unknown. This study aims to evaluate the effects of chronic DEHP exposure on innate immunosenescence in Caenorhabditis elegans. We show that the length of the exposure period significantly impacts DEHP-induced age-related declines, which is linked to immunosenescence and oxidative stress. We found that the DEHP-caused immunosenescence is accompanied with downregulation of an antimicrobial gene lys-7 as well as an enhancement of the nuclear translocation of HLH-30, an orthologue of mammalian transcription factor EB (TFEB). Moreover, DEHP exposure increases the expression of riok-1, a human RIO kinase homolog, which is associated with DEHP-induced HLH-30/TFEB translocation. Our findings suggest that early-life and chronic exposure to DEHP, mostly due to parent compound rather than its metabolite mono(2-ethylhexyl) phthalate (MEHP), may weaken the innate immunity in C. elegans and may enhance susceptibility to infections or promote immunosenescence in aged populations.

Innovative therapeutic strategies for cardiovascular disease.

As a significant non-communicable disease, cardiovascular disease is the leading cause of death for both men and women, comprises almost twenty percent of deaths in most racial and ethnic groups, can affect greater than twenty-five million individuals worldwide over the age of twenty, and impacts global economies with far-reaching financial challenges. Multiple factors can affect the onset of cardiovascular disease that include high serum cholesterol levels, elevated blood pressure, tobacco consumption and secondhand smoke exposure, poor nutrition, physical inactivity, obesity, and concurrent diabetes mellitus. Yet, addressing any of these factors cannot completely eliminate the onset or progression of cardiovascular disorders. Novel strategies are necessary to target underlying cardiovascular disease mechanisms. The silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1), a histone deacetylase, can limit cardiovascular injury, assist with stem cell development, oversee metabolic homeostasis through nicotinamide adenine dinucleotide (NAD+) pathways, foster trophic factor protection, and control cell senescence through the modulation of telomere function. Intimately tied to SIRT1 pathways are mammalian forkhead transcription factors (FoxOs) which can modulate cardiac disease to reduce oxidative stress, repair microcirculation disturbances, and reduce atherogenesis through pathways of autophagy, apoptosis, and ferroptosis. AMP activated protein kinase (AMPK) also is critical among these pathways for the oversight of cardiac cellular metabolism, insulin sensitivity, mitochondrial function, inflammation, and the susceptibility to viral infections such as severe acute respiratory syndrome coronavirus that can impact cardiovascular disease. Yet, the relationship among these pathways is both intricate and complex and requires detailed insight to successfully translate these pathways into clinical care for cardiovascular disorders.

Discovery of a new class of reversible TEA domain transcription factor inhibitors with a novel binding mode.

The TEA domain (TEAD) transcription factor forms a transcription co-activation complex with the key downstream effector of the Hippo pathway, YAP/TAZ. TEAD-YAP controls the expression of Hippo-responsive genes involved in cell proliferation, development, and tumorigenesis. Hyperactivation of TEAD-YAP activities is observed in many human cancers and is associated with cancer cell proliferation, survival, and immune evasion. Therefore, targeting the TEAD-YAP complex has emerged as an attractive therapeutic approach. We previously reported that the mammalian TEAD transcription factors (TEAD1-4) possess auto-palmitoylation activities and contain an evolutionarily conserved palmitate-binding pocket (PBP), which allows small-molecule modulation. Since then, several reversible and irreversible inhibitors have been reported by binding to PBP. Here, we report a new class of TEAD inhibitors with a novel binding mode. Representative analog TM2 shows potent inhibition of TEAD auto-palmitoylation both in vitro and in cells. Surprisingly, the co-crystal structure of the human TEAD2 YAP-binding domain (YBD) in complex with TM2 reveals that TM2 adopts an unexpected binding mode by occupying not only the hydrophobic PBP, but also a new side binding pocket formed by hydrophilic residues. RNA-seq analysis shows that TM2 potently and specifically suppresses TEAD-YAP transcriptional activities. Consistently, TM2 exhibits strong antiproliferation effects as a single agent or in combination with a MEK inhibitor in YAP-dependent cancer cells. These findings establish TM2 as a promising small-molecule inhibitor against TEAD-YAP activities and provide new insights for designing novel TEAD inhibitors with enhanced selectivity and potency.

Shared cis-regulatory modules control expression of the tandem paralogs midline and H15 in the follicular epithelium.

The posterior end of the follicular epithelium is patterned by midline (MID) and its paralog H15, the Drosophila homologs of the mammalian Tbx20 transcription factor. We have previously identified two cis-regulatory modules (CRMs) that recapitulate the endogenous pattern of mid in the follicular epithelium. Here, using CRISPR/Cas9 genome editing, we demonstrate redundant activity of these mid CRMs. Although the deletion of either CRM alone generated marginal change in mid expression, the deletion of both CRMs reduced expression by 60%. Unexpectedly, the deletion of the 5' proximal CRM of mid eliminated H15 expression. Interestingly, expression of these paralogs in other tissues remained unaffected in the CRM deletion backgrounds. These results suggest that the paralogs are regulated by a shared CRM that coordinates gene expression during posterior fate determination. The consistent overlapping expression of mid and H15 in various tissues may indicate that the paralogs could also be under shared regulation by other CRMs in these tissues.

Examining the role of EGR1 during viral infections.

Early growth response 1 (EGR1) is a multifunctional mammalian transcription factor capable of both enhancing and/or inhibiting gene expression. EGR1 can be activated by a wide array of stimuli such as exposure to growth factors, cytokines, apoptosis, and various cellular stress states including viral infections by both DNA and RNA viruses. Following induction, EGR1 functions as a convergence point for numerous specialized signaling cascades and couples short-term extracellular signals to influence transcriptional regulation of genes required to initiate the appropriate biological response. The role of EGR1 has been extensively studied in both physiological and pathological conditions of the adult nervous system where it is readily expressed in various regions of the brain and is critical for neuronal plasticity and the formation of memories. In addition to its involvement in neuropsychiatric disorders, EGR1 has also been widely examined in the field of cancer where it plays paradoxical roles as a tumor suppressor gene or oncogene. EGR1 is also associated with multiple viral infections such as Venezuelan equine encephalitis virus (VEEV), Kaposi's sarcoma-associated herpesvirus (KSHV), herpes simplex virus 1 (HSV-1), human polyomavirus JC virus (JCV), human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV). In this review, we examine EGR1 and its role(s) during viral infections. First, we provide an overview of EGR1 in terms of its structure, other family members, and a brief overview of its roles in non-viral disease states. We also review upstream regulators of EGR1 and downstream factors impacted by EGR1. Then, we extensively examine EGR1 and its roles, both direct and indirect, in regulating replication of DNA and RNA viruses.

Engineered endosymbionts that alter mammalian cell surface marker, cytokine and chemokine expression

Developing modular tools that direct mammalian cell function and activity through controlled delivery of essential regulators would improve methods of guiding tissue regeneration, enhancing cellular-based therapeutics and modulating immune responses. To address this challenge, Bacillus subtilis was developed as a chassis organism for engineered endosymbionts (EES) that escape phagosome destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to impact host cell response and function. Two synthetic operons encoding either the mammalian transcription factors Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the genome of B. subtilis expressing listeriolysin O (LLO) from Listeria monocytogenes and expressed from regulated promoters. Controlled expression of the mammalian proteins from B. subtilis LLO in the cytoplasm of J774A.1 macrophage/monocyte cells altered surface marker, cytokine and chemokine expression. Modulation of host cell fates displayed some expected patterns towards anti- or pro-inflammatory phenotypes by each of the distinct transcription factor pairs with further demonstration of complex regulation caused by a combination of the EES interaction and transcription factors. Expressing mammalian transcription factors from engineered intracellular B. subtilis as engineered endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes.

Transcription factor Sp1 regulates mitotic chromosome assembly and segregation

Aneuploidy is a pervasive feature of cancer cells that results from chromosome missegregation. Several transcription factors have been associated with aneuploidy; however, no studies to date have demonstrated that mammalian transcription factors directly regulate chromosome segregation during mitosis. Here, we demonstrate that the ubiquitously expressed transcription factor specificity protein 1 (Sp1), which we have previously linked to aneuploidy, has a mitosis-specific role regulating chromosome segregation. We find that Sp1 localizes to mitotic centromeres and auxin-induced rapid Sp1 degradation at mitotic onset results in chromosome segregation errors and aberrant mitotic progression. Furthermore, rapid Sp1 degradation results in anomalous mitotic chromosome assembly characterized by loss of condensin complex I localization to mitotic chromosomes and chromosome condensation defects. Consistent with these defects, Sp1 degradation results in reduced chromosome passenger complex activity and histone H3 serine 10 phosphorylation during mitosis, which is essential for condensin complex I recruitment and chromosome condensation. Together, these data provide the first evidence of a mammalian transcription factor acting specifically during mitosis to regulate chromosome segregation.

Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2.

ABSTRACTNFAT5 is the only known mammalian tonicity-responsive transcription factor with an essential role in cellular adaptation to hypertonic stress. It is also implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity, but the underlying mechanisms remain elusive. Here, we demonstrate that NFAT5 enters the nucleus via the nuclear pore complex. We found that NFAT5 utilizes a unique nuclear localization signal (NFAT5-NLS) for nuclear import. siRNA screening revealed that only karyopherin β1 (KPNB1), but not karyopherin α, is responsible for the nuclear import of NFAT5 via direct interaction with the NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is driven by exportin-T (XPOT), where the process requires RuvB-like AAA-type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified an unconventional tonicity-dependent nucleocytoplasmic trafficking pathway for NFAT5 that represents a critical step in orchestrating rapid cellular adaptation to change in extracellular tonicity. These findings offer an opportunity for the development of novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.