CRISPR-Cas9 potential for identifying novel therapeutic targets in muscle-invasive bladder cancer.

Gene editing technologies help identify the genetic perturbations driving tumour initiation, growth, metastasis and resistance to therapeutics. This wealth of information highlights tumour complexity and is driving cancer research towards precision medicine approaches based on an individual's tumour genetics. Bladder cancer is the 11th most common cancer in the UK, with high rates of relapse and low survival rates in patients with muscle-invasive bladder cancer (MIBC). MIBC is highly heterogeneous and encompasses multiple molecular subtypes, each with different responses to therapeutics. This evidence highlights the need to identify innovative therapeutic targets to address the challenges posed by this heterogeneity. CRISPR-Cas9 technologies have been used to advance our understanding of MIBC and determine novel drug targets through the identification of drug resistance mechanisms, targetable cell-cycle regulators, and novel tumour suppressor and oncogenes. However, the use of these technologies in the clinic remains a substantial challenge and will require careful consideration of dosage, safety and ethics. CRISPR-Cas9 offers considerable potential for revolutionizing bladder cancer therapies, but substantial research is required for validation before these technologies can be used in the clinical setting.

Tumor circadian clock strength influences metastatic potential and predicts patient prognosis in luminal A breast cancer.

Studies in shift workers and model organisms link circadian disruption to breast cancer. However, molecular circadian rhythms in noncancerous and cancerous human breast tissues and their clinical relevance are largely unknown. We reconstructed rhythms informatically, integrating locally collected, time-stamped biopsies with public datasets. For noncancerous breast tissue, inflammatory, epithelial-mesenchymal transition (EMT), and estrogen responsiveness pathways show circadian modulation. Among tumors, clock correlation analysis demonstrates subtype-specific changes in circadian organization. Luminal A organoids and informatic ordering of luminal A samples exhibit continued, albeit dampened and reprogrammed rhythms. However, CYCLOPS magnitude, a measure of global rhythm strength, varied widely among luminal A samples. Cycling of EMT pathway genes was markedly increased in high-magnitude luminal A tumors. Surprisingly, patients with high-magnitude tumors had reduced 5-y survival. Correspondingly, 3D luminal A cultures show reduced invasion following molecular clock disruption. This study links subtype-specific circadian disruption in breast cancer to EMT, metastatic potential, and prognosis.

Prolylcarboxypeptidase Alleviates Hypertensive Cardiac Remodeling by Regulating Myocardial Tissue Angiotensin II.

Background Prolonged activation of angiotensin II is the main mediator that contributes to the development of heart diseases, so converting angiotensin II into angiotensin 1-7 has emerged as a new strategy to attenuate detrimental effects of angiotensin II. Prolylcarboxypeptidase is a lysosomal pro-X carboxypeptidase that is able to cleave angiotensin II at a preferential acidic pH optimum. However, insufficient attention has been given to the cardioprotective functions of prolylcarboxylpeptidase. Methods and Results We established a CRISPR/CRISPR-associated protein 9-mediated global prolylcarboxylpeptidase-knockout and adeno-associated virus serotype 9-mediated cardiac prolylcarboxylpeptidase overexpression mouse models, which were challenged with the angiotensin II infusion (2 mg/kg per day) for 4 weeks, aiming to investigate the cardioprotective effect of prolylcarboxylpeptidase against hypertensive cardiac hypertrophy. Prolylcarboxylpeptidase expression was upregulated after 2 weeks of angiotensin II infusion and then became downregulated afterward in wild-type mouse myocardium, suggesting its compensatory function against angiotensin II stress. Moreover, angiotensin II-treated prolylcarboxylpeptidase-knockout mice showed aggravated cardiac remodeling and dampened cardiac contractility independent of hypertension. We also found that prolylcarboxylpeptidase localizes in cardiomyocyte lysosomes, and loss of prolylcarboxylpeptidase led to excessive angiotensin II levels in myocardial tissue. Further screening demonstrated that hypertrophic prolylcarboxylpeptidase-knockout hearts showed upregulated extracellular signal-regulated kinases 1/2 and downregulated protein kinase B activities. Importantly, adeno-associated virus serotype 9-mediated restoration of prolylcarboxylpeptidase expression in prolylcarboxylpeptidase-knockout hearts alleviated angiotensin II-induced hypertrophy, fibrosis, and cell death. Interestingly, the combination of adeno-associated virus serotype 9-mediated prolylcarboxylpeptidase overexpression and an antihypertensive drug, losartan, likely conferred more effective protection than a single treatment protocol to mitigate angiotensin II-induced cardiac dysfunction. Conclusions Our data demonstrate that prolylcarboxylpeptidase protects the heart from angiotensin II-induced hypertrophic remodeling by controlling myocardial angiotensin II levels.

Subtype-specific circadian clock dysregulation modulates breast cancer biology, invasiveness, and prognosis.

Studies in shift workers and model organisms link circadian disruption to breast cancer. However, molecular rhythms in non-cancerous and cancerous human breast tissues are largely unknown. We reconstructed rhythms informatically, integrating locally collected, time-stamped biopsies with public datasets. For non-cancerous tissue, the inferred order of core-circadian genes matches established physiology. Inflammatory, epithelial-mesenchymal transition (EMT), and estrogen responsiveness pathways show circadian modulation. Among tumors, clock correlation analysis demonstrates subtype-specific changes in circadian organization. Luminal A organoids and informatic ordering of Luminal A samples exhibit continued, albeit disrupted rhythms. However, CYCLOPS magnitude, a measure of global rhythm strength, varied widely among Luminal A samples. Cycling of EMT pathway genes was markedly increased in high-magnitude Luminal A tumors. Patients with high-magnitude tumors had reduced 5-year survival. Correspondingly, 3D Luminal A cultures show reduced invasion following molecular clock disruption. This study links subtype-specific circadian disruption in breast cancer to EMT, metastatic potential, and prognosis.

Disruptions in endocytic traffic contribute to the activation of the NLRP3 inflammasome.

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and is regulated by subcellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endolysosomal markers and for the inositol lipid PI4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod, driving enhanced inflammasome activation and cytokine secretion. Together, these data suggest that NLRP3 can sense disruptions in the trafficking of endosomal cargoes, which may explain in part the spatial activation of the NLRP3 inflammasome. These data highlight mechanisms that could be exploited in the therapeutic targeting of NLRP3.

RAC1B function is essential for breast cancer stem cell maintenance and chemoresistance of breast tumor cells.

Breast cancer stem cells (BCSC) are presumed to be responsible for treatment resistance, tumor recurrence and metastasis of breast tumors. However, development of BCSC-targeting therapies has been held back by their heterogeneity and the lack of BCSC-selective molecular targets. Here, we demonstrate that RAC1B, the only known alternatively spliced variant of the small GTPase RAC1, is expressed in a subset of BCSCs in vivo and its function is required for the maintenance of BCSCs and their chemoresistance to doxorubicin. In human breast cancer cell line MCF7, RAC1B is required for BCSC plasticity and chemoresistance to doxorubicin in vitro and for tumor-initiating abilities in vivo. Unlike Rac1, Rac1b function is dispensable for normal mammary gland development and mammary epithelial stem cell (MaSC) activity. In contrast, loss of Rac1b function in a mouse model of breast cancer hampers the BCSC activity and increases their chemosensitivity to doxorubicin treatment. Collectively, our data suggest that RAC1B is a clinically relevant molecular target for the development of BCSC-targeting therapies that may improve the effectiveness of doxorubicin-mediated chemotherapy.

On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members.

CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.

HES1 protein oscillations are necessary for neural stem cells to exit from quiescence.

Quiescence is a dynamic process of reversible cell cycle arrest. High-level persistent expression of the HES1 transcriptional repressor, which oscillates with an ultradian periodicity in proliferative neural stem cells (NSCs), is thought to mediate quiescence. However, it is not known whether this is due to a change in levels or dynamics. Here, we induce quiescence in embryonic NSCs with BMP4, which does not increase HES1 level, and we find that HES1 continues to oscillate. To assess the role of HES1 dynamics, we express persistent HES1 under a moderate strength promoter, which overrides the endogenous oscillations while maintaining the total HES1 level within physiological range. We find that persistent HES1 does not affect proliferation or entry into quiescence; however, exit from quiescence is impeded. Thus, oscillatory expression of HES1 is specifically required for NSCs to exit quiescence, a finding of potential importance for controlling reactivation of stem cells in tissue regeneration and cancer.

Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network.

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.

A Transgenic Line That Reports CSF1R Protein Expression Provides a Definitive Marker for the Mouse Mononuclear Phagocyte System.

The proliferation, differentiation, and survival of cells of the mononuclear phagocyte system (MPS; progenitors, monocytes, macrophages, and classical dendritic cells) are controlled by signals from the M-CSF receptor (CSF1R). Cells of the MPS lineage have been identified using numerous surface markers and transgenic reporters, but none is both universal and lineage restricted. In this article, we report the development and characterization of a CSF1R reporter mouse. A FusionRed (FRed) cassette was inserted in-frame with the C terminus of CSF1R, separated by a T2A-cleavable linker. The insertion had no effect of CSF1R expression or function. CSF1R-FRed was expressed in monocytes and macrophages and absent from granulocytes and lymphocytes. In bone marrow, CSF1R-FRed was absent in lineage-negative hematopoietic stem cells, arguing against a direct role for CSF1R in myeloid lineage commitment. It was highly expressed in marrow monocytes and common myeloid progenitors but significantly lower in granulocyte-macrophage progenitors. In sections of bone marrow, CSF1R-FRed was also detected in osteoclasts, CD169+ resident macrophages, and, consistent with previous mRNA analysis, in megakaryocytes. In lymphoid tissues, CSF1R-FRed highlighted diverse MPS populations, including classical dendritic cells. Whole mount imaging of nonlymphoid tissues in mice with combined CSF1R-FRed/Csf1r-EGFP confirmed the restriction of CSF1R expression to MPS cells. The two markers highlight the remarkable abundance and regular distribution of tissue MPS cells, including novel macrophage populations within tendon and skeletal muscle and underlying the mesothelial/serosal/capsular surfaces of every major organ. The CSF1R-FRed mouse provides a novel reporter with exquisite specificity for cells of the MPS.

LRRC8A is essential for hypotonicity-, but not for DAMP-induced NLRP3 inflammasome activation.

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1ß and IL-18. The NLRP3 inflammasome is frequently associated with the damaging inflammation of non-communicable disease states and is considered an attractive therapeutic target. However, there is much regarding the mechanism of NLRP3 activation that remains unknown. Chloride efflux is suggested as an important step in NLRP3 activation, but which chloride channels are involved is still unknown. We used chemical, biochemical, and genetic approaches to establish the importance of chloride channels in the regulation of NLRP3 in murine macrophages. Specifically, we identify LRRC8A, an essential component of volume-regulated anion channels (VRAC), as a vital regulator of hypotonicity-induced, but not DAMP-induced, NLRP3 inflammasome activation. Although LRRC8A was dispensable for canonical DAMP-dependent NLRP3 activation, this was still sensitive to chloride channel inhibitors, suggesting there are additional and specific chloride sensing and regulating mechanisms controlling NLRP3.

Inflammation is a critical part of a healthy immune system, which protects us against harmful pathogens (such as bacteria or viruses) and works to restore damaged tissues. In the immune cells of our body, the inflammatory process can be activated through a group of inflammatory proteins that together are known as the NLRP3 inflammasome complex. While inflammation is a powerful mechanism that protects the human body, persistent or uncontrolled inflammation can cause serious, long-term damage. The inappropriate activation of the NLRP3 inflammasome has been implicated in several diseases, including Alzheimer's disease, heart disease, and diabetes. The NLRP3 inflammasome can be activated by different stimuli, including changes in cell volume and exposure to either molecules produced by damaged cells or toxins from bacteria. However, the precise mechanism through which the NLRP3 becomes activated in response to these stimuli was not clear. The exit of chloride ions from immune cells is known to activate the NLRP3 inflammasome. Chloride ions exit the cell through proteins called anion channels, including volume-regulated anion channels (VRACs), which respond to changes in cell volume. Green et al. have found that, in immune cells from mice grown in the lab called macrophages, VRACs are the only chloride channels involved in activating the NLRP3 inflammasome when the cell's volume changes. However, when the macrophages are exposed to molecules produced by damaged cells or toxins from bacteria, Green et al. discovered that other previously unidentified chloride channels are involved in activating the NLRP3 inflammasome. These results suggest that it might be possible to develop drugs to prevent the activation of the NLRP3 inflammasome that selectively target specific sets of chloride channels depending on which stimuli are causing the inflammation. Such a selective approach would minimise the side effects associated with drugs that generically suppress all NLRP3 activity by directly binding to NLRP3 itself. Ultimately, this may help guide the development of new, targeted anti-inflammatory drugs that can help treat the symptoms of a variety of diseases in humans.

Analysis of chromatin organization and gene expression in T cells identifies functional genes for rheumatoid arthritis.

Genome-wide association studies have identified genetic variation contributing to complex disease risk. However, assigning causal genes and mechanisms has been more challenging because disease-associated variants are often found in distal regulatory regions with cell-type specific behaviours. Here, we collect ATAC-seq, Hi-C, Capture Hi-C and nuclear RNA-seq data in stimulated CD4+ T cells over 24 h, to identify functional enhancers regulating gene expression. We characterise changes in DNA interaction and activity dynamics that correlate with changes in gene expression, and find that the strongest correlations are observed within 200 kb of promoters. Using rheumatoid arthritis as an example of T cell mediated disease, we demonstrate interactions of expression quantitative trait loci with target genes, and confirm assigned genes or show complex interactions for 20% of disease associated loci, including FOXO1, which we confirm using CRISPR/Cas9.

A Human Stem Cell Model of Fabry Disease Implicates LIMP-2 Accumulation in Cardiomyocyte Pathology.

Here, we have used patient-derived induced pluripotent stem cell (iPSC) and gene-editing technology to study the cardiac-related molecular and functional consequences of mutations in GLA causing the lysosomal storage disorder Fabry disease (FD), for which heart dysfunction is a major cause of mortality. Our in vitro model recapitulated clinical data with FD cardiomyocytes accumulating GL-3 and displaying an increased excitability, with altered electrophysiology and calcium handling. Quantitative proteomics enabled the identification of >5,500 proteins in the cardiomyocyte proteome and secretome, and revealed accumulation of the lysosomal protein LIMP-2 and secretion of cathepsin F and HSPA2/HSP70-2 in FD. Genetic correction reversed these changes. Overexpression of LIMP-2 directly induced the secretion of cathepsin F and HSPA2/HSP70-2, implying causative relationship, and led to massive vacuole accumulation. In summary, our study has revealed potential new cardiac biomarkers for FD, and provides valuable mechanistic insight into the earliest pathological events in FD cardiomyocytes.

Human TNF-Luc reporter mouse: A new model to quantify inflammatory responses.

Tumour necrosis factor (TNF) is a key cytokine during inflammatory responses and its dysregulation is detrimental in many inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. Here, we used a bacterial artificial chromosome (BAC) construct that expresses luciferase under the control of the human TNF locus to generate a novel transgenic mouse, the hTNF.LucBAC strain. In vitro stimulation of hTNF.LucBAC cells of different origin revealed a cell specific response to stimuli demonstrating the integrated construct's ability as a proxy for inflammatory gene response. Lipopolysaccharide was the most potent luciferase inducer in macrophages, while TNF was a strong activator in intestinal organoids. Lipopolysaccharide-induced luciferase activity in macrophages was downregulated by inhibitors of NF-κB pathway, as well as by Interleukin-10, a known anti-inflammatory cytokine. Moreover, the transgene-dependent luciferase activity showed a positive correlation to the endogenous murine soluble TNF secreted to the culture medium. In conclusion, the hTNF.LucBAC strain is a valuable tool for studying and screening molecules that target TNF synthesis and will allow further functional studies of the regulatory elements of the TNF locus.

Quantitative analysis of competitive cytokine signaling predicts tissue thresholds for the propagation of macrophage activation.

Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor κB (NF-κB) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-κB signaling in the cells and their size. We compared the rates of TNF-α production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF-α signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF-α response and cellular NF-κB signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF-α propagation was constrained by competitive uptake of TNF-α from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues while avoiding out-of-context responses at longer distances.

CRISPR/Cas9 mediated mutation of mouse IL-1α nuclear localisation sequence abolishes expression.

Inflammation is a host defense process against infection. Inflammatory mediators include cytokines of the interleukin-1 family, such as IL-1α and IL-1ß. Unlike IL-1ß, IL-1α carries an N-terminal nuclear localisation sequence (NLS) and is trafficked to the nucleus. The importance of IL-1α nuclear localisation is poorly understood. Here, we used CRISPR/Cas9 to make inactivating mutations to the NLS on the Il1a gene. A colony of NLS mutant mice was successfully generated with precise knock-in mutations to incapacitate NLS function. NLS mutant mice had no gross changes in immunophenotype or inflammatory response but, surprisingly, failed to express IL-1α. We deduced that, in making specific mutations in the Il1a gene, we also mutated a long-noncoding (lnc)RNA in the complementary strand which has cis-regulatory transcriptional control of the Il1a gene itself. The mutations generated in the Il1a gene also result in mutation of the lncRNA sequence and a predicted alteration of its secondary structure, potentially explaining a subsequent failure to function as a transcriptional activator of Il1a expression. Thus, lncRNA secondary structure may regulate IL-1α expression. Our results serve as a cautionary note that CRISPR -mediated genome editing without full knowledge of genomic context can result in unexpected, yet potentially informative observations.

The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration.

OBJECTIVES: The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. METHODS: Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. RESULTS: Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1ß but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. CONCLUSIONS: We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1ß instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation.

Dynamic NF-κB and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation.

Dynamic cellular systems reprogram gene expression to ensure appropriate cellular fate responses to specific extracellular cues. Here we demonstrate that the dynamics of Nuclear Factor kappa B (NF-κB) signalling and the cell cycle are prioritised differently depending on the timing of an inflammatory signal. Using iterative experimental and computational analyses, we show physical and functional interactions between NF-κB and the E2 Factor 1 (E2F-1) and E2 Factor 4 (E2F-4) cell cycle regulators. These interactions modulate the NF-κB response. In S-phase, the NF-κB response was delayed or repressed, while cell cycle progression was unimpeded. By contrast, activation of NF-κB at the G1/S boundary resulted in a longer cell cycle and more synchronous initial NF-κB responses between cells. These data identify new mechanisms by which the cellular response to stress is differentially controlled at different stages of the cell cycle.

Role of Estrogen Response Element in the Human Prolactin Gene: Transcriptional Response and Timing.

The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering, we have manipulated a 160-kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located -1189 bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing Luc under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to 17ß-estradiol (E2) treatment compared with cells expressing Luc from the wild-type (WT) ERE hPRL-Luc promoter (ERE-WT). The -1189 ERE controls not only the response to E2 treatment but also the acute transcriptional response to TNFα, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response after TNFα treatment, the acute phase of which was blocked after treatment with the estrogen receptor antagonist 4-hydroxy-tamoxifen. Unexpectedly, we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, real-time bioluminescence imaging showed that transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the -1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFα and, crucially, that cycles of hPRL promoter activity are independent of estrogen receptor binding.