Exogenous Reporter Gene Research Articles

Dissection of 3D chromosome organization in Streptomyces coelicolor A3(2) leads to biosynthetic gene cluster overexpression

The soil-dwelling filamentous bacteria, Streptomyces, is widely known for its ability to produce numerous bioactive natural products. Despite many efforts toward their overproduction and reconstitution, our limited understanding of the relationship between the host's chromosome three dimension (3D) structure and the yield of the natural products escaped notice. Here, we report the 3D chromosome organization and its dynamics of the model strain, Streptomyces coelicolor, during the different growth phases. The chromosome undergoes a dramatic global structural change from primary to secondary metabolism, while some biosynthetic gene clusters (BGCs) form special local structures when highly expressed. Strikingly, transcription levels of endogenous genes are found to be highly correlated to the local chromosomal interaction frequency as defined by the value of the frequently interacting regions (FIREs). Following the criterion, an exogenous single reporter gene and even complex BGC can achieve a higher expression after being integrated into the chosen loci, which may represent a unique strategy to activate or enhance the production of natural products based on the local chromosomal 3D organization.

A New Method for Programmable RNA Editing Using CRISPR Effector Cas13X.1.

Type VI CRISPR-Cas13 is the only CRISPR system that can bind and cleave RNA without DNase activity. We used the newly discovered, smaller Cas13X.1 protein to construct an editing system in mammalian cells, aiming to break the delivery restrictions of CRISPR-Cas13 system in vivo and promote the application of Cas13X system in clinical therapy. We employed exogenous fluorescence reporter gene mCherry and endogenous gene transketolase (TKT) closely related to cancer cell metabolism as target genes to evaluate the Cas13X.1 system. The recombinant plasmids targeting exogenous gene mCherry and endogenous gene TKT were constructed based on Cas13X.1 backbone plasmid. The editing efficiency, protein expression level, downstream gene transcript level and safety of Cas13X.1 system were evaluated. Both TKT transcripts of endogenous genes and mCherry transcripts of exogenous genes were significantly degraded by Cas13X.1 system with a knockdown efficiency up to 50%. At the same time, Cas13X.1 down-regulated the expression of the corresponding protein level in the editing of transcripts. In addition, the transcripts of key metabolic enzymes related to TKT were also down-regulated synchronously, suggesting that the degradation of TKT transcripts by Cas13X.1 system affected the main metabolic pathways related to TKT. The morphology, RNA integrity and apoptosis of cells loaded with Cas13X.1 system were not affected. The Cas13X.1 system we constructed had strong RNA knockdown ability in mammalian cells with low cellular toxicity. Compared with other CRISPR-Cas13 systems, Cas13X.1 system with smaller molecular weight has more advantages in vivo delivery. The Cas13X.1 system targeting TKT transcripts also provides an alternative method for the study of anti-cancer therapy.

Antigen-Dependent Inducible T-Cell Reporter System for PET Imaging of Breast Cancer and Glioblastoma.

For the past several decades, chimeric antigen receptor T-cell therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells invivo. Methods: Using synthetic biology, we engineered T cells with a chimeric receptor synthetic intramembrane proteolysis receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette on recognition of specific tumor markers. We then applied a SNIPR-based PET reporter system to 2 cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors, respectively. Results: Antigen-specific reporter induction of the SNIPR PET T cells was confirmed invitro using green fluorescent protein fluorescence, luciferase luminescence, and the HSV-TK PET reporter with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG). T cells associated with their target antigens were successfully imaged using PET in dual-xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with more than 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. Conclusion: The main innovation found in this work was PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

Comparison of the frequencies of ENU-induced point mutations in male germ cells and inherited germline mutations in their offspring

BackgroundGene mutations induced in germ cells may be transmitted to the next generation and cause adverse effects such as genetic diseases. Certain mutations may result in infertility or death in early development. Thus, the mutations may not be inheritable. However, the extent to which point mutations in male germ cells are transmitted to the next generation or eliminated during transmission is largely unknown. This study compared mutation frequencies (MFs) in sperm of N-ethyl-N-nitrosourea (ENU)-treated gpt delta mice and de novo MFs in the whole exome/genome of their offspring.ResultsMale gpt delta mice were treated with 10, 30, and 85 mg/kg of ENU (i.p., weekly × 2) and mated with untreated females to generate offspring. We previously reported a dose-dependent increase in de novo MFs in the offspring estimated by whole exome sequencing (WES) (Mutat. Res., 810, 30–39, 2016). In this study, gpt MFs in the sperm of ENU-treated mice were estimated, and the MFs per reporter gene were converted to MFs per base pair. The inherited de novo MFs in the offspring (9, 26 and 133 × 10− 8/bp for 10, 30, and 85 mg/kg ENU-treated groups, respectively) were comparable to those of the converted gpt MFs in the sperm of ENU-treated fathers (6, 16, and 69 × 10− 8/bp). It indicated that the gpt MFs in the ENU-treated father’s sperm were comparable to the inherited de novo MFs in the offspring as estimated by WES. In addition, de novo MFs in the offspring of 10 mg/kg ENU-treated and control fathers were estimated by whole genome sequencing (WGS), because WES was not sufficiently sensitive to detect low background MF. The de novo MF in the offspring of the ENU-treated fathers was 6 × 10− 8/bp and significantly higher than that of the control (2 × 10− 8/bp). There were no significant differences in de novo MFs between gene-coding and non-coding regions. WGS analysis was able to detect ENU-induced characteristic de novo base substitutions at a low dose group.ConclusionsDespite a difference between exome/genome and exogenous reporter genes, the results indicated that ENU-induced point mutations in male germ cells could be transmitted to the next generation without severe selection.

The Influence of Chromosomal Environment on X-Linked Gene Expression in Drosophila melanogaster.

Sex chromosomes often differ from autosomes with respect to their gene expression and regulation. In Drosophila melanogaster, X-linked genes are dosage compensated by having their expression upregulated in the male soma, a process mediated by the X-chromosome-specific binding of the dosage compensation complex (DCC). Previous studies of X-linked gene expression found a negative correlation between a gene’s male-to-female expression ratio and its distance to the nearest DCC binding site in somatic tissues, including head and brain, which suggests that dosage compensation influences sex-biased gene expression. A limitation of the previous studies, however, was that they focused on endogenous X-linked genes and, thus, could not disentangle the effects of chromosomal position from those of gene-specific regulation. To overcome this limitation, we examined the expression of an exogenous reporter gene inserted at many locations spanning the X chromosome. We observed a negative correlation between the male-to-female expression ratio of the reporter gene and its distance to the nearest DCC binding site in somatic tissues, but not in gonads. A reporter gene’s location relative to a DCC binding site had greater influence on its expression than the local regulatory elements of neighboring endogenous genes, suggesting that intra-chromosomal variation in the strength of dosage compensation is a major determinant of sex-biased gene expression. Average levels of sex-biased expression did not differ between head and brain, but there was greater positional effect variation in the brain, which may explain the observed excess of endogenous sex-biased genes located on the X chromosome in this tissue.

An androgen-independent mechanism underlying the androgenic effects of 3-methylcholanthrene, a potent aryl hydrocarbon receptor agonist

Aryl hydrocarbon receptor (AhR) and androgen receptor (AR) are ligand-activated transcription factors with profound cross-talk between their signal transduction pathways. Previous studies have shown that AhR agonists activate the transcription of AR-regulated genes in an androgen-independent manner; however, the underlying mechanism remains unclear. To decipher this mechanism, we evaluated the effects of 3-methylcholanthrene (3MC), a potent AhR agonist, on the transcription of AR-regulated genes in three AR-expressing cell lines. 3MC induced the expression of not only three representative AR-regulated chromosomal genes but also the exogenous AR-responsive luciferase reporter gene. No significant difference in the 3MC-induced luciferase activity was detected in the presence of SKF-525A, a non-specific inhibitor of CYP enzymes. The androgenic effects of 3MC were diminished by AhR and AR knockdown. Following 3MC treatment, the amount of nuclear AhR and AR increased synchronously. Co-immunoprecipitation revealed that AhR and AR formed a complex in the nucleus of cells treated with 3MC. AR was recruited to the proximal promoter and distal enhancer regions of the PSA gene upon the addition of 3MC. We propose that AhR activated by 3MC forms a complex with unliganded AR which translocates from the cytoplasm to the nucleus. Nuclear AR now binds the transcriptional regulatory region of AR-regulated genes and activates the transcription.

Synthesis and Evaluation of Caged siRNAs with Single cRGD Modification for Photoregulating RNA Interference.

We designed and synthesized caged siRNAs with photolabile linker and single cRGD peptide modifications for the photoregulation of gene expression. Photolabile linker and cRGD were inserted at 5' terminus of siRNAs to obtain cRGD-modified caged siRNAs. All these caged siRNAs could be activated through light activation to release the native siRNAs and further achieve the photoregulation of gene silencing of two exogenous reporter genes (firefly luciferase and green fluorescent protein, GFP) and one endogenous gene (the mitosis motor protein, Eg5). The intracellular distribution and cellular uptake pathways of these caged siRNAs were also investigated. Tumor-bearing mice were further used to demonstrate the photoregulation of gene silencing with cRGD-modified caged siRNAs in vivo. Overall, the data support the use of this new generation of caged siRNAs in cancer therapy.

Distal and proximal hypoxia response elements cooperate to regulate organ-specific erythropoietin gene expression.

While it has been well-established that distal hypoxia response elements (HRE) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HRE has not yet been described. Hepatic Epo expression is regulated by an HRE located downstream of the EPOgene, but this 3' HRE is not essential for renal EPO gene expression. We previously identified a 5' HRE and could show that both HRE direct exogenous reporter gene expression. Here, we show that, whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HRE contribute to endogenous Epo induction. Moreover, two novel putative HRE were identified in the EPO promoter. In hepatoma cells, HIF interacted mainly with the distal 3' HRE, but in neuronal cells, HIF most strongly bound the promoter, bound to a lesser extent the 3' HRE, and did not bind the 5' HRE. Interestingly, mutation of either of the two distal HRE abrogated HIF binding to the 3' and promoter HRE. These results suggest that a canonical functional HRE can recruit multiple transcription factors (not necessarily HIF) to mediate HIF binding to different distant HRE in an organ-specific manner.

The Development and Use of Reporter Influenza B Viruses.

Influenza B viruses (IBVs) are major contributors to total human influenza disease, responsible for ~1/3 of all infections. These viruses, however, are relatively less studied than the related influenza A viruses (IAVs). While it has historically been assumed that the viral biology and mechanisms of pathogenesis for all influenza viruses were highly similar, studies have shown that IBVs possess unique characteristics. Relative to IAV, IBV encodes distinct viral proteins, displays a different mutational rate, has unique patterns of tropism, and elicits different immune responses. More work is therefore required to define the mechanisms of IBV pathogenesis. One valuable approach to characterize mechanisms of microbial disease is the use of genetically modified pathogens that harbor exogenous reporter genes. Over the last few years, IBV reporter viruses have been developed and used to provide new insights into the host response to infection, viral spread, and the testing of antiviral therapeutics. In this review, we will highlight the history and study of IBVs with particular emphasis on the use of genetically modified viruses and discuss some remaining gaps in knowledge that can be addressed using reporter expressing IBVs.

Gene activation by a CRISPR-assisted trans enhancer.

The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted trans enhancer, for activating gene expression at high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, CMV enhancer DNA was recruited to target genes in trans by two systems: dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GAL4/sgRNA-UAS-CMV. The former recruited trans enhancer by annealing between two short complementary oligonucleotides at the ends of the sgRNA and trans enhancer. The latter recruited trans enhancer by binding between GAL4 fused to dCas9 and UAS sequence of trans enhancer. The trans enhancer activated gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter genes and variant endogenous genes in various cells, with much higher activation efficiency than that of current dCas9 activators.

A Living, Single Cell View of Myc's Effects on Transcription

How does c-MYC act on transcription events in single living cells to cause a global amplification of gene expression? To answer this question, we employ two recently-developed imaging techniques: 1) single-molecule FISH to quantify RNA in fixed cells, and 2) live cell imaging with the MS2-PP7 stem loop system to observe real-time RNA production at a given gene locus. Our results from both the exogenous reporter and endogenous gene show that MYC increases the duration of transcription events— this results in greater gene expression, but also an increase in its heterogeneity. These findings provide living, single cell evidence of MYC as a global amplifier of gene expression, and suggests the mechanism is by stabilizing the active period of a gene. We speculate that the added consequence of MYC's propensity to increase gene expression heterogeneity could drive cells to transiently populate pathological states and phenotypes that may ultimately make them susceptible to cancer. In addition, we seek to assess the direct effects of MYC on gene transcription. We engineered a photo-inducible version of MYC (Pi-MYC) to achieve spatial and temporal control of MYC within living cells. We demonstrate that Pi-MYC has oncogenic capability similar to wildtype MYC by coordinating with mutant RAS to transform mouse fibroblasts and inducing them to exhibit anchorage-independent growth. This tool shall allow us to follow the direct activity of genes in response to MYC perturbation.

Three-Component Repurposed Technology for Enhanced Expression: Highly Accumulable Transcriptional Activators via Branched Tag Arrays.

In the past few years, several types of artificial transcriptional activator, based on CRISPR-Cas9, have been developed and refined. Of these, in synergistic activation mediator and SunTag systems, the effector proteins, expressed in trans, can be recruited to the target sites via the MS2 RNA-binding system and GCN4-scFv antibody system, respectively. Here, we report a strong transcriptional activation system achieved by fusing GCN4 repeat to MS2 coat protein to accumulate numbers of activators, fused to scFv antibodies. By targeting the CDH1 gene, we show that our novel system, named “TREE,” results in a greater effect of activating exogenous reporter and endogenous gene. Moreover, by targeting another gene, RANKL, we consistently show the superiority of the TREE system with fewer single-guide RNAs compared to conventional systems. Our TREE system is a promising tool for transcriptional activation and can potentially contribute to other dCas9-mediated technologies such as epigenome editing and chromosome visualization.

Caged siRNAs with Single cRGD Modification for Photoregulation of Exogenous and Endogenous Gene Expression in Cells and Mice.

RNA interference (RNAi) mediated gene silencing holds significant promise in gene therapy. It is very important to manually regulate the activity of small interference RNAs (siRNAs) in the controllable mode. Here, we designed and synthesized a series of caged siRNAs through bioconjugation of cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD) peptide to the 5' end of siRNA through a photolabile linker. These cRGD modified caged siRNAs allowed for precise light-regulation of gene expression of two exogenous reporter genes (firefly luciferase and green fluorescent protein, GFP) and an endogenous gene (the mitosis motor protein, Eg5) in the integrin αvβ3 positive cells. This kind of bioconjugate further enabled photochemical activation of siRNA activity, and the target gene silencing was successfully achieved in tumor-bearing mice by intratumoral injection. This study also suggested that photomodulation of target gene expression using single cRGD caged siRNA at the 5' end of antisense strand RNA inhibited siRNA activity probably due to three factors: (1) trapping of cRGD modified siRNA in endosome and lysosome, (2) the steric hindrance of cRGD, (3) the binding of cRGD to its corresponding receptor.

Genome-wide mutation detection by interclonal genetic variation

Genetic toxicology assays estimate mutation frequencies by phenotypically screening for the activation or inactivation of endogenous or exogenous reporter genes. These reporters can only detect mutations in narrow areas of the genome and their use is often restricted to certain in vitro and in vivo models. Here, we show that Interclonal Genetic Variation (ICGV) can directly identify mutations genome-wide by comparing sequencing data of single-cell clones derived from the same source or organism. Upon ethyl methanesulfonate (EMS) exposure, ICGV detected greater levels of mutation in a dose- and time-dependent manner in E. coli. In addition, ICGV was also able to identify a ∼20-fold increase in somatic mutations in T-cell clones derived from an N-ethyl-N-nitrosourea (ENU)-treated rat vs. a vehicle-treated rat. These results demonstrate that the genetic differences of single-cell clones can be used for genome-wide mutation detection.

Re-direction of carbon flux to key precursor malonyl-CoA via artificial small RNAs in photosynthetic Synechocystis sp. PCC 6803

BackgroundPhotosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO2. Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts.ResultsAiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium Synechocystis sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in Escherichia coli. Both regulatory tools functioned well in regulating exogenous reporter gene lacZ and endogenous glgC gene in Synechocystis sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD730 nm) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch.ConclusionsIn this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for Synechocystis sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.

Single Molecule Analysis of Transcription in Live Cells Reveals the Gene Regulatory Function of MYC In Vivo

We seek to determine how the MYC oncogene modulates gene transcription kinetics with single-molecule imaging techniques.Thirty-five years after the discovery of MYC and despite intensive research effort, there is still little understanding of how this transcription factor acts or why it is an oncogene. Several models have recently emerged that cast doubt on the prevailing view of MYC as a gene-specific transcription factor and instead envision its oncogenic mechanism as a global amplifier or invader of enhancer regions. We seek to evaluate these hypotheses of MYC function and shed light on the in-vivo behavior of this transcription factor by determining how MYC modulates the kinetics of transcription events in single cells using single-molecule RNA fluorescence in-situ hybridization (RNA FISH) and live cell imaging of transcription.Our initial results testing the effect of MYC overexpression on an exogenous reporter gene in U2-OS cells shows that MYC increases the saturation level of the reporter dose response, indicating that MYC is able to act as a general amplifier without the need of enhancer regions. Unexpectedly however, the positive correlation between MYC and reporter RNA levels in the population is not reflected at the single cell level. This data suggests that static measurements of RNA abundance in single cells - such FISH or RNA-Seq - needs to be interpreted within a framework that explicitly takes into account the dynamic nature of transcription events. Further, as a way to directly assess the effect of the MYC protein on the transcription behavior of reporter genes, we engineered a photo-activatable version of MYC (PA-MYC) to achieve spatial and temporal control of the transcription factor within living cells. This shall allow us to follow the direct response of the transcription in response to MYC perturbation.

CRISPR/Cas9-Mediated Targeted Knockin of Exogenous Reporter Genes in Zebrafish.

Genome editing technologies such as ZFN, TALEN, and CRISPR/Cas9 efficiently induce DNA double-stranded breaks (DSBs) at a targeted genomic locus, often resulting in a frameshift-mediated target gene disruption. It remains difficult to perform targeted integration of exogenous genes by genome editing technologies. DSBs can be restored through DNA repair mechanisms, such as non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). It is well known that HR facilitates homology-dependent integration of donor DNA template into a targeted locus. Recently, both NHEJ-mediated and MMEJ-mediated targeted integrations of exogenous genes have been developed in zebrafish. This chapter summarizes the application of CRISPR/Cas9-mediated knock-in technology in zebrafish.

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.

128. Targeting a High-Expression FVIII Transgene to Exogenous Locations in the Genome without Disrupting Endogenous Gene Expression

The technology that is now available for targeted genome editing consists of artificial nucleases that create double stranded breaks at specific genomic locations that can then be corrected by intrinsic cellular repair processes. Genome editing can be achieved by exploiting the repair mechanism of homologous recombination by providing a donor vector containing the gene(s) of interest flanked by sequences homologous to the site of the double strand break. To date, gene addition in this manner is targeted to defined inert safe harbor loci. We developed an innovative targeting strategy in which homologous recombination could be utilized to target a transcriptionally active genomic locus without disrupting expression of the endogenous gene. In this manner, essentially any location in the genome becomes a potential safe harbor for homologous recombination-mediated genome editing. One advantage to this strategy is that it allows for transgene expression to be under the precise control of endogenous regulatory elements, rather than using heterologous promoters. Therefore, the optimal locus with the most advantageous regulatory elements to drive transgene expression can be selected for gene addition. Pilot experiments confirmed the expression of exogenous reporter genes (such as the green fluorescent protein or the more therapeutically relevant biologically inert surface selectable marker delta NGFR) from either an exogenous ubiquitous promoter or from the exogenous promoter due to the addition of a T2A element incorporated downstream of the endogenous gene. Notably, expression of the endogenous gene was confirmed to be unaltered. These experiments were performed at three separate genomic locations. To demonstrate the feasibility of this strategy therapeutically however, a high-expression FVIII chimeric transgene (HPFVIII) was incorporated into two distinctly active genomic loci: the IL2R gamma chain locus and the adenosine deaminase locus. The donor vectors utilized contained the cDNA for the endogenous gene locus (either the IL2R gamma chain gene or the adenosine deaminase gene) followed by a ubiquitous promoter intended to drive the expression of HPFVIII, all of which was flanked by one kilobase arms of homology. Both the myeloid erythroid-leukemic K562 cell line and the acute T cell leukemic Jurkat cell line were modified upon nucleofection and confirmed to be targeted by sequencing. Single cell clones were produced and assessed for HPFVIII expression by a one-stage APTT based coagulation assay. Clones were found to express therapeutic levels of FVIII from both loci in both cell types (1 Unit / 2 million cells / 24 hrs). These experiments set the stage for targeting of HPFVIII into hematopoietic stem and progenitor CD34+ cells for the purpose of treating Hemophilia A.

Androgen ablation elicits PP1-dependence for AR stabilization and transactivation in prostate cancer.

Previous reports have documented protein phosphatase 1 (PP1) as an essential androgen receptor (AR) activator. However, more systemic studies are needed to further define PP1 effects on AR, particularly in the settings of prostate cancer cells and under conditions mimicking androgen ablation. PP1 effects on AR protein expression, degradation, ubiquitination, and stabilization were examined in non-prostate cancer cells, followed by validation on exogenous settings in androgen-sensitive (LNCaP and VCaP) and castration-resistant (C4-2) prostate cancer cells. Effects of PP1 on AR protein expression, on AR-mediated transcription of exogenous reporter and endogenous gene, and on LNCaP and C4-2 cell proliferation were monitored under androgen-containing versus androgen-depleted conditions to assess the effects of PP1 on AR responsiveness to androgen. In this report, we determined that PP1 functions to stabilize AR proteins that exclusively undergo the proteasome-dependent degradation, and the stimulatory effects of PP1 were predominantly mediated by the AR ligand-binding domain (LBD). Consistently, PP1 enhances AR protein stability by disrupting the LBD-mediated and K48-linked ubiquitination cascade. We further validated the above findings in the prostate cancer cells by showing that PP1 inhibition can increase ubiquitin- and proteasome-dependent degradation of endogenous AR under androgen deprivation. Significantly, we found that PP1 could markedly activate AR transcriptional activities under conditions mimicking androgen ablation and that androgen sensitivity was substantially evoked by PP1 inhibition in the prostate cancer cell lines. As summarized in a simplified model, our studies defined an essential PP1-mediated pathway for AR protein stabilization that can compensate the loss of androgen and established a mechanistic link between PP1 and androgen responsiveness. The amplified PP1-dependence for AR activation under the androgen ablated conditions provides a rationale to therapeutically target the PP1-AR module in the castration-resistant prostate cancer (CRPC). Our findings also suggested an alternative AR-targeting compounds screening strategy that aims to circumvent PP1-AR interaction.