Endogenous Promoter Research Articles

Abstract 2853: Transposon-activated POU5F1B promotes colorectal cancer growth and metastasis

Abstract The human genome contains some 4 million transposable element (TE)-derived sequences, which collectively influence gene expression from early embryogenesis to adulthood. The illegitimate production of oncogene-encoding TE-driven transpochimeric gene transcripts (TcGTs) has been noted in tumors, but their participation in the oncogenic process has seldom been demonstrated. Here we describe how the aberrant de-repression of a primate-specific LTR66 endogenous retroviral promoter generates a TcGT overexpressing the great-ape-restricted POU5F1B retrogene in ~65% of 286 colorectal cancers (CRC) patients, correlating with more advanced tumor stages and shorter relapse-free and overall survival. We further demonstrate that the POU5F1B protein stimulates the clonogenic and proliferation capacity of human CRC cell lines in vitro, and their tumorigenic and metastatic potential in mouse xenotransplantation models. Although POU5F1B is a retrotransposition-mediated derivative of the POU5F1/OCT4 transcription factor, its product is a predominantly cytoplasmic protein enriched in membranes that associates with mediators of signal transduction, notably the ERBB2 receptor tyrosine kinase and several of its known interactors. POU5F1B overexpression results in activating genes involved in signaling, and conditioned medium of POU5F1B-overexpressing cells enhances the clonogenic potential of CRC cells in trans. As POU5F1B is encoded by an apparently non-essential gene only lowly expressed in normal tissues, and as POU5F1B-containing TcGTs are detected in other tumors besides CRC, it represents an attractive target for the development of cancer therapies. Citation Format: Laia Simó-Riudalbas, Evarist Planet, Sandra Offner, Julien Duc, Laurence Abrami, Sagane Dind, Alexandre Coudray, Mairene Coto-Llerena, Salvatore Piscuoglio, Claus L. Andersen, Jesper Bertram Bramsen, Didier Trono. Transposon-activated POU5F1B promotes colorectal cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2853.

Exploration and characterization of hypoxia-inducible endogenous promoters in Aspergillus niger.

Aspergillus niger is widely used for the efficient production of organic acids and enzyme preparations. However, this organism lacks basic genetic elements for dynamic control, especially inducible promoters that can respond to specific environmental signals. Since these are desirable for better adaptation of fermentation to large-scale industrial production, herein, we have identified the two first hypoxia-inducible promoters in A. niger, PsrbB and PfhbA. Their performance under high or low oxygen conditions was monitored using two reporter proteins, green fluorescent protein (EGFP) and β-glucuronidase (GUS). For comparison, basal expression of the general strong promoter PgpdA was lower than PsrbB but higher than PfhbA. However, under hypoxia, both promoters showed higher expression than under hyperoxia, and these values were also higher than those observed for PgpdA. For PsrbB, strength under hypoxia was ~2-3 times higher than under hyperoxia (for PfhbA, 3-9 times higher) and ~2.5-5 times higher than for PgpdA (for PfhbA, 2-3 times higher). Promoter truncation analysis showed that the PsrbB fragment -1024 to -588 bp is the core region that determines hypoxia response. KEY POINTS: The first identification of two hypoxia-inducible promoters in A. niger is a promising tool for modulation of target genes under hypoxia. Two reporter genes revealed a different activity and responsiveness to hypoxia of PfhbA and PsrbB promoters, which is relevant for the development of dynamic metabolic regulation of A. niger fermentation. PsrbB promoter truncation and bioinformatics analysis is the foundation for further research.

Heme Regulates the Expression in Erythroblasts of Placenta Growth Factor, a Contributor to Pulmonary Hypertension in Sickle Cell Disease, through the Oxidant Response Transcription Factor NRF2

Patients with sickle cell disease (SCD) have elevated plasma levels of placenta growth factor (PGF), which promotes expression of the pulmonary vasoconstrictor endothelin-1 (ET-1) contributing to pulmonary hypertension, an important age-related and life-limiting complication of SCD. In SCD patients, markers of high iron burden are associated with the highest PGF levels, leading us to the investigation of a mechanism where excessive heme induces PGF expression. Our lab's published work has demonstrated a significant heme-bound iron (hemin) stimulation of the PGF promoter in human K562 cells and in primary human erythroid cells. Our initial experiments with N-acetylcysteine and the nonspecific oxidant hydrogen peroxide, suggested to us a role of oxidant stress in PGF regulation, hypothetically through the NRF2 antioxidant response mechanism.We conducted gene expression analysis by quantitative real time PCR for PGF in human K562 erythroblastoid cells, erythroid-differentiated human and mouse primary bone marrow cells before and after exposure to hemin. We tested the effect of Nrf2 agonists and an antagonist. We analyzed expression of heme oxygenase-1 as a positive control, and expression of several Nrf2 family members, as well as baseline genes. We performed siRNA gene knockdown experiments in cultured cells, chromatin immunoprecipitation experiments and intravenous injection of hemin (120 μmol/kg) in the tail veins of wild type and Nrf2 null mice, followed by assay of plasma PGF by ELISA and qPCR of bone marrow.We find that heme regulates the expression of several members of the Nrf2 family, which in turn may appear to affect PGF expression. More specifically, the antioxidant transcription factory family members NRF2, MafF, MafG and BACH1 are transcriptionally activated in a specific temporal pattern after heme exposure. NFE2 and MafK transcripts were simultaneously repressed by heme treatment. The well-characterized non-oxidative NRF2 agonist sulforaphane activated the endogenous PGF promoter and the NRF2 inhibitor brusatol inhibited heme induction of the endogenous PGF promoter. Specific gene knockdown results using siRNA supports additive roles for NRF2 and its heterodimeric partner MafG in activating the PGF promoter in response to heme, presumably as a heterodimer. We verified direct binding of NRF2 on the PGF promoter by chromatin immunoprecipitation experiments. While PGF regulation shows quite many common aspects to HO-1 regulation (heme and oxidative stress response), we find that protoporphyrin induces HO-1 (known to require BACH-1), but not PGF.In addition to the experimental data from our human K562 cell culture models, we have found corroborating results in primary erythroid-differentiated human and murine hematopoietic cells. In vivo, intravenous injection of heme in wild type mice produces robust secretion of PGF protein into plasma within 3 hours (192 ± 16 vs. 527 ± 62 pg/mL, P <0.0001, mean ± SEM, one-way ANOVA with Sidak's multiple comparison test) and this heme induction of plasma PGF protein is blunted in the Nrf2 null mice (527 ± 62 vs. 343 ± 21 pg/mL, P=0.004).Our results support a mechanism in which accelerated heme exposure in SCD promotes robust expression of PGF in erythroblasts during erythroid differentiation, through a pathway that involves EKLF, NRF2 and MafG, culminating in expression of the vascoconstrictor endothelin-1. This mechanism helps to explain the clinical observation that adults with SCD heavily exposed to red cell transfusion are more likely to develop high endothelin levels and pulmonary hypertension, as a potential consequence of excess heme trafficking from the turnover of transfused red cells. These results might inspire greater adherence to existing approved therapies to chelate iron in SCD. [Display omitted] DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties. Kato:Bayer: Research Funding; Global Blood Therapeutics: Consultancy; MAST Therapeutics: Research Funding; Novartis: Consultancy.

A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction As a Developmental Susceptibility to ETV6-RUNX1

Childhood acute lymphoblastic leukemia (cALL) is clinically distinct from that in adults with higher incidence, better prognosis, a distinct mutational spectrum and that the pre-leukemic initiating events frequently occur in utero . One hypothesis for this difference is that childhood cancers arise in transient cell compartments unique to early human development. We have explored this in ETV6-RUNX1-associated cALL, where evidence from twin studies and neonatal heel prick testing has shown that this translocation occurs in utero and is an initiating event . Consistent with this, epidemiological evidence and changes in placental structure suggest that leukemic initiation occurs in the first trimester when both extra-embryonically and intra-embryonically-derived waves of hematopoiesis coexist. We therefore characterized B-lymphoid development in first trimester human embryos aiming to identify a candidate compartment vulnerable to the pre-leukemic initiating effect of ETV6-RUNX1. Using CD19 as a marker associated with definitive B lineage restriction in adult hematopoiesis, we established that the first CD19+ B cell progenitors emerged in the human fetal liver (FL) at Carnegie Stage (CS) 17 and were distinct from their cord blood or adult bone marrow counterparts in that the majority expressed surface IL7 receptor. Analysis of IL7R expression also uncovered a CD19-IL7R+ candidate B progenitor compartment. CD19-IL7R+ cells produced CD19+ B cells in vitro, possessed IGH DJ recombination, but also exhibited myeloid (but not megakaryocyte-erythroid) potential. Single cell analysis revealed co-expression of lymphoid and myeloid lineage programs in CD19-IL7R+ progenitors at CS20, whereas at CS17 their transcriptional programs appeared markedly more myeloid, indicating that lymphoid potential is first acquired in CD19-IL7R+ progenitors in this developmental window. Myeloid cells derived from CS20 IL7R progenitors harbored IGH DJ rearrangements, confirming their origins from a RAG-expressing lymphoid progenitor. We tested whether these distinct aspects of embryonic lymphoid development could be modeled using human pluripotent stem cells (hPSCs). In vitro B cell differentiation of hPSCs produced IL7R expressing CD19+ B cells as well as a CD19-IL7R+ progenitor that also “switched” from myeloid to B-myeloid programming during culture. At the global transcriptional level the hPSC-derived lymphoid hierarchy maps closely with that from the embryo, with both separating from the adult, suggesting that hPSCs provide a developmentally relevant model of early fetal B lymphopoiesis. We next used CRIPSR-directed homologous recombination to knock in the cDNA of RUNX1 into intron V of ETV6 resulting in expression of ETV6-RUNX1 under the endogenous ETV6 promoter. ETV6-RUNX1 hPSCs displayed a partial block in B lymphoid differentiation at the CD19-IL7R+ progenitor to proB cell transition and competitive co-culture demonstrated a selective advantage to the ETV6-RUNX1+ CD19-IL7R+ progenitors over their wild type counterparts. Single cell transcriptional analysis showed that the ETV6-RUNX1+ CD19+ B cells that do emerge express an abnormal B-myeloid gene expression signature akin to that seen in the CD19-IL7R+ progenitor. Consistent with this programming, when grown in myeloid cytokines, ETV6-RUNX1+ CD19+ B cells exhibited both a survival advantage and an aberrant ability to form IGH DJ rearranged macrophage colonies. Both the transcriptional and functional phenotypes were dependent on ETV6-RUNX1, as demonstrated by their reversion upon cre-mediated excision of the knock-in cassette. Our data support a model whereby ETV6-RUNX1 inhibits effective resolution of a lympho-myeloid program present in early fetal CD19-IL7R+ progenitors, impeding B lineage differentiation and resulting in the production of B cells with aberrant myeloid gene expression and potential. Such programming may explain the relatively high levels of myeloid antigen expression and lineage promiscuity seen in ETV6-RUNX1 and other cALLs. This hPSC model of ETV6-RUNX1 pre-leukemia will afford systematic evaluation of the contribution of second hit mutations seen in cALL and offers a tractable platform for drug screening. In conclusion we propose that a novel IL7R+ lympho-myeloid progenitor in the human embryonic fetal liver is a candidate target cell for in utero pre-leukemic initiation in childhood B-ALL. DisclosuresNo relevant conflicts of interest to declare.

Aberrant HCK Survival Signaling By Mutated MYD88 Requires PAX5 in Waldenstrom's Macroglobulinemia and ABC DLBCL Cells

Activating mutations in MYD88 are present in 95% and 40% of patients with WM and ABC DLBCL, respectively. MYD88 mutations are known to trigger growth and survival of WM and ABC DLBCL cells through BTK/IRAK1/IRAK4 directed NF-kB signaling (Ngo et al, Nature 2011; Yang et al, Blood 2013). Recently, we identified that mutated MYD88 can trigger the SRC family member HCK, a kinase that is downregulated in later stages of B-cell ontogeny (Yang et al, Blood 2016). HCK promotes malignant cell survival through activation of BTK, as well as AKT and ERK1/2, whereas knockdown of HCK leads to decreased survival in WM and ABC DLBCL cells. The regulatory mechanisms that promote mutated MYD88 directed HCK signaling are unknown, and may permit therapeutic advances for MYD88 mutated diseases. To clarify whether HCK expression is regulated by TLRs / MYD88 or BCR signaling, we performed activation studies using triggers of TLR9 (ODN-2006) and TLR4 (LPS-EP) signaling, and BCR (anti-IgM/IgG) signaling in MYD88 wild-type or mutated B-cell lymphoma cells. Stimulation of TLRs but not BCR increased HCK transcription, particularly in wild-type MYD88 expressing B-cell lymphoma cells thereby demonstrating the importance of TLR/MYD88 signaling in driving HCK expression. To better understand the mechanism for the aberrant upregulation of HCK transcription in MYD88 mutated B-cell lymphoma cells, we performed promoter-binding TFs profiling, and observed that among the TFs, PAX5 signal was reduced the most by the addition of HCK promoter into BCWM.1 cell nuclear extracts. The signals of many other TFs that can be regulated by NF-kB (AP1, C\\EBP, Myb, NF-kB), MAPK (AP1, C\\EBP, CREB, HNF4, NF-1) and STAT3 signaling were also reduced. These results indicate that PAX5 and mutated MYD88 directed TFs including NF-kB, AP-1 and STAT3 bind to endogenous HCK promoter sequence. HCK promoter binding consensus sequence analysis and ChIP studies confirmed the presence of PAX5, as well as AP-1, NF-kB and STAT3 binding sites on the HCK promoter. Knockdown of PAX5 reduced HCK mRNA level in BCWM.1 and TMD-8 cells indicating an activating function of PAX5 in the regulation of HCK expression. The deletion of AP-1, NF-kB and STAT3 binding sites on HCK promoter reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 in HCK gene expression in MYD88 mutated cells. Treatment of BCWM.1 and TMD-8 cells with inhibitors of AP-1, NF-kB and STAT3 decreased HCK promoter activities by luciferase reporter assay, as well as endogenous HCK mRNA transcription levels in MYD88 mutated cells. Our data indicate PAX5 along with mutated MYD88 triggered transcription factors NF-kB, AP-1 and STAT3 orchestrate aberrant HCK expression. The findings provide a framework for efforts directed at blocking MYD88 triggered HCK survival signaling in MYD88 mutated B-cell lymphomas. DisclosuresCastillo:Abbvie: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

MiR-126 Promotes Leukemogenesis in Inv(16) Acute Myeloid Leukemia

MicroRNAs (miRNAs) are short non-coding RNA molecules that inhibit the expression of multiple target proteins. MiRNA expression profiles can predict prognosis and are associated with distinct cytogentic abnormalities in acute myeloid leukemia (AML). Core-binding factor (CBF) AML, including inv(16)(p13q22)/t(16;16)(p13.1;q22) and t(8;21) chromosomal rearrangements, express significantly higher levels of miRNA (miR)-126 than non-CBF AML. We and others have shown that miR-126 is highly expressed in hematopoietic stem cells and leukemic stem cell (LSC)-enriched subpopulations and functions to promote quiescence. We previously reported that miR-126 expression is associated with an AML LSC-gene expression signature and independently predicts poor prognosis in cytogenetically normal AML. However, the role of miR-126 and the mechanism of dysregulation in inv(16) AML remains unknown.Inv(16) chromosomal rearrangement generates a leukemogenic fusion oncogene CBFB - MYH11 (CM). We utilized a conditional Cbfb-MYH11 knock-in (Cbfb56M/+ / Mx1-Cre) mouse that can be induced to express CM from the endogenous Cbfb promoter, thus mimicking human inv(16) AML. Time sequential miRNA-seq analysis revealed that miR-126-3p and miR-126-5p expression was significantly up-regulated (log fold change =2.09 and 2.36 respectively; p< 0.0001) early (1 month) and increased progressively throughout leukemia progression (log fold change = 4.11 and 4.45; p< 0.0001). Increased expression of miR-126 was observed in multiple preleukemic progenitor subpopulations, including LSK (Lin-Sca1+ckit+) and pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin-ckit+Sca1-CD16/32-/loCD105-CD150+) in CM mice.To determine the functional contribution of miR-126 to inv(16) leukemogenesis, we crossed our CM knock-in mice with a conditional miR-126 knockout. Preliminary survival results indicate that CM/miR-126△/+ (heterozygote) exhibit significantly prolonged survival compared to CM mice, with a median survival of 266 vs. 159 days, respectively (p<0.05). Survival monitoring is ongoing for a cohort of CM/miR-126△/△(homozygote) mice. To date, this cohort is already demonstrating significantly enhanced survival compared to CM mice (p<0.05).We hypothesize that high miR-126 expression might contribute to survival and quiescence of CM AML LSK, which is enriched for leukemia-initiating cells. Thus, we performed lentiviral-mediated miR-126 knockdown in CM AML LSK cells and observed increased apoptosis and reduced quiescence in vitro . To assess the role of miR-126 in leukemia progression in vivo, we designed a novel CpG-anti-miR-126 inhibitor. This compound comprises an inhibitory anti-miR-126 RNA component that is linked to a Class A CpG motif for targeted cell delivery. We showed that the CpG-anti-miR-126 is efficiently taken up (60-90%) by AML blasts and LSK cells and can achieve approximately 50-80% knock-down of miR-126 in vitro and in vivo. Mice transplanted with CMAML and treated with CpG-anti-miR-126 (5 mg/kg, 5 times/week for 5 weeks) had a significant delay in leukemia progression and enhanced survival (median survival 92 days vs 35 days; n=9-10, p=0.0339), supporting the notion that high miR-126 contributes to leukemia growth. We further evaluated the effects of combining CpG-anti-miR-126 treatment with chemotherapy, adopting a regimen consisted of Cytarabine (Ara-C; i.p. 100 mg/kg for 5 days) and daunorubicin (DNR; i.v. 3 mg/kg for 3 days). We observed significantly improved survival in mice treated with CpG-anti-miR-126 combined with Ara-C and DNR compared to CpG-scrambled combined with Ara-C and DNR (median survival 183 days vs. 64 days; n=9-10; p=0.0305). These studies demonstrate a key role for miR-126 in promoting leukemogenesis of inv(16) AML. Studies to elucidate the mechanism underlying miR-126 dysregulation and function in inv(16) AML are ongoing. DisclosuresNo relevant conflicts of interest to declare.

Analysis of expression characteristics of soybean leaf and root tissue-specific promoters in Arabidopsis and soybean

The characterization of tissue-specific promoters is critical for studying the functions of genes in a given tissue/organ. To study tissue-specific promoters in soybean, we screened tissue-specific expressed genes using published soybean RNA-Seq-based transcriptome data coupled with RT-PCR analysis. We cloned the promoters of three genes, GmADR1, GmBTP1, and GmGER1, and constructed their corresponding β-Glucuronidase (GUS) promoter-GUS reporter vectors. We generated transgenic Arabidopsis plants and examined the expression patterns of these promoters by GUS staining and RT-PCR analysis. We also transformed the promoter-GUS reporter vectors into soybean to obtain hairy roots, and examined promoter expression by GUS staining. We found a root-specific expression pattern of GmADR1 and GmBTP1 in both Arabidopsis and soybean, and the promoter of GmGER1 showed a leaf-specific pattern in transgenic Arabidopsis plants. To test the potential utility of these promoters in soybean improvement by transgenic means, we used the GmADR1 promoter to drive expression of a salt resistance gene in soybean, GmCaM4, by generating transgenic soybean plants. We found that the transgenic plants had significantly enhanced salt tolerance compared to non-transformed wild-type, suggesting that introducing endogenous promoters by transgenic means can drive the expression of functional genes in specific tissues and organs in soybean.

CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes

AbstractXMEN disease, defined as “X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect,” is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.

A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.

Improving the Heterologous Production of Fungal Peroxygenases through an Episomal Pichia pastoris Promoter and Signal Peptide Shuffling System.

Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.

Function Enhancement of a Metabolic Module via Endogenous Promoter Replacement for Pseudomonas sp. JY-Q to Degrade Nicotine in Tobacco Waste Treatment.

Nicotine-degrading Pseudomonas sp. JY-Q is a preferred strain utilized in reconstituted tobacco process for tobacco waste treatment. However, its efficiency of nicotine metabolism still requires to be improved via genomic technology such as promoter engineering based on genomic information. Concerning upstream module of nicotine metabolic pathway, we found that two homologous genes of nicotine dehydrogenase (nicA2 and nox) coexisted in strain JY-Q. However, the transcriptional amount of nox was 20-fold higher than that of nicA2. Thus, the nicA2 expression required improvement. Combinatorial displacement was accomplished for two predicted endogenous promoters, named as PnicA2 and Pnox for nicA2 and nox, respectively. The mutant with Pnox as the promoters for both nicA2 and nox exhibited the best nicotine metabolic capacity which increased by 66% compared to the wild type. These results suggested that endogenous promoter replacement is also feasible for function improvementof metabolic modules and strain enhancement of biodegradation capacity to meet real environment demand.

Genome editing efficiency of four Drosophila suzukii endogenous U6 promoters.

The invasive spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) has caused serious economic losses to the fruit industry. The conventional control methods have many limitations and genetic engineering technologies such as CRISPR/Cas9-mediated gene drive are promising approaches. In the CRISPR/Cas9 system, the transcriptional regulatory elements play an important role in the activities of gRNA. Thus, in order to improve the genome editing efficiency of the CRISPR/Cas9 system in D. suzukii, we cloned and tested four endogenous U6 promoters to drive mutagenesis of the white gene. Our results showed that all the four promoters could be used with variable efficiency. The promoter DsU6-3 had the highest genome editing efficiency among the four DsU6 promoters. Compared with the DsU6-3 promoter, the DmU6:3 promoter showed lower efficiency to drive mutagenesis in D. suzukii. These findings expand the range of promoters available to express gRNAs in D. suzukii, facilitating the basic and applied research on this important pest.

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.

Generation of a Clta‐Reporter Mouse Model for in vivo imaging of Endocytosis

Endocytosis via different pathways is a fundamental cellular process in all eukaryotic organisms and is essential to higher eukaryotic life. One of the first and best characterized pathways is clathrin-mediated endocytosis (CME) that uses clathrin-coated vesicles for the uptake of material from the cell surface. Over the recent years more and more proteins that are part of the CME machinery as well as new cargoes were described. This led to important insights into the whole pathway. However, there is still a lack of a good understanding of how these different components work together in vivo. Especially the differential physiological role of the clathrin light chains a and b in health and disease is still not clear and under debate. Using the CRISPR/Cas9 technique, we generated a Clathrin-eGFP knock-in reporter mouse model to study CME in vivo using intravital microscopy. To prevent overexpression artefacts, the fusion protein is expressed under the control of the endogenous promoter, as eGFP was introduced in-frame in front of the clta stop codon. The Clta-eGFP fusion protein is expressed in all tissues examined and is able to interact with known interaction partners. Two-photon intravital microscopy in kidney proximal tubules of Clta-eGFP reporter mice showed that albumin uptake is not distinguishable from WT littermates.With this reporter mouse model, we are able to visualize the endocytic process in vivo by intravital microscopy and these mice are therefore a suitable and powerful tool for basic research about CME processes in vivo.

A molecular toolkit for the green seaweed Ulva mutabilis.

The green seaweed Ulva mutabilis is an ecologically important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed U. mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors, and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50%-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed.

Targeting the Apoa1 locus for liver-directed gene therapy

Clinical application of somatic genome editing requires therapeutics that are generalizable to a broad range of patients. Targeted insertion of promoterless transgenes can ensure that edits are permanent and broadly applicable while minimizing risks of off-target integration. In the liver, the Albumin (Alb) locus is currently the only well-characterized site for promoterless transgene insertion. Here, we target the Apoa1 locus with adeno-associated viral (AAV) delivery of CRISPR-Cas9 and achieve rates of 6% to 16% of targeted hepatocytes, with no evidence of toxicity. We further show that the endogenous Apoa1 promoter can drive robust and sustained expression of therapeutic proteins, such as apolipoprotein E (APOE), dramatically reducing plasma lipids in a model of hypercholesterolemia. Finally, we demonstrate that Apoa1-targeted fumarylacetoacetate hydrolase (FAH) can correct and rescue the severe metabolic liver disease hereditary tyrosinemia type I. In summary, we identify and validate Apoa1 as a novel integration site that supports durable transgene expression in the liver for gene therapy applications.

Low efficacy of recombinant SV40 in Ugt1a1-/- mice with severe inherited hyperbilirubinemia.

In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy.

Integration of metabolic pathway manipulation and promoter engineering for the fine-tuned biosynthesis of malic acid in Bacillus coagulans.

Bacillus coagulans, a thermophilic facultative anaerobe, is a favorable chassis strain for the biosynthesis of desired products. In this study, B. coagulans was converted into an efficient malic acid producer by metabolic engineering and promoter engineering. Promoter mapping revealed that the endogenous promoter Pldh was a tandem promoter. Accordingly, a promoter library was developed, covering a wide range of relative transcription efficiencies with small increments. A reductive tricarboxylic acid pathway was established in B. coagulans by introducing the genes encoding pyruvate carboxylase (pyc), malate dehydrogenase (mdh), and phosphoenolpyruvate carboxykinase (pckA). Five promoters of various strengths within the library were screened to fine-tune the expression of pyc to improve the biosynthesis of malic acid. In addition, genes involved in the competitive metabolic pathways were deleted to focus the substrate and energy flux toward malic acid. Dual-phase fed-batch fermentation was performed to increase the biomass of the strain, further improving the titer of malic acid to 25.5 g/L, with a conversion rate of 0.3 g/g glucose. Our study is apioneer research using promoter engineering and genetically modified B. coagulans for the biosynthesis of malic acid, providing an effective approach for the industrialized production of desired products using B. coagulans.

CTP Synthase 2 From Arabidopsis thaliana Is Required for Complete Embryo Development.

Pyrimidine de novo synthesis is an essential pathway in all organisms. The final and rate-limiting step in the synthesis of the nucleotide cytidine triphosphate (CTP) is catalyzed by CTP synthase (CTPS), and Arabidopsis harbors five isoforms. Single mutant lines defective in each one of the four isoforms do not show apparent phenotypical alterations in comparison to wild-type plants. However, Arabidopsis lines that contain T-DNA insertions in the CTPS2 gene were unable to produce homozygous offspring. Here, we show that CTPS2 exhibits a distinct expression pattern throughout embryo development, and loss-of-function mutants are embryo lethal, as siliques from +/ctps2 plants contained nearly 25% aborted seeds. This phenotype was rescued by complementation with CTPS2 under control of its endogenous promoter. CTPS2::GFP lines revealed expression only in the tip of columella cells in embryo root tips of the heart and later stages. Furthermore, CTPS2 expression in mature roots, most pronounced in the columella cells, shoots, and vasculature tissue of young seedlings, was observed. Filial generations of +/ctps2 plants did not germinate properly, even under external cytidine supply. During embryo development, the CTPS2 expression pattern resembled the established auxin reporter DR5::GFP. Indeed, the cloned promoter region we used in this study possesses a repeat of an auxin response element, and auxin supply increased CTPS2 expression in a cell-type-specific manner. Thus, we conclude that CTPS2 is essential for CTP supply in developing embryos, and loss-of-function mutants in CTPS2 are embryo lethal.

The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.