Original Research: Expression Profile of CAPZA3 And TR-KIT Genes in Men with Globozoospermia & Asthenoteratospermia that Undergo ICSI Protocol

Objective(s): Sperm-mediated oocyte activation depends upon suitable expression and assembly of sperm-borne oocyte-activating factors (SOAFs) during spermiogenesis. Several factors have been considered as candidates for oocyte activation in recent years. Globozoospermia is a severe sperm morphology disorder that is a rare type of teratozoospermia with an incidence of 0.1% among infertile individuals. testis-specific genes including CAPZA3 [capping protein (actin filament) muscle Z-line, alpha, which is considered as a nominee for sperm associated oocyte activating factors, an actin-capping protein, controlling actin polymerization during spermiogenesis. They contain a common bidirectional promoter. Another gene TR-KIT (a truncated form of the KIT receptor) which is a major sperm-associated oocyte-activating factor. The objective of this study was to investigate the expression profile of CAPZA3 and TR-KIT mRNA, in men with total globozoospermia, Asthenoteratospermia, and fertile individuals. Materials and Methods: Semen samples were collected from three groups including 25 fertile men, 20 Asthenoteratospermia and 12 Globozoospermia that undergo intra-cytoplasmic sperm injection (ICSI), Expression of CAPZA3 and TR-KIT were assessed by Real time PCR. Results: Individuals with Globozoospermia have presented significantly lower expression of CAPZA3 and TR-KIT mRNA when compared with fertile men. Asthenoteratospermia (male factor) showed significantly lower expression of CAPZA3 mRNA, whereas non-significantly of TR-KIT mRNA. Levels of CAPZA3 and TR-KIT mRNA in the spermatozoa of fertile men were significantly higher than the corresponding values of the globozoospermic and Asthenoteratospermia subjects. Conclusion: Analysis mRNA of CAPZA3 gene maybe assist the researcher to identify individuals with a lack of ability to induce oocyte activation and make them a candidate for artificial oocyte activation and help researcher to identify genetic defects associated with failed fertilization. whereas, mRNA of TR-KIT gene appears inability to induce oocyte activation

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high affinity heterodimer formation and enzymatic function

In a reaction that requires processing by E1 and E2 enzymes, the E3 ligase complex DTX3L-PARP9 ADP-ribosylates the carboxyl terminus of Ubiquitin and prevents its conjugation to substrate protein. This provides an NAD+-sensitive mechanism for regulating mono-Ub modification of certain substrates. DTX3L and PARP9 are coordinatedly expressed from a single, bidirectional promoter and can be isolated as a protein complex from multiple cell types. We used purified DTX3L and PARP9, prepared as individual proteins, to investigate how the complex assembles and how complex formation is related to E3 activity. We find that DTX3L binds PARP9 with nanomolar affinity and with an apparent stoichiometry of 1:1. Using a combination of deletion mutants and cross-linking mass spectrometry, we show the interaction involves the D3 domain of DTX3L (230 - 510) and a central region of PARP9 (491 - 630). We found evidence that the DTX3L-PARP9 heterodimer can assemble into a high molecular weight oligomer dependent on the D1 (1 - 100) and D2 (101 - 200) domains of DTX3L. We also show that ADP-ribosylation of Ubiquitin by DTX3L-PARP9 is reversible by several macrodomain-type hydrolases. Our data helps to provide a structural framework for understanding how ubiquitination and ADP-ribosylation are mediated by DTX3L-PARP9.

Co-regulation and function of FOXM1/RHNO1 bidirectional genes in cancer

The FOXM1 transcription factor is an oncoprotein and a top biomarker of poor prognosis in human cancer. Overexpression and activation of FOXM1 is frequent in high-grade serous carcinoma (HGSC), the most common and lethal form of human ovarian cancer, and is linked to copy number gains at chromosome 12p13.33. We show that FOXM1 is co-amplified and co-expressed with RHNO1, a gene involved in the ATR-Chk1 signaling pathway that functions in the DNA replication stress (RS) response. We demonstrate that FOXM1 and RHNO1 are head-to-head (i.e. bidirectional) genes (BDG) regulated by a bidirectional promoter (BDP) (named F/R-BDP). FOXM1 and RHNO1 each promote oncogenic phenotypes in HGSC cells, including clonogenic growth, DNA homologous recombination repair (HR), and poly-ADP ribosylase (PARP) inhibitor resistance. FOXM1 and RHNO1 are one of the first examples of oncogenic BDG, and therapeutic targeting of FOXM1/RHNO1 BDG is a potential therapeutic approach for ovarian and other cancers.

A Small Natural Light-induced Bidirectional Promoter of Rapeseed (Brassica Napus)

A bidirectional promoter is an intergenic region located between a pair of adjacent and oppositely transcribed genes (‘head-to-head’ genes) that concurrently promote both genes expression. In the genome of Brassica napus, we identified a bidirectional promoter (265bp long), named PBn265. PBn265 was located between the transcription initiation codons (ATG) of two genes that encode the homeodomain protein SHH2, and chloroplast GROUP II intron splicing factor CFM3. Its bidirectional promoting activity was verified by transient expression of Nicotiana benthamiana leaf tissue via Agrobacterium-mediated transformation of PBn265-F::EGFP and PBn265-R::mCherry. The expression of both reporter genes, EGFP linked to one end and GUS on the other end of the PBn265 sequence, was observed in the various tissues of the transgenic Arabidopsis thaliana using histochemical staining and fluorescence microscopy. Furthermore, we also found that the promoting activity of this sequence was regulated by illumination. Considering its short sequence length and light inducible regulation, this promoter likely has application potential in bioengineering and agricultural molecular breeding.

Therapeutic Angiogenesis by a “Dynamic Duo”: Simultaneous Expression of HGF and VEGF165 by Novel Bicistronic Plasmid Restores Blood Flow in Ischemic Skeletal Muscle

Therapeutic angiogenesis is a promising strategy for relief of ischemic conditions, and gene delivery was used to stimulate blood vessels’ formation and growth. We have previously shown that intramuscular injection of a mixture containing plasmids encoding vascular endothelial growth factor (VEGF)165 and hepatocyte growth factor (HGF) leads to restoration of blood flow in mouse ischemic limb, and efficacy of combined delivery was superior to each plasmid administered alone. In this work, we evaluated different approaches for co-expression of HGF and VEGF165 genes in a panel of candidate plasmid DNAs (pDNAs) with internal ribosome entry sites (IRESs), a bidirectional promoter or two independent promoters for each gene of interest. Studies in HEK293T culture showed that all plasmids provided synthesis of HGF and VEGF165 proteins and stimulated capillary formation by human umbilical vein endothelial cells (HUVEC), indicating the biological potency of expressed factors. Tests in skeletal muscle explants showed a dramatic difference and most plasmids failed to express HGF and VEGF165 in a significant quantity. However, a bicistronic plasmid with two independent promoters (cytomegalovirus (CMV) for HGF and chicken b-actin (CAG) for VEGF165) provided expression of both grow factors in skeletal muscle at an equimolar ratio. Efficacy tests of bicistronic plasmid were performed in a mouse model of hind limb ischemia. Intramuscular administration of plasmid induced significant restoration of perfusion compared to an empty vector and saline. These findings were supported by increased CD31+ capillary density in animals that received pHGF/VEGF. Overall, our study reports a first-in-class candidate gene therapy drug to deliver two pivotal angiogenic growth factors (HGF and VEGF165) with properties that provide basis for future development of treatment for an unmet medical need—peripheral artery disease and associated limb ischemia.

Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon

Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.

Effects of activation of the LINE-1 antisense promoter on the growth of cultured cells

Long interspersed element 1 (LINE-1, or L1) is a retrotransposon that constitutes ~ 17% of the human genome. Although ~ 6000 full-length L1s spread throughout the human genome, their biological significance remains undetermined. The L1 5′ untranslated region has bidirectional promoter activity with a sense promoter driving L1 mRNA production and an antisense promoter (ASP) driving the production of L1-gene chimeric RNAs. Here, we stimulated L1 ASP activity using CRISPR-Cas9 technology to evaluate its biological impacts. Activation of the L1 ASP upregulated the expression of L1 ASP-driven ORF0 and enhanced cell growth. Furthermore, the exogenous expression of ORF0 also enhanced cell growth. These results indicate that activation of L1 ASP activity fuels cell growth at least through ORF0 expression. To our knowledge, this is the first report demonstrating the role of the L1 ASP in a biological context. Considering that L1 sequences are desilenced in various tumor cells, our results indicate that activation of the L1 ASP may be a cause of tumor growth; therefore, interfering with L1 ASP activity may be a potential strategy to suppress the growth.

Abstract 13202: LncRNA-MAP3K4 Regulates Vascular Inflammation Through a P38 MAPK Signaling Pathway and Cis -modulation of MAP3K4

Introduction: Long non-coding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in vascular inflammation remains poorly understood. Hypothesis: Identification of inflammation-responsive lncRNAs expressed in the aortic intima may provide novel mechanistic insights in vascular inflammation. Methods: Using RNA-Seq profiling to identify a lncRNA derived specifically from the aortic intima of atherosclerotic mice, we discovered an inflammation-responsive lncRNA, lncRNA-MAP3K4, and evaluated its role in the mechanisms mediating vascular cell inflammation. Results: Aortic expression of lncRNA-MAP3K4 , an intima-enriched and polyadenylated lncRNA , was reduced by 50% with atherosclerotic progression and by 75% following LPS-induced endotoxemia in mice. GapmeR-mediated silencing of lncRNA-MAP3K4 potently reduced mRNA and protein expression of adhesion molecules or chemokines (e.g. ICAM-1, E-selectin, MCP-1) in endothelial cells via a p38-MAPK pathway, and decreased PBMC adhesion to endothelium by 40%. Moreover, lncRNA-MAP3K4 knockdown also reduced inflammatory markers in vascular smooth muscle cells and macrophages. Analyzing the lncRNA-MAP3K4 locus, we found MAP3K4, an upstream kinase of the MAPK cascade, shared the promoter region with lncRNA-MAP3K4. In vitro and in vivo, lncRNA-MAP3K4 and MAP3K4 showed parallel inflammation-responsive expression patterns. lncRNA-MAP3K4 knockdown reduced mRNA and protein expression of MAP3K4 in cis in vessel wall cell types. ChIP-seq data showed chromatin modifications and bidirectional promoter characteristics in the lncRNA-MAP3K4/ MAP3K4 promoter region. MAP3K4 knockdown showed a similar anti-inflammation phenotype as lncRNA-MAP3K4 via a p38-MAPK pathway and cooperativity with lncRNA-MAP3K4 . Conclusions: Deficiency of lncRNA-MAP3K4 markedly reduced inflammation in vascular cells via a p38-MAPK pathway and cis -regulation of MAP3K4 from a shared bidirectional promoter. This study illustrated a divergently transcribed lncRNA/protein-coding gene pair involved in vascular inflammation and more broadly informs a better understanding of mammalian genome regulatory mechanisms in vascular disease states.

Molecular Characterization of a Pathogen-Inducible Bidirectional Promoter from Hot Pepper (Capsicum annuum)

In hot pepper, the sesquiterpene phytoalexin capsidiol is catalyzed by the two final-step enzymes, a sesquiterpene cyclase (EAS) and a hydroxylase (EAH), which are genetically linked and present as head-to-head orientation in the genome. Transcriptomic analysis revealed that a subset of EAS and EAH is highly induced following pathogen infection, suggesting the coregulation of EAS and EAH by a potential bidirectional activity of the promoter (pCaD). A series of the nested deletions of pCaD in both directions verified the bidirectional promoter activity of the pCaD. Promoter deletion analysis revealed that the 226 bp of the adjacent promoter region of EAS and GCC-box in EAH orientation were determined as critical regulatory elements for the induction of each gene. Based on promoter analyses, we generated a set of synthetic promoters to maximize reporter gene expression within the minimal length of the promoter in both directions. We found that the reporter gene expression was remarkably induced upon infection with Phytophthora capsici, Phytophthora infestans, and bacterial pathogen Pseudomonas syringae pv. tomato DC3000 but not with necrotrophic fungi Botrytis cinerea. Our results confirmed the bidirectional activity of the pCaD located between the head-to-head oriented phytoalexin biosynthetic genes in hot pepper. Furthermore, the synthetic promoter modified in pCaD could be a potential tool for pathogen-inducible expression of target genes for developing disease-resistant crops. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .