Subunit Promoter Research Articles

Changes in pituitary gonadotropin subunits and hypothalamic Kiss-1 gene expression by administration of sex steroids in ovary-intact female rats.

We examined how the sex steroids influence the synthesis of gonadotropins. The effects of sex steroids estradiol (E2), progesterone (P4), and dihydrotestosterone (DHT) in pituitary gonadotroph cell model (LβT2 cells) in vitro and ovary-intact rats in vivo were examined. The effects of sex steroids on Kiss1 gene expression in the hypothalamus were also examined in ovary-intact rats. In LβT2 cells, E2 increased common glycoprotein alpha (Cga) and luteinizing hormone beta (Lhb) subunit promoter activity as well as their mRNA expression. Although gonadotropin subunit promoter activity was not modulated by P4, Cga and Lhb mRNA expression was increased by P4. DHT inhibited Cga and Lhb mRNA expression with a concomitant decrease in their promoter activity. During the 2-week administration of exogenous E2 to ovary-intact rats, the estrous cycle determined by vaginal smears was disrupted. P4 or DHT administration completely eliminated the estrous cycle. Protein expression of all three gonadotropin subunits within the pituitary gland was inhibited by E2 or P4 treatment in vivo; however, DHT reduced Cga expression but did not modulate Lhb or follicle-stimulating hormone beta subunit expression. E2 administration significantly repressed Kiss1 mRNA expression in a posterior hypothalamic region that included the arcuate nucleus. P4 and DHT did not modulate Kiss1 mRNA expression in this region. In contrast, P4 administration significantly inhibited Kiss1 mRNA expression in the anterior region of the hypothalamus that included the anteroventral periventricular nucleus. The expression of gonadotropin-releasing hormone (Gnrh) mRNA in the anterior hypothalamic region, where the preoptic area is located, appeared to be decreased by treatment with E2 and P4. Our findings suggest that sex steroids have different effects in the hypothalamus and pituitary gland.

A synthetic light-inducible photorespiratory bypass enhances photosynthesis to improve rice growth and grain yield

Bioengineering of photorespiratory bypasses is an effective strategy for improving plant productivity by modulating photosynthesis. In previous work, two photorespiratory bypasses, the GOC and GCGT bypasses, increased photosynthetic rates but decreased seed-setting rate in rice (Oryza sativa), probably owing to excess photosynthate accumulation in the stem. To solve this bottleneck, we successfully developed a new synthetic photorespiratory bypass (called the GMA bypass) in rice chloroplasts by introducing Oryza sativa glycolate oxidase 1 (OsGLO1), Cucurbita maxima malate synthase (CmMS), and Oryza sativa ascorbate peroxidase7 (OsAPX7) into the rice genome using a high-efficiency transgene stacking system. Unlike the GOC and GCGT bypass genes driven by constitutive promoters, OsGLO1 in GMA plants was driven by a light-inducible Rubisco small subunit promoter (pRbcS); its expression dynamically changed in response to light, producing a more moderate increase in photosynthate. Photosynthetic rates were significantly increased in GMA plants, and grain yields were significantly improved under greenhouse and field conditions. Transgenic GMA rice showed no reduction in seed-setting rate under either test condition, unlike previous photorespiratory-bypass rice, probably reflecting proper modulation of the photorespiratory bypass. Together, these results imply that appropriate engineering of the GMA bypass can enhance rice growth and grain yield without affecting seed-setting rate.

Activity of Arabidopsis Rubisco small subunit promoter in various tissues of chickpea.

The activity of a green tissue-specific promoter of the Rubisco small subunit gene fromArabidopsis(AraSSU) was studied using transgenic chickpea lines. We generated transgenic chickpea lines expressing anAraSSUpromoter-drivencry2Aagene throughthe Agrobacterium-mediatedtransformation method. Lines withAraSSUexpressed the gene in all green tissues at high levels (> 90ng/mg of fresh weight tissue) compared to lines generated usingCaMV35S(< 10ng/mg FW). We used vertical cross sections of various tissues of homozygous progeny using microtome for immunolocalization. The immunolocalization showed the expression of thecry2Aagene in the green mesophyll cells of the leaves of bothAraSSUand CaMV35 chickpea lines. Moreover, the accumulation ofAraSSU-regulatedCry2Aa protein was also observed in vascular tissues, including enucleate sieve elements and their companion cells. However, no expression was observed in the roots ofAraSSUlines. In the case ofCaMV35lines, the transgene expression was observed in all the tissues. Since our data indicated that theAraSSUpromoter is active in non-green tissues such as vascular bundles. Therefore, we validated this by RT-PCR. We found Cry2Aa RNA transcripts in leaves, stems without epidermis (for vascular tissues), and roots with and without epidermis. Thus,the AraSSUpromoter is active in all above-ground tissues of the chickpea plant.

Epigenetic regulation of oxidative and inflammatory phenotypes in women with gestational diabetes and offspring

Abstract Background Hyperglycemia-induced oxidative stress and inflammation are potent drivers of atherosclerotic cardiovascular disease (ASCVD). Gestational diabetes (GDM) is characterized by chronic hyperglycemia during pregnancy and may represent a clinical model to study the mechanisms of oxidative stress and inflammation induced by hyperglycemia. GDM is associated with a range of adverse perinatal and long-term outcomes for both mother and offspring. In this perspective, it is emerging a putative association between maternal GDM and offspring's epigenetic trait. Purpose To investigate the link between histone modifications, oxidative stress and inflammatory phenotype as well as the transmission of epigenetic signatures to the offspring. Methods We analyzed peripheral blood mononuclear cells (PBMC) from GDM and control mothers as well as human umbilical vein endothelial cells (HUVEC) and cord blood mononuclear cells (CBMC) isolated from newborn umbilical cords obtained at delivery from both groups. Histone methyltransferase MLL1-dependent trimethylation of histone 3 at lysine 4 amino residue (H3K4me3) on NF-kB p65 subunit promoter region was assessed by chromatin immunoprecipitation (ChIP) and real-time qPCR in HUVEC, PBMC and CBMC, respectively. MLL1 and downstream inflammatory and redox genes were determined by real-time qPCR and immunocytochemistry in the presence and in the absence of MLL1 inhibitor MM-102. Measurement of reactive oxygen species (ROS) was performed by electron spin resonance spectroscopy. Results For the first time, we demonstrated a significant increase of MLL1 expression with subsequent MLL1-induced upregulation of NF-kB p65 gene via H3K4me3 in GDM as compared to control cells. MLL1-driven epigenetic remodeling of NF-kB p65 promoter is upstream to the activation of inflammatory pathway. Indeed, treatment with MM-102 decreased H3K4me3 and blunted expression of NF-kB p65 as well as VCAM-1, MCP-1 and IL-6 genes. We also found that expression of ROS scavenger aldehyde dehydrogenase 2 is reduced, whereas pro-oxidant nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX4 is upregulated. Interestingly, the increased ROS generation observed in GDM is involved in the upregulation of MLL1 as shown by the restoring effect of antioxidant vitamin C on MLL1 expression levels. Conclusions Our results suggest that a complex interplay between oxidative stress and histone modifications are responsible for the GDM maternal inflammatory and oxidative phenotypes and its transmission to the offspring. The deciphering of epigenetic-induced chromatin remodelling opens the perspective for pharmacological reprogramming of adverse chromatin changes to reduce the burden of early development of metabolic phenotypes and ASCVD. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University G. d'Annunzio MIUR fundings Schematic figure

Epigenetic Regulation of Gonadotropin Hormone Beta Subunit Gene Expression

Gonadotropin releasing hormone (GnRH) from the hypothalamus regulates the synthesis and secretion of gonadotropin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH regulates steroidogenesis in both sexes and ovulation in females, while FSH stimulates folliculogenesis in females and spermatogenesis in males. LH and FSH are heterodimers of a common alpha subunit and a unique beta subunit, which provides biological specificity and is the rate limiting factor in hormone synthesis. Immediate early genes, early growth response 1 (Egr1) and fos proto-oncogene (cFos) are critical in the induction of LH-beta and FSH-beta subunits by GnRH, respectfully. However, gaps exist in our understanding of developmental initiation and hormonal regulation of gonadotropin gene expression. Specifically, epigenetic mechanisms that may play a role in beta subunit transcriptional regulation are unknown. The aim of this work was to identify transcriptional cofactors that are recruited to gonadotropin beta-subunit promoters with or without GnRH. Transcription factors interact with cofactors that recruit chromatin remodeling enzymes in order to regulate transcription. Identification of cofactors may explain tight regulation of gonadotropin hormone levels in reproductive physiology. Previous studies identified regions on the beta-subunit promoters that are necessary for GnRH responsiveness. These regions were used to pull down interacting proteins that bind to these response elements using nuclear extracts from the immortalized mature gonadotrope cell line, LβT2. Using a discovery proteomics approach, we identified different transcriptional cofactors that are recruited to beta subunit promoters with or without GnRH. Approximately 1500-2000 proteins were identified per pulldown. As expected, proteins known to interact with beta subunit promoters, such as Egr1, cFos and cJun were identified in the DNA pulldown experiments as positive controls. We identified 63 proteins unique for LH-beta promoter under control conditions and 60 unique for FSH-beta promoter, of which 7 proteins for LH-beta and 8 proteins for FSH-beta may play a role as corepressors. We further identified 97 proteins that were pulled down with the LH-beta promoter following GnRH treatment, of which 9 proteins were also pulled down with Egr1 as potential coactivator candidates. We also identified 72 proteins that were pulled down with the FSH-beta promoter following GnRH treatment, of which 6 proteins were pulled down with cFos as potential coactivator candidates. Functional studies to identify roles of these cofactors in gonadotropin hormone expression are in progress. The identification of epigenetic regulators will allow for better understanding of the transcriptional regulation of gonadotropin beta-subunit gene expression, which is critical for reproductive function.

LncRNA LOXL1-AS1 is transcriptionally activated by JUND and contributes to osteoarthritis progression via targeting the miR-423-5p/KDM5C axis

AimsThis study focused on investigating the potential role of long non-coding RNA (lncRNA) lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1) in the progression of osteoarthritis (OA). Materials and methodsqRT-PCR assay was applied to detect gene expression, while western blot was performed to measure levels of apoptosis-related proteins. CCK-8, colony formation and TUNEL assays were conducted to explore the functional role of LOXL1-AS1 in OA. ChIP assay was utilized to assess the affinity between JunD proto-oncogene, AP-1 transcription factor subunit (JUND) and LOXL1-AS1 promoter. Mechanism experiments were implemented to investigate the underlying molecular mechanism of LOXL1-AS1. Key findingsLOXL1-AS1 was up-regulated in OA cartilage tissues. Silencing LOXL1-AS1 hampered proliferation and inflammation, yet promoting apoptosis in chondrocytes. LOXL1-AS1 was transcriptionally activated by JUND1. LOXL1-AS1 sequestered miR-423-5p and abolished miR-423-5p-mediated repression on lysine demethylase 5C (KDM5C), thus promoted the development of OA. SignificanceLncRNA LOXL1-AS1 is transcriptionally activated by JUND and facilitates the proliferation and inflammation of chondrocytes via elevating miR-423-5p-mediated KDM5C in OA, which may provide potential therapeutic target for OA.

SRXN1 Is Necessary for Resolution of GnRH-Induced Oxidative Stress and Induction of Gonadotropin Gene Expression.

A defining characteristic of the hypothalamus-pituitary-gonad reproductive endocrine axis is the episodic secretion of the pituitary gonadotropin hormones LH and FSH by the anterior pituitary gonadotropes. Hormone secretion is dictated by pulsatile stimulation, with GnRH released by hypothalamic neurons that bind and activate the G protein-coupled GnRH receptor expressed by gonadotropes. Hormone secretion and synthesis of gonadotropins are influenced by the amplitude and frequency of GnRH stimulation; variation in either affects the proportion of LH and FSH secreted and the differential regulation of hormone subunit gene expression. Therefore, proper decoding of GnRH signals is essential for appropriate gonadotropin synthesis and secretion. The GnRH receptor robustly activates downstream signaling cascades to facilitate exocytosis and stimulate gene expression and protein synthesis. It is necessary to rapidly quench signaling to preserve sensitivity and adaptability to changing pulse patterns. Reactive oxygen species (ROS) generated by receptor-activated oxidases fulfill the role of rapid signaling intermediates that facilitate robust and transient signaling. However, excess ROS can be detrimental and, unchecked, can confuse signal interpretation. We demonstrate that sulfiredoxin (SRXN1), an ATP-dependent reductase, is essential for normal responses to GnRH receptor signaling and plays a central role in resolution of ROS induced by GnRH stimulation. SRXN1 expression is mitogen-activated protein kinase dependent, and knockdown reduces Lhb and Fshb glycoprotein hormone subunit mRNA and promoter activity. Loss of SRXN1 leads to increased basal and GnRH-stimulated ROS levels. We conclude that SRXN1 is essential for normal responses to GnRH stimulation and plays an important role in ROS management.

Overexpression of the DOF-Type Transcription Factor Enhances Lipid Synthesis in Chlorella vulgaris

In the present study, DNA binding with one finger (DOF)-type transcription factors were screened from the Chlorella vulgaris genome database. The candidate DOF transcription factor was endogenously overexpressed in C. vulgaris to improve neutral lipid production. The protein expression vector contains the heat shock protein 70 and ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit promoters and self-cleaving 2A peptide to reduce the transgene-silencing effect of C. vulgaris. A total of 74 phleomycin-resistant transformants were obtained. Under nitrogen-deficient conditions, the transformant CvDOF#3 showed approximately 1.5-fold higher neutral lipid content per cell compared to the original strain and also showed a His-tagged DOF candidate protein expression of 0.6%. Microscopic observations revealed that CvDOF#3 cells were larger in size. However, the observed differences in average cell diameter between CvDOF#3 and control cells were not statistically significant. These results indicated that the protein expression vector harboring the dual promoters and the 2A peptide, when used in combination with enzymatic cell wall degradation and glass bead transformation, could be useful for transgene and protein expression in C. vulgaris. Further experiment is necessary to confirm the expression efficiency of the HSP70 and RBCS dual promoter and 2A peptide strategy after construction of homologous recombination system in C. vulgaris. Our findings suggested that the overexpression of the endogenous DOF-type transcription factor can be used for improving the lipid content in C. vulgaris.

SIN3B promotes integrin αV subunit gene transcription and cell migration of hepatocellular carcinoma.

Paired amphipathic helix protein (SIN3B) is a transcription corepressor for many genes. Here we show a different regulation mechanism of integrin αV gene expression by SIN3B in human hepatocellular carcinoma (HCC). We first observed a close relationship between Integrin αV and SIN3B expressions in HCC patients and tumor cell lines with different metastatic potentials. Overexpression of SIN3B significantly accelerated the cell migration rate of SMMC-7721, but failed when integrin αV expression was silenced. Interestingly, SIN3B stimulated integrin αV subunit promoter activity only in the presence of sulfatide. Importantly, SIN3B was identified in the complex with sulfatide by mass spectrometry. Fat blot assay indicated that SIN3B specifically interacted with sulfatide. Molecular modeling suggested that sulfatide induced the conformational change of SIN3B from compacted α-helices to a relaxed β-sheet in PAH2 domain. The data of immunoprecipitation and ChIP assay indicated that altered SIN3B lost the binding affinity with MAD1 and HDAC2, which reduced the recruitment of HDAC2 on integrin αV gene promoter and prevented the deacetylation of the histone 3. In conclusion, this study demonstrated that SIN3B promoted the transcriptional activation of the integrin αV subunit gene promoter by reducing interaction with HDAC2.

Potential role for histone deacetylation in chronic diazepam-induced downregulation of α1-GABAA receptor subunit expression.

Corroborating evidence indicate that the downregulation of GABAA receptor subunit expression may underlie tolerance to the anticonvulsant and anxiolytic actions of benzodiazepine (BZ) ligands that act as full allosteric modulators (FAMs) of GABA actions at a variety of GABAA receptor subtypes. We and others have shown that 10‐14 days treatment with increasing doses of diazepam (a FAM) resulted in anticonvulsant tolerance and decreased the expression of the α1 GABAA receptor subunit mRNA and protein in frontal cortex. In addition, we have also shown that long‐term treatment with imidazenil, a partial allosteric modulator of GABA action at selective GABAA receptor subtypes, fail to change the expression of the α1 subunit mRNA or induce tolerance to its anticonvulsant or anxiolytic action. However, little is known regarding the potential role of epigenetic mechanisms on long‐term BZ‐induced downregulation of GABAA receptor subunit. Therefore, we examined the role of histone acetylation and DNA methylation mechanisms on long‐term diazepam‐induced downregulation of the α1 subunit mRNA expression in rat frontal cortex. We found that 10 days treatment with increasing doses of diazepam but not imidazenil decreased the expression of the α1 GABAA receptor subunit mRNA and promoter acetylation in frontal cortex. In addition, we also found that 10 days treatment with diazepam but not imidazenil increased the expression of histone deacetylase (HDAC) 1 and 2 in frontal cortex. Thus, the increased expression of HDAC1 and HDAC2 (class 1 HDACs) and consequently increased histone deacetylation mechanism of this class 1 HDACs, may underlie long‐term diazepam‐induced decreased expression of the α1 GABAA receptor subunit mRNA in frontal cortex.

The effects of sodium butyrate on ethanol-seeking behavior and H3K9 acetylation in NMDA receptor 2B subunit promotor region in the hippocampus of rats

Objective To investigate the effects of sodium butyrate on ethanol-seeking behavior and H3K9 acetylation levels in NMDA receptor 2B subunit(NR2B)promoter region in the hippocampus of Wistar rats. To explore the epigenetic mechanism underlying ethanol-seeking behavior. Methods According to random number table, 48 male Wistar rats were divided into saline group, sodium butyrate group, ethanol group and sodium butyrate + ethanol group, with 12 rats in each group and administered by intraperitioneal injection respectively. Conditioned place preference(CPP)was used to evaluate the ethanol-seeking behavior. Using Western-blot, real-time PCR and chromatin immunoprecipitation assays, the expression of NR2B protein, NR2BmRNA and the relative level acetylated H3K9 in NR2B promoter region in the hippocampus were determined respectively. Results The CPP test and the CPP score in each group were different(P 0.05). Compared with the ethanol group, CPP test of sodium butyrate + ethanol group was significantly higher(P 0.05). Compared with the ethanol group, the expression of NR2B protein, NR2BmRNA and acetylated level H3K9 in NR2B promoter region in the hippocampus of ethanol group, these of sodium butyrate + ethanol group were significantly higher(P<0.05). The CPP score were positively correlated with the expression of NR2B protein(r=0.474, P<0.05). The expression of NR2B protein were positively correlated with the expression of NR2BmRNA(r=0.468, P<0.05). The expression of NR2BmRNA were positively correlated with the expression of H3K9 acetylation in NR2B promoter region(r=0.596, P<0.05), and the CPP score were positively correlated with the expression of H3K9 acetylation in NR2B promoter region (r=0.542, P<0.05). Conclusion The increasing acetylation level of H3K9 in NR2B promoter region in the hippocampus may be one of the epigenetic mechanisms of promoting ethanol-seeking behavior, and H3K9 deacetylation in NR2B promoter region in the hippocampus is likely to be a new target for controlling ethanol dependence. Key words: Alcohol; Sodium butyrate; Histone acetylation; NMDA receptor 2B subunit

BKCa channel activity and vascular contractility alterations with hypertension and aging via β1 subunit promoter methylation in mesenteric arteries

Promoter methylation is a key mechanism in the epigenetic reprogramming of gene expression patterns. Here, we investigated the contribution of DNA methylation and the associated expression and function of large-conductance Ca2+-activated K+ (BKCa) channel in mesenteric arteries when hypertension was superimposed on aging. Male Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) at young (12 weeks), adult (36 weeks) and old (64 weeks) life stages were used. BKCa channel currents, BKCa channel activity in regulating vascular tone, and BKCa channel β1 subunit (BKβ1) function and expression were greater in mesenteric arteries from SHR than from age-matched WKY controls. Consistently, hypertension decreased CpG methylation of the BKβ1 promoter at all ages. Furthermore, aging triggered an increase in BKβ1 promoter methylation in both old WKY and SHR, with concomitant suppression of the β1 subunit and BKCa channel activity. Aging enhanced β1 gene promoter methylation and subunit expression without changing BKCa channel function between young and adult WKY animals. In contrast, aging did not alter CpG methylation but facilitated BKCa channel currents and upregulated BKβ1 expression and function in adult SHR. Taken together, our results provide compelling evidence that hypertension and aging exert opposing effects on DNA methylation at BKβ1 promoter, subsequently resulting in different β1 expression levels and functional modulation of mesenteric arterial contractility. Such information is useful in revealing the epigenetic regulation of BKCa channel function in vascular smooth muscle and in comprehensively understanding the molecular mechanisms in vascular physiology and pathophysiology.

The Rubisco Chaperone BSD2 May Regulate Chloroplast Coverage in Maize Bundle Sheath Cells.

In maize (Zea mays), Bundle Sheath Defective2 (BSD2) plays an essential role in Rubisco biogenesis and is required for correct bundle sheath (BS) cell differentiation. Yet, BSD2 RNA and protein levels are similar in mesophyll (M) and BS chloroplasts, although Rubisco accumulates only in BS chloroplasts. This raises the possibility of additional BSD2 roles in cell development. To test this hypothesis, transgenic lines were created that overexpress and underexpress BSD2 in both BS and M cells, driven by the cell type-specific Rubisco Small Subunit (RBCS) or Phosphoenolpyruvate Carboxylase (PEPC) promoters or the ubiquitin promoter. Genetic crosses showed that each of the transgenes could complement Rubisco deficiency and seedling lethality conferred by the bsd2 mutation. This was unexpected, as RBCS-BSD2 lines lacked BSD2 in M chloroplasts and PEPC-BSD2 lines expressed half the wild-type BSD2 level in BS chloroplasts. We conclude that BSD2 does not play a vital role in M cells and that BS BSD2 is in excess of requirements for Rubisco accumulation. BSD2 levels did affect chloroplast coverage in BS cells. In PEPC-BSD2 lines, chloroplast coverage decreased 30% to 50%, whereas lines with increased BSD2 content exhibited a 25% increase. This suggests that BSD2 has an ancillary role in BS cells related to chloroplast size. Gas exchange showed decreased photosynthetic rates in PEPC-BSD2 lines despite restored Rubisco function, correlating with reduced chloroplast coverage and pointing to CO2 diffusion changes. Conversely, increased chloroplast coverage did not result in increased Rubisco abundance or photosynthetic rates. This suggests another limitation beyond chloroplast volume, most likely Rubisco biogenesis and/or turnover rates.

Expression patterns of the native Shrunken-2 promoter in Sorghum bicolor visualised through use of the GFP reporter gene.

The AGPase large subunit (shrunken-2) promoter was demonstrated to be active in the placentochalaza and endosperm of developing grain as well as the root tips in transgenic sorghum. The temporal and spatial expression patterns of the Sorghum bicolor Shrunken-2 (Sh2) promoter were evaluated using the green fluorescence protein reporter gene (gfp) in transgenic sorghum, within the context of upregulating starch biosynthesis in the developing grain. GFP fluorescence was analysed throughout development in various tissue types using confocal laser scanning microscopy techniques. Sh2 promoter activity was first detected in the placentochalaza region of the developing caryopsis and apoplasm adjacent to the nucellar epidermis at 7days post anthesis (dpa) where fluorescence remained relatively constant until 17dpa. Fluorescence in this region weakened by 20dpa and disappeared by 25dpa. Expression was also detected in the developing endosperm, but not until 12dpa, continuing until 25dpa. Whilst the endosperm expression was expected, the fluorescence detected in the placentochalaza was completely unexpected. Although transcript presence does not mean the resulting biochemistry is also present, these preliminary findings may suggest alternate spatial activity of ADP-glucose pyrophosphorylase prior to uptake by the developing grain. Sh2 promoter activity was also unexpectedly detected in the root tips at all developmental time points. Sh2 promoter activity was not detected in any reproductive floral tissue (both pre and post anthesis) or in pollen. Similarly, no expression was detected in leaf tissue at any stage.

Growth Arrest and DNA-damage-inducible Protein 45β-mediated DNA Demethylation of Voltage-dependent T-type Calcium Channel 3.2 Subunit Enhances Neuropathic Allodynia after Nerve Injury in Rats.

Growth arrest and DNA-damage-inducible protein 45β reactivates methylation-silenced neural plasticity-associated genes through DNA demethylation. However, growth arrest and DNA-damage-inducible protein 45β-dependent demethylation contributes to neuropathic allodynia-associated spinal plasticity remains unclear. Adult male Sprague-Dawley rats (654 out of 659) received a spinal nerve ligation or a sham operation with or without intrathecal application of one of the following: growth arrest and DNA-damage-inducible protein 45β messenger RNA-targeted small interfering RNA, lentiviral vector expressing growth arrest and DNA-damage-inducible protein 45β, Ro 25-6981 (an NR2B-bearing N-methyl-D-aspartate receptor antagonist), or KN-93 (a calmodulin-dependent protein kinase II antagonist) were used for behavioral measurements, Western blotting, immunofluorescence, dot blots, detection of unmodified cytosine enrichment at cytosine-phosphate-guanine site, chromatin immunoprecipitation quantitative polymerase chain reaction analysis, and slice recordings. Nerve ligation-enhanced growth arrest and DNA-damage-inducible protein 45β expression (n = 6) in ipsilateral dorsal horn neurons accompanied with behavioral allodynia (n = 7). Focal knockdown of growth arrest and DNA-damage-inducible protein 45β expression attenuated ligation-induced allodynia (n = 7) by reducing the binding of growth arrest and DNA-damage-inducible protein 45β to the voltage-dependent T-type calcium channel 3.2 subunit promoter (n = 6) that decreased expression of and current mediated by the voltage-dependent T-type calcium channel 3.2 subunit (both n = 6). In addition, NR2B-bearing N-methyl-D-aspartate receptors and calmodulin-dependent protein kinase II act in an upstream cascade to increase growth arrest and DNA-damage-inducible protein 45β expression, hence enhancing demethylation at the voltage-dependent T-type calcium channel 3.2 subunit promoter and up-regulating voltage-dependent T-type calcium channel 3.2 subunit expression. Intrathecal administration of Ro 25-6981, KN-93, or a growth arrest and DNA-damage-inducible protein 45β-targeting small interfering RNA (n = 6) reversed the ligation-induced enrichment of unmodified cytosine at the voltage-dependent T-type calcium channel 3.2 subunit promoter by increasing the associated 5-formylcytosine and 5-carboxylcytosine levels. By converting 5-formylcytosine or 5-carboxylcytosine to unmodified cytosine, the NR2B-bearing N-methyl-D-aspartate receptor, calmodulin-dependent protein kinase II, or growth arrest and DNA-damage-inducible protein 45β pathway facilitates voltage-dependent T-type calcium channel 3.2 subunit gene demethylation to mediate neuropathic allodynia.

Interaction between kisspeptin and adenylate cyclase-activating polypeptide 1 on the expression of pituitary gonadotropin subunits: a study using mouse pituitary lbetaT2 cells.

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.

Notch-Mediated Epigenetic Regulation of Voltage-Gated Potassium Currents.

Ventricular arrhythmias often arise from the Purkinje-myocyte junction and are a leading cause of sudden cardiac death. Notch activation reprograms cardiac myocytes to an induced Purkinje-like state characterized by prolonged action potential duration and expression of Purkinje-enriched genes. To understand the mechanism by which canonical Notch signaling causes action potential prolongation. We find that endogenous Purkinje cells have reduced peak K+ current, Ito, and IK,slow when compared with ventricular myocytes. Consistent with partial reprogramming toward a Purkinje-like phenotype, Notch activation decreases peak outward K+ current density, as well as the outward K+ current components Ito,f and IK,slow. Gene expression studies in Notch-activated ventricles demonstrate upregulation of Purkinje-enriched genes Contactin-2 and Scn5a and downregulation of K+ channel subunit genes that contribute to Ito,f and IK,slow. In contrast, inactivation of Notch signaling results in increased cell size commensurate with increased K+ current amplitudes and mimics physiological hypertrophy. Notch-induced changes in K+ current density are regulated at least in part via transcriptional changes. Chromatin immunoprecipitation demonstrates dynamic RBP-J (recombination signal binding protein for immunoglobulin kappa J region) binding and loss of active histone marks on K+ channel subunit promoters with Notch activation, and similar transcriptional and epigenetic changes occur in a heart failure model. Interestingly, there is a differential response in Notch target gene expression and cellular electrophysiology in left versus right ventricular cardiac myocytes. In summary, these findings demonstrate a novel mechanism for regulation of voltage-gated potassium currents in the setting of cardiac pathology and may provide a novel target for arrhythmia drug design.

Homologous promoter derived constitutive and chloroplast targeted expression of synthetic cry1Ac in transgenic chickpea confers resistance against Helicoverpa armigera

The insecticidal crystal protein derived from gram positive soil bacterium Bacillus thuringiensis plays an important role in controlling lepidopteran infestation. The present study seeks to protect chickpea plants from Helicoverpa armigera infestation by over expressing cry1Ac. Homologous Ubiquitin and RuBisCO small subunit (rbcS) promoters were used to transcribe cry1Ac in transgenic chickpea both constitutively and in a tissue specific manner through Agrobacterium mediated transformation of chickpea var. ICCV89314. Expressed Cry1Ac was specifically targeted to the chloroplast rich tissues using transit peptide sequence. After monitoring transgene integration by Southern hybridization, transgenic chickpea lines were further analyzed by western blot, ELISA and insect bioassay. Expression of cry1Ac in chickpea under the control of above two promoters conferred a high level of protection against pod borer infestation, where chloroplast targeting system was found to be more efficient in controlling this particular devastating lepidopteran pest.

Metabolic engineering of Dunaliella salina for production of ketocarotenoids.

Dunaliella is a commercially important marine alga producing high amount of β-carotene. The use of Dunaliella as a potential transgenic system for the production of recombinant proteins has been recently recognized. The present study reports for the first time the metabolic engineering of carotenoid biosynthesis in Dunaliella salina for ketocarotenoid production. The pathway modification included the introduction of a bkt gene from H. pluvialis encoding β-carotene ketolase (4,4'β-oxygenase) along with chloroplast targeting for the production of ketocarotenoids. The bkt under the control of Dunaliella Rubisco smaller subunit promoter along with its transit peptide sequence was introduced into the alga through standardized Agrobacterium-mediated transformation procedure. The selected transformants were confirmed using GFP and GUS expression, PCR and southern blot analysis. A notable upregulation of the endogenous hydroxylase level of transformants was observed where the BKT expression was higher in nutrient-limiting conditions. Carotenoid analysis of the transformants through HPLC and MS analysis showed the presence of astaxanthin and canthaxanthin with maximum content of 3.5 and 1.9µg/g DW, respectively. The present study reports the feasibility of using D. salina for the production of ketocarotenoids including astaxanthin.

Selective transgene expression in cerebellar Purkinje cells and granule cells using adeno-associated viruses together with specific promoters

Adeno-associated virus (AAV) is a powerful tool for gene delivery into the brain and has been used for transgene expression in the cerebellar cortex. Although the efficacies of different AAV serotypes to transduce cerebellar Purkinje cells were examined, it has been difficult to achieve cell-type specific transgene expression. Here we used AAV serotype 1 with two specific promoters, namely, Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) and the minimum region of the GABAA receptor α6 subunit (GABRα6) promoters, and compared their expression patterns in the cerebellar cortex with the expression patterns of ubiquitous promoters that are often used for AAV-mediated expression. Whereas AAV with ubiquitous promoters, the cytomegalovirus early enhancer/chicken β-actin promoter, and a small fragment of the synapsin-1 gene promoter caused ubiquitous expression in all cerebellar neurons tested, AAV with the CaMKIIα promoter injected into 10-day-old mice enabled selective expression in Purkinje cells. Furthermore, we developed AAV with the GABRα6 promoter, and succeeded for the first time to express the transgene exclusively in granule cells. Fresh cerebellar slices of mice injected with these AAVs were applicable for physiological experiments, such as patch clamp recording, optogenetic imaging, and stimulation. Thus, these AAV vectors are useful tools towards understanding the basic properties of cerebellar neurons or mechanisms of cerebellar functions. Particularly, selective expression in Purkinje or granule cells is useful for analyses using genetically-modified animals, such as knockout mice.