General Control Non-derepressible 5 Research Articles

Gibberellin Signaling through RGA Suppresses GCN5 Effects on Arabidopsis Developmental Stages.

Histone acetyltransferases (HATs) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar, and floral development, and plant environmental response. The flowers of gcn5 plants display a reduced stamen length and exhibit male sterility relative to the wild-type plants. We show that these effects may arise from gibberellin (GA)-signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The repressor GA (RGA) DELLA protein represses plant growth, inflorescence, and flower and seed development. Our molecular data indicate that GCN5 is required for the activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of the RGA and GCN5; the RGA can partially suppress GCN5 action during the whole plant life cycle. The reduced elongation of the stamen filament of gcn5-6 mutants is reversed in the rga-t2;gcn5-6 double mutants. RGAs suppress the GCN5 effect on the gene expression and histone acetylation of GA catabolism and GA signaling. Interestingly, the RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream of GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is partially mediated by RGA and GA signaling.

Talazoparib enhances the quinacrine-mediated apoptosis in patient-derived oral mucosa CSCs by inhibiting BER pathway through the modulation of GCN5 and P300.

The presence of cancer stem cells (CSCs) in the tumor microenvironment (TME) is majorly responsible for the development and recurrence of cancer. Earlier reports suggested that upon DNA damage, poly-(ADP-ribose) polymerase-1 (PARP-1) helps in chromatin modulation and DNA repair process, thereby promoting CSC survival. But whether a combination of DNA damaging agents along with PARP inhibitors can modulate chromatin assembly, inhibit DNA repair processes, and subsequently target CSCs is not known. Hence, we have investigated the effect of nontoxic bioactive compound quinacrine (QC) and a potent PARP inhibitor Talazoparib in patient-derived oral mucosa CSCs (OM-CSCs) and in vivo xenograft mice preclinical model systems. Data showed that QC + Talazoparib inhibited the PARP-1-mediated chromatin remodelers' recruitment and deregulated HAT activity of GCN5 (general control nonderepressible-5) and P300 at DNA damage site, thereby preventing the access of repair proteins to the damaged DNA. Additionally, this combination treatment inhibited topoisomerase activity, induced topological stress, and induced apoptosis in OM-CSCs. Similar results were observed in an in vivo xenograft mice model system. Collectively, the data suggested that QC + Talazoparib treatment inhibited BER pathway, induced genomic instability and triggered apoptosis in OM-CSCs through the deregulation of PARP-1-mediated chromatin remodelers (GCN5 and P300) activity. Schematic representation of QC + Talazoparib-induced apoptosis in oral mucosa CSCs. (1) Induction of DNA damage takes place after QC treatment (2) PARP1-mediated PARylation at the site of DNA damage, which recruits multiple chromatin remodelers (3) Acetylation at the histone tails relax the structure of chromatin and recruits the BER pathway proteins at the site of DNA damage. (4) BER pathway activated at the site of DNA damage. (5) CSCs survive after successful repair of DNA damage. (6) Treatment of QC-treated CSCs with PARP inhibitor Talazoparib (7) Inhibition of PARylation results in failure of chromatin remodelers to interact with PARP1. (8) Inhibition of acetylation status leads to chromatin compaction. (9) BER pathway proteins are not recruited at the site of DNA damage, resulting in inhibition of BER pathway and accumulation of unrepaired DNA damage, leading to apoptosis and cell death.

Quantitative Acetylomics Reveals Substrates of Lysine Acetyltransferase GCN5 in Adult and Aging Drosophila.

Protein lysine acetylation is a dynamic post-translational modification (PTM) that regulates a wide spectrum of cellular events including aging. General control nonderepressible 5 (GCN5) is a highly conserved lysine acetyltransferase (KAT). However, the acetylation substrates of GCN5 in vivo remain poorly studied, and moreover, how lysine acetylation changes with age and the contribution of KATs to aging remain to be addressed. Here, using Drosophila, we perform label-free quantitative acetylomic analysis, identifying new substrates of GCN5 in the adult and aging process. We further characterize the dynamics of protein acetylation with age, which exhibits a trend of increase. Since the expression of endogenous fly Gcn5 progressively increases during aging, we reason that, by combining the substrate analysis, the increase in acetylation with age is triggered, at least in part, by GCN5. Collectively, our study substantially expands the atlas of GCN5 substrates in vivo, provides a resource of protein acetylation that naturally occurs with age, and demonstrates how individual KAT contributes to the aging acetylome.

Heat shock factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat shock response

Despite its pivotal roles in biology, how the transcriptional activity of c-MYC is tuned quantitatively remains poorly defined. Here, we show that heat shock factor 1 (HSF1), the master transcriptional regulator of the heat shock response, acts as a prime modifier of the c-MYC-mediated transcription. HSF1 deficiency diminishes c-MYC DNA binding and dampens its transcriptional activity genome wide. Mechanistically, c-MYC, MAX, and HSF1 assemble into a transcription factor complex on genomic DNAs, and surprisingly, the DNA binding of HSF1 is dispensable. Instead, HSF1 physically recruits the histone acetyltransferase general control nonderepressible 5 (GCN5), promoting histone acetylation and augmenting c-MYC transcriptional activity. Thus, we find that HSF1 specifically potentiates the c-MYC-mediated transcription, discrete from its canonical role in countering proteotoxic stress. Importantly, this mechanism of action engenders two distinct c-MYC activation states, primary and advanced, which may be important to accommodate diverse physiological and pathological conditions.

Histone Acetyltransferase GCN5 Affects Auxin Transport during Root Growth by Modulating Histone Acetylation and Gene Expression of PINs.

General Control Non-Derepressible 5 (GCN5) is a histone acetyltransferase that targets multiple genes and is essential for the acetylation of Lysine residues in the N-terminal tail of histone H3 in Arabidopsis. GCN5 interacts with the transcriptional coactivator Alteration/Deficiency in Activation 2b (ADA2b), which enhances its activity functioning in multiprotein complexes, such as the Spt-Ada-Gcn5-Acetyltransferase complex (SAGA). Mutations in GCN5 and ADA2b result in pleiotropic phenotypes, including alterations in the growth of roots. Auxin is known to regulate root development by modulating gene expression patterns. Auxin moves polarly during plant growth via the Pin-formed (PIN) auxin efflux transport proteins. The effect of GCN5 and ADA2b on auxin distribution at different stages of early root growth (4 to 7 days post-germination) was studied using the reporter lines DR5rev::GFP and PIN1::PIN1-GFP. In wild-type plants, auxin efflux transporter PIN1 expression increases from the fourth to the seventh day of root growth. The PIN1 expression was reduced in the roots of gcn5-1 and ada2b-1 compared to the wild type. The expression of PIN1 in ada2b-1 mutants is confined only to the meristematic zone, specifically in the stele cells, whereas it is almost abolished in the elongation zone. Gene expression analysis showed that genes associated with auxin transport, PIN1, PIN3 and PIN4, are downregulated in gcn5-1 and ada2b-1 mutants relative to the wild type. As a result, auxin accumulation was also reduced in gcn5-1 and ada2b-1 compared to wild-type roots. Furthermore, acetylation of Lysine 14 of histone H3 (H3K14) was also affected in the promoter and coding region of PIN1, PIN3 and PIN4 genes during root growth of Arabidopsis in gcn5 mutants. In conclusion, GCN5 acts as a positive regulator of auxin distribution in early root growth by modulating histone H3 acetylation and the expression of auxin efflux transport genes.

GCN5 participates in KLF4-VEGFA feedback to promote endometrial angiogenesis

SummaryEndometrial angiogenesis is necessary for good endometrial receptivity. Krüppel-like factor 4 (KLF4) is a transcription factor that is essential for regulating angiogenesis. Here we found that vascular endothelial growth factor A (VEGFA) can form a positive feedback loop with KLF4 to promote the proliferation and migration of human endometrial microvascular endothelial cells (HEMECs) and inhibit cell apoptosis. General control non-derepressible 5 (GCN5) is also time-dependent on VEGFA and participates in the KLF4-VEGFA loop. In addition, we found that GCN5 is a succinyltransferase that modulates the succinylation of histones and nonhistones. GCN5 interacts with KLF4 and is recruited to the KLF4-binding site of the VEGFA promoter to succinylate H3K79, which initiates gene transcription epigenetically. For nonhistones, GCN5 succinylates KLF4 that is activated by ERK signaling in HEMECs treated with VEGFA to increase its transcription activity. These results demonstrate KLF4-VEGFA positive feedback loop is regulated by epigenetics, which contributes to endometrial angiogenesis.

SMAD4 transcriptionally activates GCN5 to inhibit apoptosis and promote osteogenic differentiation in dexamethasone-induced human bone marrow mesenchymal stem cells

BackgroundSteroid-induced osteonecrosis of the femoral head (SONFH) is a serious complication caused by long-term or excessive use of glucocorticoids (GCs). General control non-derepressible 5 (GCN5) has been reported to be lowly expressed in bone tissue. Therefore, this paper attempts to investigate the role of GCN5 in SONFH and identify the potential regulatory mechanism. Experimental designFollowing human bone mesenchymal stem cells (hBMSCs) being stimulated with dexamethasone (Dex), GCN5 expression was detected using RT-qPCR and western blotting. Then, GCN5 was overexpressed and cell viability was assessed by cell counting kit and lactate dehydrogenase kit. Cell apoptosis was determined with terminal deoxynucleotidyl transferase dUTPnickendlabeling (TUNEL) and the expression of apoptosis-related proteins was evaluated using western blotting. Alkaline phosphatase (ALP) staining and alizarin red staining were adopted for the analysis of osteogenic differentiation. Additionally, the relationship between small mothers against decapentaplegic protein 4 (SMAD4) and GCN5 was predicted by hTFtarget website and verified by luciferase reporter- and chromatin immunoprecipitation (ChIP) assays. Subsequently, SMAD4 was silenced to determine cell viability, apoptosis and osteogenic differentiation in Dex-induced hBMSCs with GCN5 upregulation. ResultsGCN5 expressed lower in hBMSCs exposed to Dex. GCN5 overexpression elevated cell viability, attenuated apoptosis and promoted osteogenic differentiation of hBMSCs. Additionally, SMAD4 transcriptionally activated GCN5 and upregulated GCN5 expression. While SMAD4 knockdown reversed the protective effects of GCN5 overexpression on Dex-induced cell viability loss, apoptosis increase and osteogenic differentiation inhibition in hBMSCs. ConclusionsSMAD4 transcriptionally activated GCN5 to inhibit apoptosis and promote osteogenic differentiation in Dex-induced hBMSCs.

Acetylation of the influenza A virus polymerase subunit PA in the N-terminal domain positively regulates its endonuclease activity.

The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 invitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity invitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.

Sublytic C5b-9 triggers glomerular mesangial cell proliferation via enhancing FGF1 and PDGFα gene transcription mediated by GCN5-dependent SOX9 acetylation in rat Thy-1 nephritis.

Rat Thy-1 nephritis (Thy-1N) is an animal model of human mesangioproliferative glomerulonephritis (MsPGN), accompanied by glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) deposition. Although sublytic C5b-9 formed on GMC membrane could induce cell proliferation, the mechanism is still unclear. In this study, we first demonstrated that the level of SRY related HMG-BOX gene 9 (SOX9), general control nonderepressible 5 (GCN5), fibroblast growth factor 1 (FGF1) and platelet-derived growth factor α (PDGFα) was all elevated both in the renal tissues of Thy-1N rats (in vivo) and in the GMCs (in vitro) with sublytic C5b-9 stimulation. Then, we not only discovered that sublytic C5b-9 caused GMC proliferation through increasing SOX9, GCN5, FGF1 and PDGFα expression, but also proved that SOX9 and GCN5 formed a complex and combined with FGF1 and PDGFα promoters, leading to FGF1 and PDGFα gene transcription. More importantly, GCN5 could mediate SOX9 acetylation at lysine 62 (K62) to enhance SOX9 binding to FGF1 or PDGFα promoter and promote FGF1 or PDGFα synthesis and GMC proliferation. Besides, the experiments in vivo also showed that FGF1 and PDGFα expression, GMC proliferation and urinary protein secretion in Thy-1N rats were greatly reduced by silencing renal SOX9, GCN5, FGF1 or PDGFα gene. Furthermore, the renal tissues of MsPGN patients also exhibited positive expression of these genes mentioned above. Collectively, our findings indicate that GCN5, SOX9 and FGF1/PDGFα can form an axis and play an essential role in sublytic C5b-9-triggered GMC proliferation, which might provide a novel insight into the pathogenesis of Thy-1N and MsPGN.

The Transcriptional Adaptor Protein ADA3a Modulates Flowering of Arabidopsis thaliana.

Histone acetylation is directly related to gene expression. In yeast, the acetyltransferase general control nonderepressible-5 (GCN5) targets histone H3 and associates with transcriptional co-activators alteration/deficiency in activation-2 (ADA2) and alteration/deficiency in activation-3 (ADA3) in complexes like SAGA. Arabidopsis thaliana has two genes encoding proteins, designated ADA3a and ADA3b, that correspond to yeast ADA3. We investigated the role of ADA3a and ADA3b in regulating gene expression during flowering time. Specifically, we found that knock out mutants ada3a-2 and the double mutant ada3a-2 ada3b-2 lead to early flowering compared to the wild type plants under long day (LD) conditions and after moving plants from short days to LD. Consistent with ADA3a being a repressor of floral initiation, FLOWERING LOCUS T (FT) expression was increased in ada3a mutants. In contrast, other genes involved in multiple pathways leading to floral transition, including FT repressors, players in GA signaling, and members of the SPL transcriptional factors, displayed reduced expression. Chromatin immunoprecipitation analysis revealed that ADA3a affects the histone H3K14 acetylation levels in SPL3, SPL5, RGA, GAI, and SMZ loci. In conclusion, ADA3a is involved in floral induction through a GCN5-containing complex that acetylates histone H3 in the chromatin of flowering related genes.

In-vitro acetylation of SARS-CoV and SARS-CoV-2 nucleocapsid proteins by human PCAF and GCN5

Recently, the novel coronavirus (SARS-CoV-2), which has spread from China to the world, was declared a global public health emergency, which causes lethal respiratory infections. Acetylation of several proteins plays essential roles in various biological processes, such as viral infections. We reported that the nucleoproteins of influenza virus and Zaire Ebolavirus were acetylated, suggesting that these modifications contributed to the molecular events involved in viral replication. Similar to influenza virus and Ebolavirus, the coronavirus also contains single-stranded RNA, as its viral genome interacts with the nucleocapsid (N) proteins. In this study, we report that SARS-CoV and SARS-CoV-2 N proteins are strongly acetylated by human histone acetyltransferases, P300/CBP-associated factor (PCAF), and general control nonderepressible 5 (GCN5), but not by CREB-binding protein (CBP) in vitro. Liquid chromatography-mass spectrometry analyses identified 2 and 12 acetyl-lysine residues from SARS-CoV and SARS-CoV-2 N proteins, respectively. Particularly in the SARS-CoV-2 N proteins, the acetyl-lysine residues were localized in or close to several functional sites, such as the RNA interaction domains and the M-protein interacting site. These results suggest that acetylation of SARS-CoV-2 N proteins plays crucial roles in their functions.

Elevated GCN5 expression confers tamoxifen resistance by upregulating AIB1 expression in ER-positive breast cancer.

Approximately 70% of breast cancers are estrogen receptor (ER)-positive and treated with endocrine therapy. A commonly used treatment agent, tamoxifen, shows high efficacy for improving prognosis. However, approximately one-third of patients treated with tamoxifen develop resistance to this drug. Here, we investigated the function of general control non-derepressible 5 (GCN5) and its downstream effectors in tamoxifen-resistant (TamR) breast cancer. TamR-MCF7 breast cancer cells maintained high GCN5 levels due to its attenuated proteasomal degradation. GCN5 overexpression upregulated amplified in breast cancer 1 (AIB1) expression, resulting in decreased p53 stability and tamoxifen resistance. Conversely, the sensitivity of GCN5-AIB1-overexpressing MCF7 cells to tamoxifen was restored by forced p53 expression. An in vivo study demonstrated a positive correlation between GCN5 and AIB1 and their contribution to tamoxifen resistance. We concluded that GCN5 promotes AIB1 expression and tamoxifen resistance in breast cancer by reducing p53 levels, suggesting the utility of GCN5 and its downstream effectors as therapeutic targets to either prevent or overcome tamoxifen resistance in breast cancer.

A Comprehensive Online Database for Exploring ∼20,000 Public Arabidopsis RNA-Seq Libraries

A Comprehensive Online Database for Exploring ∼20,000 Public Arabidopsis RNA-Seq Libraries

Retracted: Ras-PI3K-AKT signaling promotes the occurrence and development of uveal melanoma by downregulating H3K56ac expression.

Uveal melanoma (UM) is an intraocular malignant tumor characterized by rapid progression and recurrence. The current conventional treatments are unsatisfactory. Histone acetylation at H3 lysine 56 (H3K56ac) has been reported to be a tumor suppressor in breast cancer. However, whether H3K56ac prevents the occurrence and development of UM remains uninvestigated. The study aimed to explore the regulatory effect of H3K56ac on Ras-PI3K-AKT induced UM cells proliferation and migration. The vectors of pEGFP-RasWT , pEGFP-K-Ras G12V/Y40C , and pEGFP-N1 were transfected into MP46 cells, and protein levels of phosphorylated AKT Ser473 and H3K56ac were examined using western blot analysis. The effect of H3K56ac on cell proliferation and migration were studied using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, colony formation, and Transwell assays. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and chromatin immunoprecipitation assays were performed to determine the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) downstream genes. Further, the regulatory effects of silent mating type information regulation 2 homolog-1 (SIRT1), general control nonderepressible 5 (GCN5), and mouse double minute 2 homolog (MDM2) on Ras-PI3K-AKT affected H3K56ac expression were also investigated. H3K56ac expression was specifically downregulated by Ras-PI3K-AKT activation pathway. H3K56ac inhibited the tumorigenic effect of Ras-PI3K-AKT on MP46 cells viability, colony formation, and migration, as well as participated in regulating the transcription of PI3K/AKT downstream genes. SIRT1 silence recovered H3K56ac expression, and reversed the tumorigenic effect of Ras-PI3K-AKT activation on MP46 cells. Downregulation of H3K56ac induced by Ras-PI3K-AKT activation was found to be associated with MDM2-mediated the degradation of GCN5. The results demonstrated that Ras-PI3K-AKT signaling promoted UM cells proliferation and migration via downregulation of H3K56ac expression, which might be related to MDM2-mediated the degradation of GCN5.

ALT cancer cells are specifically sensitive to lysine acetyl transferase inhibition.

Some cancer cells elongate their telomeres through the ALT (alternative lengthening of telomeres) pathway, which is based on homologous recombination for the addition of telomere repeats without telomerase activity. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF), two homologous lysine acetyltransferases, exert opposite effects on the ALT pathway, inhibiting or favoring it respectively. Here we show that ALT cells are particularly sensitive to the inhibition of acetyltransferases activities using Anacardic Acid (AA). AA treatment recapitulates the effect of PCAF knockdown on several ALT features, suggesting that AA decreased the ALT mechanism through the inhibition of lysine transferase activity of PCAF, but not that of GCN5. Furthermore, AA specifically sensitizes human ALT cells to radiation as compared to telomerase-positive cells suggesting that the inhibition of lysine acetyltransferases activity may be used to increase the radiotherapy efficiency against ALT cancers.

GCN5 inhibition prevents IL-6-induced prostate cancer metastases through PI3K/PTEN/Akt signaling by inactivating Egr-1.

General control non-derepressible 5 (GCN5) is ectopically expressed in different types of human cancer and association with the carcinogenesis, development, and poor prognosis of cancers. The present study was aimed to investigate the potential role and related mechanisms of GCN5 in IL-6–treated prostate cancer (PCa) cell. The results showed that an elevated GCN5 expression was stimulated by IL-6. Knockdown of GCN5 significantly inhibited IL-6–driven proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Moreover, early growth response-1 (Egr-1) expression was elevated by IL-6 treatment and GCN5 siRNA down-regulated the expression of Egr-1. Furthermore, overexpression of Egr-1 attenuated the effects of GCN5 silence on cell proliferation, migration, invasion, and EMT in PCa. Besides, knockdown of GCN5 resulted in the down-regulation of p-Akt and up-regulation of PTEN, which was partly impeded by Egr-1 overexpression. The effects of GCN5 overexpression on cell proliferation and invasion were suppressed by LY294002, In conclusion, these data demonstrated the negative effect of up-regulated GCN5 in IL-6-induced metastasis and EMT in PCa cells through PI3K/PTEN/Akt signaling pathway down-regulating Egr-1 expression.

Modulating PCAF/GCN5 Immune Cell Function through a PROTAC Approach.

P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain. Consistent with reported roles for these proteins in immune function, we find that PCAF-deficient macrophages exhibit a markedly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). Investigating the potential to target this pathway pharmacologically, we show that chemical inhibition of the PCAF/GCN5 bromodomains is insufficient to recapitulate the diminished inflammatory response of PCAF-deficient immune cells. However, by generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), we identify small molecules able to degrade PCAF/GCN5 and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data illustrate the power of the PROTAC approach in the context of multidomain proteins, revealing a novel anti-inflammatory therapeutic opportunity for targeting PCAF/GCN5.

Synergistic action of GCN5 and CLAVATA1 in the regulation of gynoecium development in Arabidopsis thaliana.

In Arabidopsis thaliana the CLAVATA1 (CLV1) receptor and GENERAL CONTROL NON DEREPRESSIBLE 5 (GCN5) histone acetyltransferase both regulate inflorescence meristem size and affect the expression of the meristem-promoting transcription factor WUSCHEL (WUS). Single and multiple mutants of GCN5 and CLAVATA members, were analysed for their gynoecium development, using morphological, physiological, genetic and molecular approaches. The clv1-1gcn5-1 double mutants exhibited novel phenotypes including elongated gynoecia with reduced valves and enlarged stigma and style, indicating a synergistic action of CLAVATA signaling and GCN5 action in the development of the gynoecium. Reporter line and gene expression analysis showed that clv1-1gcn5-1 plants have altered auxin and cytokinin response, distribution and ectopic overexpression of WUS. WUS expression was found in the style of wild-type gynoecia stage 10-13, suggesting a possible novel role for WUS in the development of the style. CLV1 and GCN5 are regulators of apical-basal and mediolateral polarity of the Arabidopsis gynoecium. They affect gynoecium morphogenesis through the negative regulation of auxin biosynthesis and promotion of polar auxin transport. They also promote cytokinin signaling in the carpel margin meristem and negatively regulate it at the stigma. Finally, they synergistically suppress WUS at the centre of the gynoecium.

Universal mRNA Translation Enhancement with Gold Nanoparticles Conjugated to Oligonucleotides with a Poly(T) Sequence.

DNA-conjugated gold nanoparticles (AuNPs) have been shown to enhance the translation of mRNA. However, the specific sequence on the DNA dictates the specific mRNA to be enhanced. This study describes poly(thymine)-functionalized AuNPs (AuNP-p(T)DNA) capable of enhancing the translation of any mRNA template that is incorporated into pcDNA6 vector with bovine growth hormone (BGH) polyadenylation signal (P(A)). We demonstrated this by incorporating four genes: green fluorescence protein (GFP), general control nonderepressible 5 (GCN5), cAMP-responsive element binding protein 1 (CREB1), and X-box-binding protein 1-spliced (XBP-1S) separately into pcDNA6 vector with BGH P(A) before their expression in HeLa lysate. The addition of AuNP-p(T)DNA to HeLa lysate containing GFP, GCN5, CREB1, and XBP-1S mRNA increased protein synthesis 1.80, 1.99, 1.95, and 2.20 times, respectively. Similar translation enhancement was also observed in a multiplex reaction containing the mRNA of three genes together in the lysate. Complementary p(T)DNA hybridization to the poly(A) tail of the mRNA was critical as the removal of either p(T)DNA or BGH P(A) in XBP-1S mRNA or the replacement of p(T)DNA with p(A)DNA reduced the translation back to baseline level. Finally, an optimum length of 25 nucleotides for the DNA oligomer and a AuNP-p(T)DNA:mRNA ratio of 0.658 achieved a 3.08-fold translation enhancement. The AuNP-p(T)DNA nanoconstruct could be incorporated into commercial cell-free protein synthesis kits as a universal translation enhancer.

Che1/AATF interacts with subunits of the histone acetyltransferase core module of SAGA complexes.

General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA- and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes.