CCAAT Box Research Articles

Identification and characterization of PsDREB2 promoter involved in tissue-specific expression and abiotic stress response from Paeonia suffruticosa.

Dehydration-responsive element-binding factor 2 (DREB2) belongs to the C-repeat-binding factor (CBF)/DREB subfamily of proteins. In this study, a 2,245 bp PsDREB2 promoter fragment was isolated from the genome of Paeonia suffruticosa. The fragment was rich in A/T bases and contained TATA box sequences, abscisic acid (ABA)-response elements, and other cis-elements, such as MYB and CAAT box. The promoter was fused with the β-glucuronidase (GUS) reporter gene to generate an expression vector. Arabidopsis thaliana was transformed with a flower dipping method. Gus activity in different tissues and organs of transgenic plants was determined via histochemical staining and quantified via GUS fluorescence. The activity of promoter regulatory elements in transgenic plants under drought, low-temperature, high-salt, and ABA stresses was analyzed. The results showed that the PsDREB2 gene promoter was expressed in the roots, stems, leaves, flowers, and silique pods but not in the seeds of transgenic Arabidopsis. Furthermore, the promoter was induced by drought, low temperature, high salt, and ABA. Hence, the PsDREB2 promoter is tissue- and stress-specific and can be used in the genetic engineering of novel peony cultivars in the future.

Cloning and functional characterization of epidermis-specific promoter MtML1 from Medicago truncatula

Epidermis-specific promoters are necessary for ectopic expression of specific functional genes such as the cuticle-related genes. Previous studies indicated that both ECERIFERUM 6 (AtCER6) and MERISTEM L1 LAYER (ATML1) promoters from Arabidopsis thaliana can drive gene expression specifically in the epidermis of shoot apical meristems (SAMs) and leaves. However, the epidermis-specific promoters from legume plants have not been reported. Here, we cloned a 5′ flanking sequence from the upstream -2150 bp to the translational start ATG codon of MtML1 gene of legume model plant Medicago truncatula. PlantCARE analysis indicated that this sequence matches the characteristics of a promoter, having TATA box and CAAT box, as well as contains some conserved elements of epidermis-specific promoters like AtCER6 and ATML1 promoters. The β-glucuronidase (GUS) histochemical analysis showed that MtML1 promoter can drive GUS gene expression in transiently transformed Nicotiana tabacum leaves under non-inducing condition. Furthermore, it can also control GUS expression in leaves and siliques rather than roots of the stably transformed Arabidopsis. More importantly, the leaf cross-section observations indicated that MtML1 exclusively expressed in the epidermis of leaves. These results suggested that MtML1 promoter performed the epidermis-specific in plant shoot. Our study establishes the foundation for driving the cuticle-related gene to express in epidermis, which may be very useful in genetic engineering of legume plants.

Epigenetic role of the nuclear factor NF-Y on ID gene family in endometrial tissues of women with endometriosis: a case control study

BackgroundA predominant difference between endometrial and normal cells is higher proliferation rate in the former cells which is benign. The genes of inhibitor of differentiation (ID) family play a major role in cell proliferation regulation which might be targeted by the nuclear transcription factor Y (NF-Y) for subsequent epigenetic modifications through the CCAAT box regulatory region. The present study was designed to investigate the epigenetic role of NF-Y on ID gene family in endometrial tissue of patients with endometriosis.Materials & methodsIn this case-control study, 20 patients with endometriosis and 20 normal women were examined for the relative expression of the NF-YA, NF-YB, NF-YC and ID genes by real-time PCR during the proliferative phase. The occupancy of NF-Y on CCAAT box region of ID genes was investigated using chromatin immunoprecipitation (ChIP) followed by real-time PCR.ResultsThe NF-YA was over-expressed in eutopic endometrium during the proliferative phase. Although the expression level of NF-YB and NF-YC were unchanged in eutopic samples, they were remarkably higher in ectopic group (P<0.05). The ID2 and ID3 genes were up-regulated in ectopic and eutopic tissues, however ID1 and ID4 genes were down-regulated in these samples (P<0.05). The ChIP analysis revealed significant enrichment of NF-Y on regulatory regions of ID2,3 genes in eutopic group, but reduced binding level of NF-Y to the ID1,3 promoters in ectopic specimens (P<0.05).ConclusionThe ability of NF-Y to regulate ID genes via CCAAT box region suggests the possible role of NF-Y transcription factor in epigenetic changes in endometrial tissues which may open novel avenues in finding new therapeutic strategies.

The Regulatory Region of Muscle-Specific Alpha Actin 1 Drives Fluorescent Protein Expression in Olive Flounder Paralichthys olivaceus.

To develop a promoter capable of driving transgene expression in non-model fish, we identified and characterized the muscle-specific alpha-actin gene in olive flounder, Paralichthys olivaceus (PoACTC1). The regulatory region of PoACTC1 includes putative regulatory elements such as a TATA box, two MyoD binding sites, three CArG boxes, and a CCAAT box. Microinjection experiments demonstrated that the regulatory region of PoACTC1, covering from -2,126 bp to +751 bp, just prior to the start codon, drove the expression of red fluorescent protein in developing zebrafish embryos and hatching olive flounder. These results suggest that the regulatory region of PoACTC1 may be useful in developing a promoter for biotechnological applications such as transgene expression in olive flounder.

Coordinated Transcriptional Regulation of Cytochrome P450 3As by Nuclear Transcription Factor Y and Specificity Protein 1.

The cytochrome P450 3A subfamily plays vital roles in the metabolism of endogenous chemicals and xenobiotics. Understanding the basal expression of CYP3A in humans and pigs is crucial for drug evaluation. In this study, we demonstrated that the basal transcriptional regulation of CYP3A genes in hepatocytes is evolutionarily conserved between humans and pigs. The basal expression of CYP3A genes is transactivated by two cis-acting elements, the CCAAT and GC boxes, located a constant distance apart in the proximal promoter region of six CYP3A genes. Mutation analysis of these two cis-acting elements suggested that they play important roles in mediating basal expression, but to different extents because of the nucleotide variations in the elements. Two transcription factors, nuclear transcription factor Y (NF-Y) and specificity protein 1 (Sp1), directly bind to these cis-acting elements in CYP3A proximal promoters in HepG2 cells and porcine hepatocytes. Furthermore, changing the distance between the NF-Y and Sp1 binding sites resulted in decreases in the promoter activity of CYP3A genes. Conclusively, our results show that human and porcine CYP3A genes are regulated by NF-Y and Sp1 in a coordinated manner, and that the distance between these two cis-acting elements is crucial for constitutive CYP3A expression.

Bioinformatic and functional analysis of promoter region of human SLC25A13 gene

The human SLC25A13 gene encodes the liver type aspartate/glutamate carrier isoform 2 (AGC2, commonly named as citrin), which plays a key role in the main NADH-shuttle of human hepatocyte. Biallelic SLC25A13 mutations result in Citrin deficiency (CD). In order to identify the important regulatory region of SLC25A13 gene and elucidate the way how potential promoter mutations affect the citrin expression, we performed promoter deletion analysis and established the reporter constructs of luciferase gene-carrying SLC25A13 promoter containing several mutations located in putative transcription factor-binding sites. The luciferase activities of all promoter constructs were measured using a Dual-Luciferase Reporter Assay System. Bioinformatic analysis showed that the promoter of SLC25A13 gene lacks TATA box and obviously typical initiator element, but contains a CCAAT box and two GC box. Promoter deletion analysis confirmed the region from −221 to −1 upstream ATG was essential for SLC25A13 to maintain the promoter activity. We utilized dual-luciferase reporter system as function analytical model to tentatively assess the effect of artificially constructed promoter mutations on citrin expression, and our analysis revealed that mutated putative CCAAT box and GC box could significantly affect the citrin expression. Our study confirmed the important SLC25A13 promoter regions that influenced citrin expression in HL7702 cells, and constructed a function analytical model. This work may be useful to further identify the pathogenic mutations leading to CD in the promoter region.

The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.

Nuclear factor Y (NF-Y) is a transcription factor trimer binding to the functionally important CCAAT box, present in promoters of growth-promoting and cell cycle-regulated genes. The regulatory nuclear factor YA (NF-YA) subunit confers sequence-specificity to the histone-like nuclear factor YB/YC dimer. NF-YA harbors 2 serines-Ser320 and Ser326-shown to be phosphorylated by cyclin-dependent kinase 2. High-throughput proteomics data indicate that they are phosphorylated in vivo. Specifically, Ser320 makes structural contacts with the DNA phosphate backbone; Ser320-P is the major NF-YA phosphorylation isoform following overexpression in HeLa cells, increasing upon mitotic arrest. EMSA with recombinant Ala and Glu mutants confirm a role of Ser320, but not Ser326, in stabilization of DNA binding. Transactivation assays of the CCAAT-dependent MDR1 and RHOB promoters show loss in transcription function for Ser320Glu and Ser320Ala NF-YA mutants. Phylogenetic analysis of NF-YA proteins indicates that Ser320 is indeed evolutionarily conserved. We conclude that phosphorylation of this residue belongs to the core mechanisms of DNA-binding control, possibly driven by the necessity to unfasten binding of or to evict NF-Y from CCAAT sites under specific conditions of growth regulation.-Bernardini, A., Lorenzo, M., Nardini, M., Mantovani, R., Gnesutta, N. The phosphorylatable Ser320 of NF-YA is involved in DNA binding of the NF-Y trimer.

Analysing structural diversity of seed storage protein gene promoters: Buckwheat a case study

Multiple sequence alignment of 5’UTR of SSP genes from accessions of Fagopyrum esculentumrevealed the invariant nature of sequences with the transcription start site at P761and TATA box located -30bp upstream the TSS. Other cis-elements identified in the sequences included the legumin box (-581, -524, -184, -135, -91), the -131 prolamin box, DOF element (-718, -649, -540,-432, -272,-225, -128) and CAAT box (-692, -530, -475, -411, -282, -168, -54). Other elements identified included those involved in abscisic acid signallingviz., ABI3 at P-470,-95,-68,RAV1 at P-694and -543and AGL15 at P-671. A comparative analysis of regulatory elements of SSP gene promoters of distantly related species the presence of five cis-regulatory elements viz. TATA BOX, E-BOX, RY- element, CAAT box and the Endosperm box, which interplay in seed specific SSP gene expression. Other modulators influencing seed specific gene expression detected in the sequences included the ABA-responsive elements ABI3, RAV1 and AGL15 which play an integral role in seed maturation. Identification of potential nucleosome binding sites in SSP gene promoters of Cicer arietinum, Brassica napus, B. campestris, Vicia faba, and Pisum sativumat positions 78, 635, 195, 112 and 152 respectively surmises the spatial fine tuning of SSP gene transcriptional regulation in these species. On the other hand, absence of nucleosome binding sites in the promoters of Fagopyrum esculentum, Zea mays, Avena sativa, Triticum aestivum and Oryza sativamay indicate relatively easier access of transcription factors to the proximal promoter, thereby providing higher level of gene expression.

Characterization and functional analysis of miR166f in drought stress tolerance in mulberry (Morus multicaulis)

MicroRNAs (miRNAs) regulate gene expression accurately and effectively at the posttranscriptional level by repressing translation or directly degrading target mRNAs. Over 40 plant miRNA family genes have been associated with abiotic stress; however, few reports have identified miRNAs and their molecular functions under abiotic stress in woody plants. Mulberry trees are ecologically and economically important perennial woody plants, but few studies have examined the stress physiology, biochemistry, and molecular biology of mulberry miRNAs. Previously, we identified miR166f in Morus alba and found that its target genes encode homeobox-leucine zipper (HD-Zip) transcription factors and histone arginine demethylase, which are involved in responses to heat, cold, drought, and salt stresses. In this study, we further characterized miR166f from mulberry, and found that it targets HD-Zip induced by drought stress. We isolated, cloned, and identified pre-miR166f, the precursor of miR166f, from the leaves of mulberry (Morus multicaulis); pre-miR166f was 91 bp in length with a minimum folding free energy of − 51.70 kcal/mol. Bioinformatics analysis showed that the 3000-bp 5′ upstream region of miR166f contained not only TATA and CAAT boxes that functioned in transcription initiation, but also had many cis-acting elements for stress responses, such as MBS, HSE, ARE, TC-rich repeats, and several hormones response elements, such as AuxRR-core, CGTCA-motif, and P-box. This analysis suggested that miR166f might participate in abiotic and biotic stress responses by regulating the expression of stress-related genes in mulberry. We constructed a miR166f binary overexpression vector, pCAMBIA-35S-GUS-miR166f, and established a transient transformation system in mulberry. Histochemical β-glucuronidase (GUS) staining of miR166f-overexpression in transient transgenic mulberry leaves showed the best effect and highest GUS gene expression compared to the wild-type control plants at day four posttransformation using an optimized concentration of transformation liquid (OD600 = 0.7). The target genes of miR166f—two HD-Zips and one histone arginine demethylase gene (JMJD6)—showed markedly lower expression levels in miR166f-overexpression transient transgenic mulberry leaves. Further investigation showed that transient transgenic mulberry trees had higher relative water content, free proline content, soluble protein content, and superoxide dismutase and peroxidase activities, and lower malondialdehyde content compared to the wild-type control plants, which suggested that overexpression of miR166f in mulberry could enhance tolerance to drought stresses in transient transgenic mulberry. The results suggested that miR166f might function as a positive regulator of drought stress tolerance in mulberry.

Correction to "CCAAT/Enhancer-Binding Protein β (Nuclear Factor for Interleukin 6) Transactivates the Human MDR1 Gene by Interaction with an Inverted CCAAT Box in Human Cancer Cells".

Correction to "CCAAT/Enhancer-Binding Protein β (Nuclear Factor for Interleukin 6) Transactivates the Human MDR1 Gene by Interaction with an Inverted CCAAT Box in Human Cancer Cells".

Identification of a Novel Function of Resveratrol and Genistein as a Regulator of β2 -Adrenergic Receptor Expression in Skeletal Muscle Cells and Characterization of Promoter Elements Required for Promoter Activation.

Modulating β2 -adrenergic receptor (β2 -AR) expression and activation is important for maintaining skeletal muscle function. In this study, two food factors, resveratrol (RSV) and genistein (GEN), that are able to regulate β2 -AR promoter activity and may improve skeletal muscle function are identified. Using luciferase reporter assay, 357 functional food factors as candidates for β2 -AR promoter activity have been screened and subsequently RSV and GEN increase β2 -AR promoter activity and β2 -AR mRNA expression. Using promoter sequence analysis, it is shown that the CCAAT box and the GC box on the β2 -AR promoter are required for the regulation of β2 -AR expression by RSV or GEN. It is also ascertained that transcription factor NF-YA binds to the CCAAT box on the β2 -AR promoter and that the amount of NF-YA bound to the CCAAT box is unchanged by RSV or GEN treatment. Finally, it is confirmed that a GEN-containing diet increases β2 -AR expression in mouse skeletal muscle and increased skeletal muscle mass. The findings show that food-derived molecules have the potential to influence skeletal muscle mass and function by regulating G protein-coupled receptor expression.

Enhancement of transgene expression by nuclear transcription factor Y and CCCTC-binding factor.

If a transgene is effectively delivered to a cell, its expression may still be limited by epigenetic mechanisms that silence the transgene. Indeed, once the transgene reaches the nucleus, it may be bound by histone proteins and condensed into heterochromatin or associated with repressor proteins that block transcription. In this study, we sought to enhance transgene expression by adding binding motifs for several different epigenetic enzymes either upstream or downstream of two promoters (CMV and EF1α). Screening these plasmids revealed that luciferase expression was enhanced 10-fold (10.4 ± 5.8) by the addition of a CCAAT box just upstream of the EF1α promoter to recruit nuclear transcription factor Y (NF-Y), while inserting a CCCTC-binding factor (CTCF) motif downstream of the EF1α promoter enhanced expression at least 14-fold (14.03 ± 6.54). ChIP assays confirmed that NF-Y and CTCF bound to the motifs that were added to each plasmid, but the presence of NF-Y and CTCF did not significantly affect the levels of histone acetylation (H3K9ac) or methylation (H3K9me3). Overall, these results show that transgene expression from the EF1α promoter can be significantly increased with motifs that recruit NF-Y or CTCF. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1581-1588, 2018.

NF-YA enters cells through cell penetrating peptides

Cell Penetrating Peptides -CPPs- are short aminoacidic stretches present in proteins that have the ability to translocate the plasma membrane and facilitate delivery of various molecules. They are usually rich in basic residues, and organized as alpha helices. NF-Y is a transcription factor heterotrimer formed by two Histone Fold Domain –HFD- subunits and the sequence-specific NF-YA. NF-YA possesses two α-helices rich in basic residues. We show that it efficiently enters cells at nanomolar concentrations in the absence of carrier peptides. Mutagenesis identified at least two separate CPPs in the A1 and A2, which overlap with previously identified nuclear localization signals (NLS). The half-life of the transduced protein is short in human cancer cells, longer in mouse C2C12 myoblasts. The internalized NF-YA is capable of trimerization with the HFD subunits and binding to the target CCAAT box. Functionality is further suggested by protein transfection in C2C12 cells, leading to inhibition of differentiation to myotubes. In conclusion, NF-YA contains CPPs, hinting at novel -and unexpected- properties of this subunit.

ICBP90 mediates Notch signaling to facilitate human hepatocellular carcinoma growth.

The Notch signaling pathway plays a key role in cell proliferation and development that is closely related to an inverted CCAAT box binding protein (ICBP90), but little is known about whether there is a correlation between Notch signaling and ICBP90. The aim of the current study was to elucidate this. MTT assay and flow cytometry were used to determine the proliferation, cell cycle and apoptosis of HepG2 or Hepa1-6 cells treated by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a specific inhibitor of the Notch pathway. RT-PCR, Western Blot and in situ immunofluorescence staining were employed to examine expression of ICBP90 in the cells. DAPT caused inhibition of the activation of the Notch signaling pathway, followed by preventing the cells at the G0/G1 phases to enter S and G2/M phases. ICBP90 and Hes-1 proteins were highly expressed in the untreated cells. The reduced levels of Notch intracellular domain (NICD) protein were observed in the DAPT-treated cells, thereby bringing about the down-regulation of ICBP90 with the increment of the DAPT dose. Consistent with this, knockdown of the Hes-1 gene, which encodes a critical transcriptional factor in the Notch pathway, also led to the attenuation of ICBP90. On the contrary, Jagged-1, a Notch ligand, facilitated ICBP90 production. Adriamycin could result in the reduction of ICBP90, which was not accompanied with the alteration of Hes-1. ICBP90 was almost fully distributed within the nuclei, but Hes-1 was visible within both the cytoplasm and nuclei. Our novel findings strongly indicate that inactivation of the Notch signaling pathway impedes hepatocellular carcinoma progress via reduction of ICBP90.

The molecular characterization, expression pattern and alternative initiation of Megalobrama amblycephala Hif prolyl hydroxylase Phd1

HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream (Megalobrama amblycephala) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation.

Analysis of Hepatitis B virus (HBV) mutations in patients from Western Saudi Arabia with chronic disease.

Extensive research has provided a link between HBV variants and the clinical complications of liver diseases. This study was performed to further investigate the relationship between HBV variants in preS, S and BCP/PC regions and disease progression in chronic hepatitis B (CHB) cases in Jeddah, Saudi Arabia. 182 CHB patients were recruited for this study. HBV DNA was amplified by PCR in the PreS, S, and BCP/PC regions. Sequences were generated from 31 and 26 treated cases in PreS and S regions respectively and from 72 cases in the BCP/PC region. The majority of cases (86.7%) were genotype D. Mutations at preS1-A2922C, X-A1624C and PC-G1887A were detected only in cases with either a high fibrosis score or hepatocellular carcinoma (HCC), while mutations at positions PC-C1982A, PC-G1951T, X-C1628T and X-A1630G were detected more frequently in HCC cases, without reaching statistical significance. Seven deletions were detected in the PreS-region. No deletions were detected in the CCAAT box. The accumulation of mutations per sample in the preS1-2 and S regions were associated with elevated ALT (p < 0.001, 0.001 and 0.001; respectively) and increased fibrosis (p = 0.018, 0.02 and 0.013; respectively). The accumulation of mutations per sample in the BCP/PC region is associated with high viral load. Occult hepatitis B infection (OBI) was identified in 5 samples. Our results add to the knowledge about HBV genotype-D variants. The accumulation of mutations per sample and OBI seem to play a role in the progression of HBV infection. G1896A was associated with the HBeAg negativity. The preS deletions did not play a role in liver disease progression.

Identification of a novel promoter from banana aquaporin family gene (MaTIP1;2) which responses to drought and salt-stress in transgenic Arabidopsis thaliana

Drought and salt stresses often affect plant growth and crop yields. Identification of promoters involved in drought and salt stress responses is of great significance for genetic improvement of crop resistance. Our previous studies showed that aquaporin can respond to drought and salt stresses, but its promoter has not yet been reported in plants. In the present study, cis-acting elements of MaAQP family member promoters were systematically analyzed in banana. Expression of MaTIP1; 2 was induced by drought and salt stresses but not sensitive to cold stress, waterlogging stress, or mechanical damage, and its promoter contained five stress-related cis-acting elements. The MaTIP1; 2 promoter (841 bp upstream of translation initiation site) from banana (Musa acuminata L. AAA group cv. Brazilian) was isolated through genome walking polymerase chain reaction, and found to contain a TATA Box, CAAT box, ABRE element, CCGTCC box, CGTCA motif, and TCA element. Transformation of the MaTIP1; 2 promoter into Arabidopsis to assess its function indicated that it responds to both drought and salt stress treatments. These results suggest that MaTIP1; 2 utilization may improve drought and salt stresses resistance of the transgenic plants by promoting banana aquaporin expression.

Identification and characterisation of a previously unknown drought tolerance-associated microRNA in barley.

Drought is the most serious abiotic stress, and causes crop losses on a worldwide scale. The present study identified a previously unknown microRNA (designated as hvu-miRX) of 21 nucleotides (nt) in length in barley. Its precursor (designated pre-miRX) and primary transcript (designated pri-miRX) were also identified, with lengths of 73 and 559nt, respectively. The identified upstream sequence of pri-miRX contained both the TATA box and the CAAT box, which are both required for initiation of transcription. Transient promoter activation assays showed that the core promoter region of pri-miRX ranged 500nt from the transcription start site. In transgenic barley overexpression of the wheat DREB3 transcription factor (TaDREB3) caused hvu-miRX to be highly expressed as compared with the same miRNA in non-transgenic barley. However, the high expression was not directly associated with TaDREB3. Genomic analysis revealed that the hvu-miRX gene was a single copy located on the short arm of chromosome 2 and appeared to be only conserved in Triticeae, but not in other plant species. Notably, transgenic barley that overexpressed hvu-miRX showed drought tolerance. Degradome library analysis and other tests showed that hvu-miRX targeted various genes including transcription factors via the cleavage mode. Our data provides an excellent opportunity to develop drought stress tolerant cereals using hvu-miRX.

An autoregulatory loop controls the expression of the transcription factor NF-Y

The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer.Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit.Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions.

Specific transcriptional and post-transcriptional regulation of the major immediate early ICP4 gene of GaHV-2 during the lytic, latent and reactivation phases.

Transcriptional and post-transcriptional mechanisms are involved in the switch between the lytic, latent and reactivation phases of the viral cycle in herpesviruses. During the productive phases, herpesvirus gene expression is characterized by a temporally regulated cascade of immediate early (IE), early (E) and late (L) genes. In alphaherpesviruses, the major product of the IE ICP4 gene is a transcriptional regulator that initiates the cascade of gene expression that is essential for viral replication. In this study, we redefine the infected cell protein 4 (ICP4) gene of the oncogenic Marek's disease virus (MDV or gallid herpesvirus 2) as a 9438 nt gene ended with four alternative poly(A) signals and controlled by two alternative promoters containing essentially ubiquitous functional response elements (GC, TATA and CCAAT boxes). The distal promoter is associated with ICP4 gene expression during the lytic and the latent phases, whereas the proximal promoter is associated with the expression of this gene during the reactivation phase. Both promoters are regulated by DNA methylation during the viral cycle and are hypermethylated during latency. Transcript analyses showed ICP4 to consist of three exons and two introns, the alternative splicing of which is associated with five predicted nested ICP4ORFs. We show that the ICP4 gene is highly and specifically regulated by transcriptional and post-transcriptional mechanisms during the three phases of the GaHV-2 viral cycle, with a clear difference in expression between the lytic phase and reactivation from latency in our model.