Binding Dynamics of a Stapled Peptide Targeting the Transcription Factor NF-Y.

Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

NF-Y subunits overexpression in gastric adenocarcinomas (STAD).

NF-Y is a pioneer transcription factor-TF-formed by the Histone-like NF-YB/NF-YC subunits and the regulatory NF-YA. It binds to the CCAAT box, an element enriched in promoters of genes overexpressed in many types of cancer. NF-YA is present in two major isoforms-NF-YAs and NF-YAl-due to alternative splicing, overexpressed in epithelial tumors. Here we analyzed NF-Y expression in stomach adenocarcinomas (STAD). We completed the partitioning of all TCGA tumor samples (450) according to molecular subtypes proposed by TCGA and ACRG, using the deep learning tool DeepCC. We analyzed differentially expressed genes-DEG-for enriched pathways and TFs binding sites in promoters. CCAAT is the predominant element only in the core group of genes upregulated in all subtypes, with cell-cycle gene signatures. NF-Y subunits are overexpressed, particularly NF-YA. NF-YAs is predominant in CIN, MSI and EBV TCGA subtypes, NF-YAl is higher in GS and in the ACRG EMT subtypes. Moreover, NF-YAlhigh tumors correlate with a discrete Claudinlow cohort. Elevated NF-YB levels are protective in MSS;TP53+ patients, whereas high NF-YAl/NF-YAs ratios correlate with worse prognosis. We conclude that NF-Y isoforms are associated to clinically relevant features of gastric cancer.

NF-Y Overexpression in Liver Hepatocellular Carcinoma (HCC).

NF-Y is a pioneer trimeric transcription factor formed by the Histone Fold Domain (HFD) NF-YB/NF-YC subunits and NF-YA. Three subunits are required for DNA binding. CCAAT-specificity resides in NF-YA and transactivation resides in Q-rich domains of NF-YA and NF-YC. They are involved in alternative splicing (AS). We recently showed that NF-YA is overexpressed in breast and lung carcinomas. We report here on the overexpression of all subunits in the liver hepatocellular carcinoma (HCC) TCGA database, specifically the short NF-YAs and NF-YC2 (37 kDa) isoforms. This is observed at all tumor stages, in viral-infected samples and independently from the inflammatory status. Up-regulation of NF-YAs and NF-YC, but not NF-YB, is associated to tumors with mutant p53. We used a deep-learning-based method (DeepCC) to extend the partitioning of the three molecular clusters to all HCC TCGA tumors. In iCluster3, CCAAT is a primary matrix found in promoters of up-regulated genes, and cell-cycle pathways are enriched. Finally, clinical data indicate that, globally, only NF-YAs, but not HFD subunits, correlate with the worst prognosis; in iCluster1 patients, however, all subunits correlate. The data show a difference with other epithelial cancers, in that global overexpression of the three subunits is reported and clinically relevant in a subset of patients; yet, they further reinstate the regulatory role of the sequence-specific subunit.

Structural Basis of Inhibition of the Pioneer Transcription Factor NF-Y by Suramin.

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo "metabolic rewiring" through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.

Small nucleolar RNA host gene 3 facilitates cell proliferation and migration in oral squamous cell carcinoma via targeting nuclear transcription factor Y subunit gamma.

Oral squamous cell carcinoma (OSCC) has been reported to be the most common oral carcinoma. Emerging evidence has revealed the key role that long noncoding RNAs (lncRNAs) play in numerous malignancies, including OSCC. LncRNA small nucleolar RNA host gene 3 (SNHG3) has been reported as an oncogenic factor in some cancers. Nonetheless, the role of SNHG3 in OSCC has never been clarified. In this study, we analyzed the expression patterns of SNHG3 in OSCC through quantitative real-time polymerase chain reaction. It was revealed that the expression level of SNHG3 was remarkably elevated in OSCC cell lines compared with the nontumor cell line. It was demonstrated by functional experiments that SNHG3 knockdown notably inhibited cell proliferation and migration in OSCC. RNA immunoprecipitation, RNA pull down, and messenger RNA (mRNA) stability test verified that SNHG3 decoyed ELAV like RNA-binding protein 1 (ELAVL1) and therefore stabilized nuclear transcription factor Y subunit gamma (NFYC) mRNA to upregulate the expression levels of NFYC in OSCC cells. At last, it was confirmed by rescue experiments that the inhibiting impacts of SNHG3 knockdown on OSCC cell proliferation and migration could be partly revived by NFYC overexpression. Besides, we validated that Wnt/ß-catenin pathway was also involved in SNHG3-regulated OSCC progression. In conclusion, SNHG3 might serve as a novel biomarker for OSCC.

Constrained Peptides with Fine-Tuned Flexibility Inhibit NF-Y Transcription Factor Assembly.

Protein complex formation depends on the interplay between preorganization and flexibility of the binding epitopes involved. The design of epitope mimetics typically focuses on stabilizing a particular bioactive conformation, often without considering conformational dynamics, which limits the potential of peptidomimetics against challenging targets such as transcription factors. We developed a peptide-derived inhibitor of the NF-Y transcription factor by first constraining the conformation of an epitope through hydrocarbon stapling and then fine-tuning its flexibility. In the initial set of constrained peptides, a single non-interacting α-methyl group was observed to have a detrimental effect on complex stability. Biophysical characterization revealed how this methyl group affects the conformation of the peptide in its bound state. Adaption of the methylation pattern resulted in a peptide that inhibits transcription factor assembly and subsequent recruitment to the target DNA.

Transcription factor Sp2 potentiates binding of the TALE homeoproteins Pbx1:Prep1 and the histone-fold domain protein Nf-y to composite genomic sites.

Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.

NF-Y Binding Site Architecture Defines a C-Fos Targeted Promoter Class.

ChIP-seq experiments detect the chromatin occupancy of known transcription factors in a genome-wide fashion. The comparisons of several species-specific ChIP-seq libraries done for different transcription factors have revealed a complex combinatorial and context-specific co-localization behavior for the identified binding regions. In this study we have investigated human derived ChIP-seq data to identify common cis-regulatory principles for the human transcription factor c-Fos. We found that in four different cell lines, c-Fos targeted proximal and distal genomic intervals show prevalences for either AP-1 motifs or CCAAT boxes as known binding motifs for the transcription factor NF-Y, and thereby act in a mutually exclusive manner. For proximal regions of co-localized c-Fos and NF-YB binding, we gathered evidence that a characteristic configuration of repeating CCAAT motifs may be responsible for attracting c-Fos, probably provided by a nearby AP-1 bound enhancer. Our results suggest a novel regulatory function of NF-Y in gene-proximal regions. Specific CCAAT dimer repeats bound by the transcription factor NF-Y define this novel cis-regulatory module. Based on this behavior we propose a new enhancer promoter interaction model based on AP-1 motif defined enhancers which interact with CCAAT-box characterized promoter regions.

Nuclear factor Y regulates ancient budgerigar hepadnavirus core promoter activity.

Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt -85 to -11 in eBHBV1 Cp was critical for the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt -64 to -50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt -58 to -54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt -21 to -17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses.

Infinity: An In-Silico Tool for Genome-Wide Prediction of Specific DNA Matrices in miRNA Genomic Loci.

MOTIVATION: miRNAs are potent regulators of gene expression and modulate multiple cellular processes in physiology and pathology. Deregulation of miRNAs expression has been found in various cancer types, thus, miRNAs may be potential targets for cancer therapy. However, the mechanisms through which miRNAs are regulated in cancer remain unclear. Therefore, the identification of transcriptional factor-miRNA crosstalk is one of the most update aspects of the study of miRNAs regulation. RESULTS: In the present study we describe the development of a fast and user-friendly software, named infinity, able to find the presence of DNA matrices, such as binding sequences for transcriptional factors, on ~65kb (kilobase) of 939 human miRNA genomic sequences, simultaneously. Of note, the power of this software has been validated in vivo by performing chromatin immunoprecipitation assays on a subset of new in silico identified target sequences (CCAAT) for the transcription factor NF-Y on colon cancer deregulated miRNA loci. Moreover, for the first time, we have demonstrated that NF-Y, through its CCAAT binding activity, regulates the expression of miRNA-181a, -181b, -21, -17, -130b, -301b in colon cancer cells. CONCLUSIONS: The infinity software that we have developed is a powerful tool to underscore new TF/miRNA regulatory networks. AVAILABILITY AND IMPLEMENTATION: Infinity was implemented in pure Java using Eclipse framework, and runs on Linux and MS Windows machine, with MySQL database. The software is freely available on the web at https://github.com/bio-devel/infinity. The website is implemented in JavaScript, PHP and HTML with all major browsers supported.

LEC1-LIKE paralog transcription factor: how to survive extinction and fit in NF-Y protein complex.

Transcription factor function is crucial for eukaryotic systems. The presence of transcription factor families in genomes represents a significant technical challenge for functional studies. To understand their function, we must understand how they evolved and maintained by organisms. Based on genome scale searches for homologs of LEAFY COTYLEDON-LIKE (L1L; AtNF-YB6), NF-YB transcription factor, we report the discovery and annotation of a complete repertoire of thirteen novel genes that belong to the L1L paralogous gene family of Solanum lycopersicum. Gene duplication events within the species resulted in the expansion of the L1L family. Sequence and structure-based phylogenetic analyses revealed two distinct groups of L1Ls in tomato. Natural selection appears to have contributed to the asymmetric evolution of paralogs. Our results point to key differences among SlL1L paralogs in the presence of motifs, structural features, cysteine composition and expression patterns during plant and fruit development. Furthermore, differences in the binding domains of L1L members suggest that some of them evolved new binding specificities. These results reveal dramatic functional diversification of L1L paralogs for their maintenance in tomato genome. Our comprehensive insights on tomato L1L family should provide the basis for further functional and genetic experimentation.

Characterization of the human mitochondrial thiamine pyrophosphate transporter SLC25A19 minimal promoter: a role for NF-Y in regulating basal transcription.

Transcriptional regulation of expression of the human mitochondrial thiamine pyrophosphate transporter (the product of the SLC25A19 gene) is unknown. To understand this regulation, we cloned and characterized the 5'-regulatory region of the SLC25A19 gene (1,080 bp). The cloned fragment was found to possess promoter activity in transiently transfected human-derived liver HepG2 cells. 5'- and 3'-deletion analysis has identified the minimal region required for basal SLC25A19 promoter activity to be between -131 and +20 (using the distal transcriptional start site as +1). The minimal promoter lacks typical TATA motif and contains two inverted CCAAT boxes (binding sites for NF-Y transcriptional factor). By means of mutational analysis, the critical role of both the upstream and downstream CCAAT boxes in basal SLC25A19 promoter activity was established; however, each of these boxes alone was found to be unable to support promoter activity. EMSA and supershift EMSA (with the use of specific antibodies against NF-Y subunits) studies, as well as chromatin immunoprecipitation assay, demonstrated the binding of NF-Y to both CCAAT boxes in vitro and in vivo, respectively. The requirement for NF-Y in SLC25A19 promoter activity in vivo was directly confirmed by the use of a dominant negative NF-YA mutant in transiently transfected HepG2 cells. These studies report for the first time the characterization of the SLC25A19 promoter and demonstrate an essential role for NF-Y in its basal activity.

CBFß stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression.

The HIV-1 accessory protein Vif hijacks a cellular Cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 (A3) family of restriction factors. Recently, the cellular transcription cofactor CBFß was shown to form a complex with CRL5-Vif and to be essential for A3 degradation and viral infectivity. We now demonstrate that CBFß is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and by activating CRL5-Vif via direct interaction. The CRL5-Vif-CBFß holoenzyme forms a well-defined heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFß. Heterodimers of CBFß and RUNX transcription factors contribute toward the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFß is mutually exclusive with RUNX heterodimerization and impacts the expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limited coding capacity uses one factor to hijack multiple host pathways.

NF-Ya protein delivery as a tool for hematopoietic progenitor cell expansion.

The clinical potential of therapeutic quantities of primary hematopoietic cells, either unmodified or altered via genetic modification, has stimulated the search for techniques that allow the production of large numbers of hematopoietic precursors, more primitive progenitors, and perhaps hematopoietic stem cells (HSC) themselves. Modifications of in vitro culture conditions to promote progenitor cell expansion have included combinations of polypeptide cytokines, small molecules, and transcription factors. Here we describe the methods for use of the transcription factor linked to a TAT-based protein transcription domain, in combination with cytokines and serum-free culture condition to stimulate the proliferation of primary cells. Human peripheral blood (PB) CD34(+) cells treated with TAT-NF-Ya fusion protein and grown in vitro for 1 month proliferate four times more than did cells in cultures that contained only cytokines, including increased production of hematopoietic cells of all maturities. These results and techniques should be suitable for multiple applications of ex vivo generation of hematopoietic cells using protein transduction.

Homologous NF-YC2 subunit from Arabidopsis and tobacco is activated by photooxidative stress and induces flowering.

The transcription factor NF-Y consists of the three subunits A, B and C, which are encoded in Arabidopsis in large gene families. The multiplicity of the genes implies that NF-Y may act in diverse combinations of each subunit for the transcriptional control. We aimed to assign a function in stress response and plant development to NF-YC subunits by analyzing the expression of NF-Y genes and exploitation of nf-y mutants. Among the subunit family, NF-YC2 showed the strongest inducibility towards oxidative stress, e.g. photodynamic, light, oxidative, heat and drought stress. A tobacco NF-YC homologous gene was found to be inducible by photooxidative stress generated by an accumulation of the tetrapyrrole metabolite, coproporphyrin. Despite the stress induction, an Arabidopsis nf-yc2 mutant and NF-YC2 overexpressors did not show phenotypical differences compared to wild-type seedlings in response to photooxidative stress. This can be explained by the compensatory potential of other members of the NF-YC family. However, NF-YC2 overexpression leads to an early flowering phenotype that is correlated with increased FLOWERING LOCUS T-transcript levels. It is proposed that NF-YC2 functions in floral induction and is a candidate gene among the NF-Y family for the transcriptional activation upon oxidative stress.

Studies on differential nuclear translocation mechanism and assembly of the three subunits of the Arabidopsis thaliana transcription factor NF-Y.

The eukaryotic transcription factor NF-Y consists of three subunits (A, B, and C), which are encoded in Arabidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combinations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF-YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.

The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes.

The heterotrimeric CCAAT-binding complex is evolutionary conserved in eukaryotic organisms. The corresponding Aspergillus nidulans CCAAT-binding factor (AnCF) consists of the subunits HapB, HapC and HapE. All of the three subunits are necessary for DNA binding. Here, we demonstrate that AnCF senses the redox status of the cell via oxidative modification of thiol groups within the histone fold motif of HapC. Mutational and in vitro interaction analyses revealed that two of these cysteine residues are indispensable for stable HapC/HapE subcomplex formation and high-affinity DNA binding of AnCF. Oxidized HapC is unable to participate in AnCF assembly and localizes in the cytoplasm, but can be recycled by the thioredoxin system in vitro and in vivo. Furthermore, deletion of the hapC gene led to an impaired oxidative stress response. Therefore, the central transcription factor AnCF is regulated at the post-transcriptional level by the redox status of the cell serving for a coordinated activation and deactivation of antioxidative defense mechanisms including the specific transcriptional activator NapA, production of enzymes such as catalase, thioredoxin or peroxiredoxin, and maintenance of a distinct glutathione homeostasis. The underlying fine-tuned mechanism very likely represents a general feature of the CCAAT-binding complexes in eukaryotes.

Regulation of mouse Scgb3a1 gene expression by NF-Y and association of CpG methylation with its tissue-specific expression.

BACKGROUND: Secretoglobin (SCGB) 3A1 is a secretory protein of small molecular weight with tumor suppressor function. It is highly expressed in lung and trachea in both human and mouse, with additional tissues expressing the protein that differ depending on the species. However, little is known about the function and transcriptional regulation of this gene in normal mouse tissues. RESULTS: By reporter gene transfection and gel mobility shift analyses, we demonstrated that expression of the mouse Scgb3a1 gene is regulated by a PU-box binding protein and a ubiquitous transcription factor NF-Y that respectively binds to the PU-boxes located at -99 to -105 bp and -158 to -164 bp, and the "CCAAT" binding sites located at -425 to -429 bp and -498 to -502 bp from the transcription start site of the gene. However, the effect of PU-box binding protein on transcriptional activation is minimal as compared to NF-Y, suggesting that NF-Y is a more critical transcription factor for mouse Scgb3a1 gene transcription. Despite that NF-Y is a ubiquitous factor, Scgb3a1 is highly expressed only in mouse lung and mtCC cells that are derived from SV40 transformed mouse Clara cells, but not in ten other mouse tissues/cells examined. Gene methylation analysis revealed that within 600 bp of the Scgb3a1 gene promoter region, there are nine CpG methylation sites present, of which two CpGs closest to the transcription start site of the gene are unmethylated in the tissues/cells expressing SCGB3A1. CONCLUSION: A ubiquitous transcription factor NF-Y binds to and activates expression of the mouse Scgb3a1 gene and tissue-specific expression of the gene is associated with CpG methylation of the promoter.

CCAAT/enhancer-binding protein (C/EBP) beta is acetylated at multiple lysines: acetylation of C/EBPbeta at lysine 39 modulates its ability to activate transcription.

Transcription factor function can be modulated by post-translational modifications. Because the transcription factor CCAAT/enhancer-binding protein (C/EBP) beta associates with the nuclear coactivator p300, which contains acetyltransferase activity, acetylation of C/EBPbeta was examined to understand its regulation and function. C/EBPbeta is acetylated by acetyltransferases p300 and p300/CREB-binding protein associated factor. Endogenous C/EBPbeta in 3T3-F442A preadipocytes is also recognized by an acetyl-lysine-specific antibody. Analysis of truncations of C/EBPbeta and peptides based on C/EBPbeta sequences identified multiple lysines within C/EBPbeta that can be acetylated. Among these, a novel acetylation site at lysine 39 of C/EBPbeta was identified. Mutation of Lys-39 to arginine or alanine impairs its acetylation and the ability of C/EBPbeta to activate transcription at the promoters for C/EBPalpha and c-fos. Different C/EBPbeta-responsive promoters require different patterns of acetylated lysines in C/EBPbeta for transcription activation. Furthermore, C/EBPbeta acetylation was increased by growth hormone, and mutation of Lys-39 impaired growth hormone-stimulated c-fos promoter activation. These data suggest that acetylation of Lys-39 of C/EBPbeta, alone or in combination with acetylation at other lysines, may play a role in C/EBPbeta-mediated transcriptional activation.

Mechanisms of transcriptional repression of cell-cycle G2/M promoters by p63.

p63 is a developmentally regulated transcription factor related to p53, which activates and represses specific genes. The human AEC (Ankyloblepharon-Ectodermal dysplasia-Clefting) and EEC (Ectrodactyly-Ectodermal dysplasia-Cleft lip/palate) syndromes are caused by missense mutations of p63, within the DNA-binding domain (EEC) or in the C-terminal sterile alpha motif domain (AEC). We show here that p63 represses transcription of cell-cycle G(2)/M genes by binding to multiple CCAAT core promoters in immortalized and primary keratinocytes. The CCAAT-activator NF-Y and DeltaNp63alpha are associated in vivo and a conserved alpha-helix of the NF-YC histone fold is required. p63 AEC mutants, but not an EEC mutant, are incapable to bind NF-Y. DeltaNp63alpha, but not the AEC mutants repress CCAAT-dependent transcription of G(2)/M genes. Chromatin immunoprecipitation recruitment assays establish that the AEC mutants are not recruited to G(2)/M promoters, while normally present on 14-3-3sigma, which contains a sequence-specific binding site. Surprisingly, the EEC C306R mutant activates transcription. Upon keratinocytes differentiation, NF-Y and p63 remain bound to G(2)/M promoters, while HDACs are recruited, histones deacetylated, Pol II displaced and transcription repressed. Our data indicate that NF-Y is a molecular target of p63 and that inhibition of growth activating genes upon differentiation is compromised by AEC missense mutations.