Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination.

The sequence-specific transcription factor NF-Y binds the CCAAT box, one of the sequence elements most frequently found in eukaryotic promoters. NF-Y is composed of the NF-YA and NF-YB/NF-YC subunits, the latter two hosting histone-fold domains (HFDs). The crystal structure of NF-Y bound to a 25 bp CCAAT oligonucleotide shows that the HFD dimer binds to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YA both binds to NF-YB/NF-YC and inserts an α helix deeply into the DNA minor groove, providing sequence-specific contacts to the CCAAT box. Structural considerations and mutational data indicate that NF-YB ubiquitination at Lys138 precedes and is equivalent to H2B Lys120 monoubiquitination, important in transcriptional activation. Thus, NF-Y is a sequence-specific transcription factor with nucleosome-like properties of nonspecific DNA binding and helps establish permissive chromatin modifications at CCAAT promoters. Our findings suggest that other HFD-containing proteins may function in similar ways.

Correction: Establishment and characterisation of a new patient-derived model of myxoid liposarcoma with acquired resistance to trabectedin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Establishment and characterisation of a new patient-derived model of myxoid liposarcoma with acquired resistance to trabectedin.

BACKGROUND: Myxoid liposarcoma is a histological subtype of liposarcoma particularly sensitive to trabectedin. In clinical use this drug does not cause cumulative toxicity, allowing prolonged treatment, generally until disease progression. No other effective therapies are available for trabectedin-resistant patients. METHODS: Through repeated in vivo treatment in athymic nude mice, we have obtained a patient-derived xenograft with acquired resistance to trabectedin. RESULTS: At basal level, the morphology of the resistant and sensitive models did not differ, in keeping with the finding that the transcriptional profiles of the resistant and sensitive tumours were very similar. After trabectedin treatment adipogenesis was induced in the parental xenograft but not in the resistant one, as assessed by pathological and molecular analysis. A defective transcription-coupled-nucleotide excision repair in the resistant tumour due to mutation of the UVSSA gene may be implicated in the mechanism of resistance. CONCLUSIONS: This is the first in vivo model of myxoid liposarcoma with acquired resistance to trabectedin. Although further studies are necessary to characterise the resistance mechanisms, this is a useful tool for studying new therapeutic strategies to overcome trabectedin resistance in patients.

NF-Y and the transcriptional activation of CCAAT promoters.

The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the "innovative" NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y - also termed CBF and CP1 - is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; Hatamochi et al., 1988; Hooft van Huijsduijnen et al, 1987; Kim & Sheffery, 1990). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements (Suzuki et al., 2001, 2004; Elkon et al., 2003; Mariño-Ramírez et al., 2004; FitzGerald et al., 2004; Linhart et al., 2005; Zhu et al., 2005; Lee et al., 2007; Abnizova et al., 2007; Grskovic et al., 2007; Halperin et al., 2009; Häkkinen et al., 2011). The same consensus, as determined by mutagenesis and SELEX studies (Bi et al., 1997), was also retrieved in ChIP-on-chip analysis (Testa et al., 2005; Ceribelli et al., 2006; Ceribelli et al., 2008; Reed et al., 2008). Additional structural features of the CCAAT box - position, orientation, presence of multiple Transcriptional Start Sites - were previously reviewed (Dolfini et al., 2009) and will not be considered in detail here.

NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations.

The CCAAT box is a frequent promoter element, as illustrated by bioinformatic analysis, and it is bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone methylations and the association of enzymatic activities. Two novel results emerged: (i) H3-H4 are present on core promoters under active conditions, with the expected cohort of 'positive' modifications; H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show that NF-Y is important for H3K36me3 deposition and for elongation, not recruitment of Pol II; (ii) H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Functional siRNA inactivation and treatment with Tranylcypromine determined that KDM1 (LSD1) plays a positive role in transcription, specifically of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. These data are the first in vivo indication of a crucial interplay between core histones and 'deviant' histone-fold such as NF-Y, leading to fine-tuning of histone methylations.

A reliable and rapid tool for plasma quantification of 18 psychotropic drugs by ESI tandem mass spectrometry.

A simple liquid chromatographic tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous analysis of 17 basic and one acid psychotropic drugs in human plasma. The method relies on a protein precipitation step for sample preparation and offers high sensitivity, wide linearity without interferences from endogenous matrix components. Chromatography was run on a reversed-phase column with an acetonitrile-H2O mixture. The quantification of target compounds was performed in multiple reaction monitoring (MRM) and by switching the ionization polarity within the analytical run. A further sensitivity increase was obtained by implementing the functionality "scheduled multiple reaction monitoring" (sMRM) offered by the recent version of the software package managing the instrument. The overall injection interval was less than 5.5 min. Regression coefficients of the calibration curves and limits of quantification (LOQ) showed a good coverage of over-therapeutic, therapeutic and sub-therapeutic ranges. Recovery rates, measured as percentage of recovery of spiked plasma samples, were ≥ 94%. Precision and accuracy data have been satisfactory for a therapeutic drug monitoring (TDM) service as for managing plasma samples from patients receiving psycho-pharmacological treatment.

The HDAC inhibitor Givinostat modulates the hematopoietic transcription factors NFE2 and C-MYB in JAK2(V617F) myeloproliferative neoplasm cells.

We investigated the mechanism of action of the histone deacetylase inhibitor Givinostat (GVS) in Janus kinase 2 (JAK2)(V617F) myeloproliferative neoplasm (MPN) cells. GVS inhibited colony formation and proliferation and induced apoptosis at doses two- to threefold lower in a panel of JAK2(V617F) MPN compared to JAK2 wild-type myeloid leukemia cell lines. By global gene expression analysis, we observed that at 6 hours, GVS modulated 293 common genes in the JAK2(V617F) cell lines HEL and UKE1, of which 19 are implicated in cell cycle regulation and 33 in hematopoiesis. In particular, the hematopoietic transcription factors NFE2 and C-MYB were downmodulated by the drug specifically in JAK2(V617F) cells at both the RNA and protein level. GVS also inhibited JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 phosphorylation, but modulation of NFE2 and C-MYB was JAK2-independent, as shown using the JAK2 inhibitor TG101209. GVS had a direct effect on the NFE2 promoters, as demonstrated by specific enrichment of associated histone H3 acetylated at lysine 9. Modulation by GVS of NFE2 was also observed in freshly isolated CD34(+) cells from MPN patients, and was accompanied by inhibition of their proliferation and differentiation toward the erythroid lineage. We conclude that GVS acts on MPN cells through dual JAK2-signal transducer and activator of transcription 5-extracellular signal-regulated kinase 1/2 inhibition and downmodulation of NFE2 and C-MYB transcription.

NF-Y affects histone acetylation and H2A.Z deposition in cell cycle promoters.

Histones post-translational modifications (PTMs) are crucial for transcriptional control, defining positive and negative chromatin territories. We previously described an extensive methylation-acetylation switch on cell cycle promoters using a single nucleosome ChIP assay. A key issue is how PTMs are locally positioned. We report an analysis on the role of the NF-Y CCAAT transcription factor on histone acetylation. Whereas H3K9 and H3K14 acetylation in core promoters is not influenced by NF-Y, H3K18ac, H3K36ac and H3K27ac are increased in the absence of NF-Y. Interestingly, NF-Y affects H2B acetylation in an opposite way: H2BK16ac is decreased and Lysine 120 acetylation, which counter-correlates with ubiquitination, increases dramatically upon NF-Y removal. KAT2A/KAT2B and subunits of the SAGA and ATAC complexes (SPT20 and ZZZ3) are differentially regulated. Finally, the deposition of H2A.Z, which maps around the TSS, is also NF-Y-dependent. In summary, NF-Y influences histone acetylation in different processes, including those involved in a methylation-acetylation switch and in the recruitment of histone variants.

An acetylation-mono-ubiquitination switch on lysine 120 of H2B.

Post-translational modifications (PTMs) of histones are crucial for transcriptional control, defining positive and negative chromatin territories. A switch of opposing functional significance between acetylation and methylation occurs on many residues. Lysine 120 of H2B is modified by two PTMs: ubiquitination, which is required for further trans-tail H3 methylations and elongation, and acetylation, whose role is less clear. ChIP-Seq with MNase I-treated chromatin indicates that H2BK120ac is present on nucleosomes immediately surrounding the TSS of transcribed or poised units, but not in core promoters. In kinetic ChIP analysis of ER-stress inducible genes, H2BK120ac precedes activation and H2B-ub deposition. Using in vitro acetylation assays, pharmacologic inhibition and RNAi, we established that KAT3 is responsible for H2BK120ac. Interestingly, the global levels of H2B-ub decreased in KAT3-inactivated cells. However, RNF20 recruitment was not impaired by KAT3-inactivation. Our data point at acetylation of Lysine 120 of H2B as an early mark of poised or active state and establish a temporal sequence between acetylation and mono-ubiquitination of this H2B residue.

Single nucleosome ChIPs identify an extensive switch of acetyl marks on cell cycle promoters.

Histones are modified by different post-translational modifications which are marks of peculiar chromatin functions. We previously evaluated histone methylations of G1/S and G2/M cell-cycle promoters at the single nucleosome level; here we report an analysis of acetylation marks, including some for which essentially nothing is known. In general, our data confirm the presence of H3/H4, but not H2A/H2B, in active core promoters. H3K14ac and H3K27ac are associated with active promoters, while H3K9ac and H3K18ac are more ambiguous, being also found under repression. Acetylation of H3K36, H3K79 and H2BK120, all residues involved in positive function through methylations or monoubiquitination, were found on repressed genes. H2Bub was present only on transcribed areas, and absent on core promoters or upstream nucleosomes. KAT2A/KAT2B and subunits of the SAGA and ATAC complexes have differential and dynamic roles: KAT2A-inactivated MEFs show a G1/S block, and KAT2B is important for G2/M. Furthermore, the precision of our analysis allows us to locate some acetylations specifically occurring upstream, downstream or in core promoters. Overall, the switch between methylations-and monoubiquitination-and acetylations on histone Lysines is a general theme on this dynamic group of genes.

An NF-Y-dependent switch of positive and negative histone methyl marks on CCAAT promoters.

BACKGROUND: Histone tails have a plethora of different post-translational modifications, which are located differently in "open" and "closed" parts of genomes. H3K4me3/H3K79me2 and H4K20me3 are among the histone marks associated with the early establishment of active and inactive chromatin, respectively. One of the most widespread promoter elements is the CCAAT box, bound by the NF-Y trimer. Two of NF-Y subunits have an H2A-H2B-like structure. PRINCIPAL FINDINGS: We established the causal relationship between NF-Y binding and positioning of methyl marks, by ChIP analysis of mouse and human cells infected with a dominant negative NF-YA: a parallel decrease in NF-Y binding, H3K4me3, H3K79me2 and transcription was observed in promoters that are dependent upon NF-Y. On the contrary, changes in the levels of H3K9-14ac were more subtle. Components of the H3K4 methylating MLL complex are not recruited in the absence of NF-Y. As for repressed promoters, NF-Y removal leads to a decrease in the H4K20me3 mark and deposition of H3K4me3. CONCLUSIONS: Two relevant findings are reported: (i) NF-Y gains access to its genomic locations independently from the presence of methyl histone marks, either positive or negative; (ii) NF-Y binding has profound positive or negative consequences on the deposition of histone methyl marks. Therefore NF-Y is a fundamental switch at the heart of decision between gene activation and repression in CCAAT regulated genes.

The histone-like NF-Y is a bifunctional transcription factor.

NF-Y is a trimeric transcription factor containing H2A/H2B-like subunits, which specifically binds to the CCAAT box, a common eukaryotic promoter element. To gain insights into NF-Y-dependent transcriptional regulation, we assessed its relationships with positive histone marks by chromatin immunoprecipitation-on-chip and correlative-profiling studies. Unbiased identification of binding sites shows that the majority of genes are bound by NF-Y in the promoter and/or within the coding region. Parallel analysis of H3K9-14ac and H3K4me3 sites indicates that NF-Y loci can be divided in two distinct clusters: (i) a large cohort contains H3K9-14ac and H3K4me3 marks and correlates with expression and (ii) a sizeable group is devoid of these marks and is found on transcriptionally silent genes. Within this class, we find that NF-Y binding is associated with negative histone marks, such as H4K20me3 and H3K27me3. NF-Y removal by a dominant negative NF-YA leads to a decrease in the transcription of expressed genes associated with H3K4me3 and H3K9-14ac, while increasing the levels of many inactive genes. These data indicate that NF-Y is embedded in positive as well as in negative methyl histone marks, serving a dual function in transcriptional regulation, as an activator or as a repressor.