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

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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.

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 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.

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.

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.

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.