Concurrent inhibition of enzymatic activity and NF-Y-mediated transcription of Topoisomerase-IIα by bis-DemethoxyCurcumin in cancer cells.

Topoisomerases-IIα (TOP2A) enzyme is essential for cell viability due to its fundamental role in DNA metabolism and in chromatin organization during interphase and mitosis. TOP2A expression is finely regulated at the transcriptional level through the binding of the CCAAT-transcription factor NF-Y to its promoter. Overexpression and/or amplification of TOP2A have been observed in many types of cancers. For this reason, TOP2A is the target of the most widely successful drugs in cancer chemotherapy, such as TOP2A poisons, which stabilize TOP2A-DNA cleavage complexes and create DSBs, leading to chromosome damage and cell death. We previously reported that the Curcumin-derivative bis-DemethoxyCurcumin (bDMC) is an anti-proliferative agent that inhibits cell growth by concomitant G1/S and G2/M arrest. Here we showed that bDMC irreversibly induces DSBs in cancer cells, but not in normal cells, by targeting TOP2A activity and expression. TOP2A ablation by siRNA corroborates its contribution to apoptosis induced by bDMC. Short-term exposure to bDMC induces retention of TOP2A-DNA intermediates, while longer exposure inhibits TOP2A transcription by affecting expression and sub-cellular localization of NF-Y subunits. ChIP analysis highlighted reduced recruitment of NF-Y to TOP2A regulatory regions, concomitantly to histone deacetylation and decreased gene transcription. Our findings suggest that the dual activity of bDMC on TOP2A represents a novel therapeutic strategy to induce persistent apoptosis in cancer cells and identify NF-Y regulation as a promising approach in anti-cancer therapy.

HSP-CBF is an NF-Y-dependent coactivator of the heat shock promoters CCAAT boxes.

The cellular response to toxic stimuli is elicited through the expression of heat shock proteins, a transcriptional process that relies upon conserved DNA elements in the promoters: the Heat Shock Elements, activated by the heat shock factors, and the CCAAT boxes. The identity of the CCAAT activator(s) is unclear because two distinct entities, NF-Y and HSP-CBF, have been implicated in the HSP70 system. The former is a conserved ubiquitous trimer containing histone-like subunits, the latter a 110-kDa protein with an acidic N-terminal. We analyzed two CCAAT-containing promoters, HSP70 and HSP40, with recombinant NF-Y and HSP-CBF using electrophoretic mobility shift assay, protein-protein interactions, transfections and chromatin immunoprecipitation assays (ChIP) assays. Both recognize a common DNA-binding protein in nuclear extracts, identified in vitro and in vivo as NF-Y. Both CCAAT boxes show high affinity for recombinant NF-Y but not for HSP-CBF. However, HSP-CBF does activate HSP70 and HSP40 transcription under basal and heat shocked conditions; for doing so, it requires an intact NF-Y trimer as judged by cotransfections with a diagnostic NF-YA dominant negative vector. HSP-CBF interacts in solution and on DNA with the NF-Y trimer through an evolutionary conserved region. In yeast two-hybrid assays HSP-CBF interacts with NF-YB. These data implicate HSP-CBF as a non-DNA binding coactivator of heat shock genes that act on a DNA-bound NF-Y.

Cloning and characterization of the histone-fold proteins YBL1 and YCL1.

Histones are among the most conserved proteins in evolution, sharing a histone fold motif. A number of additional histonic proteins exist and are involved in the process of transcriptional regulation. We describe here the identification, cloning and characterization of two small members of the H2A-H2B sub-family (YBL1 and YCL1) related to the NF-YB and NF-YC subunits of the CCAAT-binding activator NF-Y and to the TATA-binding protein (TBP) binding repressor NC2. Unlike the latters, YBL1 and YCL1 have no intrinsic CCAAT or TATA-binding capacity. In nucleosome reconstitution assays, they can form complexes with histones in solution and on DNA and they are part of relatively large complexes, as determined by glycerol gradient experiments. Our data support the idea that YBL1 and YCL1 are divergent with respect to NF-YB and NF-YC for specific functions, but have coevolved the capacity to interact with nucleosomal structures.

Dissection of the NF-Y transcriptional activation potential.

NF-Y is a trimeric CCAAT-binding factor with histone fold subunits (NF-YB/NF-YC) and bipartite activation domains located on NF-YA and NF-YC. We reconstituted the NF-Y activation potential in vivo with GAL4 DBD fusions. In the GAL4-YA configuration, activation requires co-expression of the three subunits; with GAL4-YB and GAL4-YC, transfections of the histone fold partners are sufficient, provided that the Q-rich domain of NF-YC is present. Combinations of mutants indicate that the Q-rich domains of NF-YA and NF-YC are redundant in the trimeric complex. Glutamines 101 and 102 of NF-YA are required for activity. We assayed NF-Y on different promoter targets, containing single or multiple GAL4 sites: whereas on a single site NF-Y is nearly as powerful as VP16, on multiple sites neither synergistic nor additive effects are observed. NF-Y activates TATA and Inr core elements and the overall potency is in the same range as other Q-rich and Pro-rich activation domains. These results represent the first in vivo evidence of subunit interactions studies and further support the hypothesis that NF-Y is a general promoter organizer rather than a brute activator.