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

Limits and advantages of abdominal ultrasonography in children with acute appendicitis syndrome

Background: Graded compression ultrasonography (US) has become the most popular technique used in suspected appendicitis and in our prospective study, we have evaluated its contribution to the diagnosis of acute appendicitis during the period 2010-2013. Materials and Methods: Four hundred and eighty children underwent urgent abdominal suspected of having acute appendicitis. Patients were divided into operated groups; (220 patients) and non-operated (260 patients) the final diagnosis was established on histopathological findings in the first group and on the phone interview in the second one. US was the sole imaging modality in all the non-operated patients and in 203 out of 220 operated ones. Seven children in the operated group underwent CT, while a second US was performed in 10 patients. Results: Acute appendicitis was confirmed in 188 operated patients while no one in the non-operated group returned to the hospital or was operated for appendicitis. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 79%, 78%, 95%, 39% and 79%, respectively. Negative appendectomy and perforation rates were 14% and 8%. Seventeen children in the operated group required a second diagnostic imaging: 7 CTs and 10 USs. All the seven CTs were consistent with appendicitis and 6 out of 10 USs showed ecographic signs of appendicitis. Conclusion: Our results support routine US in all the children with suspected appendicitis because it helps in reducing negative appendectomy and perforation rate. Moreover, a negative US does not justify a subsequent and immediate CT because clinical re-evaluation and a second US can clarify the diagnosis

Sigmoid volvulus in a neonate: case report and review of literature.

Sigmoid volvulus (SV) is an extremely rare cause of bowel obstruction in the newborn period. We report a neonatal case of SV misdiagnosed as small bowel volvulus. At laparotomy, the classical findings of SV were observed without gangrene. The operative procedure consisted of simple detorsion without sigmoidopexy

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

Topoisomerase-IIa (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

NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription

The expression of the high risk HPV18 E6 and E7 oncogenic proteins induces the transformation of epithelial cells, through the disruption of p53 and Rb function. The binding of cellular transcription factors to cis-regulatory elements in the viral Upstream Regulatory Region (URR) stimulates E6/E7 transcription. Here, we demonstrate that the CCAAT-transcription factor NF-Y binds to a non-canonical motif within the URR and activates viral gene expression. In addition, NF-Y indirectly up-regulates HPV18 transcription through the transactivation of multiple cellular transcription factors. NFYA depletion inhibits the expression of E6 and E7 genes and re-establishes functional p53. The activation of p53 target genes in turn leads to apoptotic cell death. Finally, we show that NF-YA loss sensitizes HPV18-positive cells toward the DNA damaging agent Doxorubicin, via p53-mediated transcriptional response

NF-YA splice variants have different roles on muscle differentiation

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors

Laparascopic-assisted nephroureterectomy for shaped urolithiasis and xanthogranulomatous pyelonephritis: Case report and review of literature

We report a case of xanthogranulomatous pyelonephritis (XGP) complicated by shaped urolithiasis, severe hydroureteronephrosis and kidney exclusion treated by laparoscopic-assisted nephroureterectomy. A 9 year-old boy was referred to us for recurrent episodes of urinary tract infection, abdominal pain and severe hydronephrosis. Abdominal CT and a Tc-99m MAG3 scan showed a non-functioning obstructed kidney with shaped urolithiasis of the distal ureter. XGP was suspected, and nephroureterectomy was performed by laparoscopic distal ureterectomy and open extraperitoneal nephrectomy. This technique avoided the need for a more extended nephrectomy incision or even a second iliac incision. It also ensured complete excision of the distal ureter with minimal risk of developing the ureteral stump syndrome, which sometimes follows nephroureterectomy. We believe that laparoscopic-assisted nephroureterectomy may be a suitable technique in those cases of difficult nephrectomy where a ureteral stump syndrome is likely to develop

Drosophila Helical factor is an inducible protein acting as an immune-regulated cytokine in S2 cells.

The innate immunity of Drosophila melanogaster is based on cellular and humoral components. Drosophila Helical factor (Hf), is a molecule previously discovered using an in silico approach and whose expression is controlled by the immune deficiency (Imd) pathway. Here we present evidence demonstrating that Hf is an inducible protein constitutively produced by the S2 hemocyte-derived cell line. Hf expression is stimulated by bacterial extracts that specifically trigger the Imd pathway. In absence of any bacterial challenge, the recombinant form of Hf can influence the expression of the antimicrobial peptides (AMPs) defensin but not drosomycin. These data suggest that in vitro Hf is an inducible and immune-regulated factor, with functions comparable to those of secreted vertebrate cytokine

Gain-of-function p53 mutants have widespread genomic locations partially overlapping with p63

p53 and p63 are transcription factors -TFs- playing master roles in the DNA-damage response and in the development and maintenance of pluristratified epithelia, respectively. p53 mutations are common in epithelial tumors and HaCaT keratinocytes harbor two p53 alleles -H179Y and R282Q- with gain-of-function (GOF) activity. Indeed, functional inactivation of mutp53 affects the growth rate of HaCaT. We investigated the strategy of mutp53, by performing ChIP-Seq experiments of mutp53 and p63 and analyzed the transcriptome after mutp53 inactivation. Mutp53 bind to 7135 locations in vivo, with a robust overlap with p63. De novo motifs discovery recovered a p53/p63RE with high information content in sites bound by p63 and mutp53/p63, but not by mutp53 alone: these sites are rather enriched in elements of other TFs. The HaCaT p63 locations are only partially overlapping with those of normal keratinocytes; importantly, and enriched in mutp53 sites which delineate a functionally different group of target genes. Our data favour a model whereby mutp53 GOF mutants act both by tethering growth-controlling TFs and highjacking p63 to new locations