Gabriele Bucci

Jmjd3 contributes to the control of gene expression in LPS-activated macrophages

Jmjd3, a JmjC family histone demethylase, is induced by the transcription factor NF‐kB in response to microbial stimuli. Jmjd3 erases H3K27me3, a histone mark associated with transcriptional repression and involved in lineage determination. However, the specific contribution of Jmjd3 induction and H3K27me3 demethylation to inflammatory gene expression remains unknown. Using chromatin immunoprecipitation‐sequencing we found that Jmjd3 is preferentially recruited to transcription start sites characterized by high levels of H3K4me3, a marker of gene activity, and RNA polymerase II (Pol_II). Moreover, 70% of lipopolysaccharide (LPS)‐inducible genes were found to be Jmjd3 targets. Although most Jmjd3 target genes were unaffected by its deletion, a few hundred genes, including inducible inflammatory genes, showed moderately impaired Pol_II recruitment and transcription. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analysed were uncoupled from measurable effects on this histone mark. These data show that Jmjd3 fine‐tunes the transcriptional output of LPS‐activated macrophages in an H3K27 demethylation‐independent manner.

Cell Reprogramming Requires Silencing of a Core Subset of Polycomb Targets

Transcription factor (TF)–induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF–induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF–induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF–dependent cell reprogramming.

Differentiation-associated microRNAs antagonize the Rb–E2F pathway to restrict proliferation

Transcriptional regulation by Rb–E2F and posttranscriptional regulation by microRNAs control the expression of cell cycle and DNA replication genes and restrict cellular proliferation.

Bone Morphogenetic Protein/SMAD Signaling Orients Cell Fate Decision by Impairing KSRP-Dependent MicroRNA Maturation

MicroRNAs (miRNAs) are essential regulators of development, physiology, and evolution, and their biogenesis is strictly controlled at multiple levels. Regulatory proteins, such as KSRP, modulate rates and timing of enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Here, we show that KSRP silencing in mesenchymal C2C12 cells produces a change in the transcriptome largely overlapping that induced by bone morphogenetic protein 2 (BMP2) signaling activation. This induces osteoblastic differentiation while preventing myogenic differentiation. KSRP silencing- and BMP2-dependent myogenic miRNA (myomiR) maturation blockade is required for osteoblastic differentiation of C2C12 cells. Our results demonstrate that phosphorylated R-SMAD proteins, the transducers of BMP2 signal, associate with phosphorylated KSRP and block its interaction with primary myomiRs. This abrogates KSRP-dependent myomiR maturation, with SMAD4, SMAD5, and SMAD9 silencing being able to rescue KSRP function. Thus, SMAD-induced blockade of KSRP-dependent myomiR maturation is critical for orienting C2C12 cell differentiation toward osteoblastic lineage.

AnnotateGenomicRegions: a web application

BackgroundModern genomic technologies produce large amounts of data that can be mapped to specific regions in the genome. Among the first steps in interpreting the results is annotation of genomic regions with known features such as genes, promoters, CpG islands etc. Several tools have been published to perform this task. However, using these tools often requires a significant amount of bioinformatics skills and/or downloading and installing dedicated software.ResultsHere we present AnnotateGenomicRegions, a web application that accepts genomic regions as input and outputs a selection of overlapping and/or neighboring genome annotations. Supported organisms include human (hg18, hg19), mouse (mm8, mm9, mm10), zebrafish (danRer7), and Saccharomyces cerevisiae (sacCer2, sacCer3). AnnotateGenomicRegions is accessible online on a public server or can be installed locally. Some frequently used annotations and genomes are embedded in the application while custom annotations may be added by the user.ConclusionsThe increasing spread of genomic technologies generates the need for a simple-to-use annotation tool for genomic regions that can be used by biologists and bioinformaticians alike. AnnotateGenomicRegions meets this demand. AnnotateGenomicRegions is an open-source web application that can be installed on any personal computer or institute server. AnnotateGenomicRegions is available at: http://cru.genomics.iit.it/AnnotateGenomicRegions.

KSRP silencing favors neural differentiation of P19 teratocarcinoma cells.

Understanding the molecular mechanisms that control the balance between multipotency and differentiation is of great importance to elucidate the genesis of both developmental disorders and cell transformation events. To investigate the role of the RNA binding protein KSRP in controlling neural differentiation, we used the P19 embryonal carcinoma cell line that is able to differentiate into neuron-like cells under appropriate culture conditions. We have recently reported that KSRP controls the differentiative fate of multipotent mesenchymal cells owing to its ability to promote decay of unstable transcripts and to favor maturation of selected micro-RNAs (miRNAs) from precursors. Here we report that KSRP silencing in P19 cells favors neural differentiation increasing the expression of neuronal markers. Further, the expression of two master transcriptional regulators of neurogenesis, ASCL1 and JMJD3, was enhanced while the maturation of miR-200 family members from precursors was impaired in KSRP knockdown cells. These molecular changes can contribute to the reshaping of P19 cells transcriptome that follows KSRP silencing. Our data suggests that KSRP function is required to maintain P19 cells in a multipotent undifferentiated state and that its inactivation can orient cells towards neural differentiation.

Retina-derived POU domain factor 1 coordinates expression of genes relevant to renal and neuronal development

Retina-derived POU domain Factor 1 (RPF-1), a member of POU transcription factor family, is encoded by POU6F2 gene, addressed by interstitial deletions at chromosome 7p14 in Wilms tumor (WT). Its expression has been detected in developing kidney and nervous system, suggesting an early role for this gene in regulating development of these organs. To investigate into its functions and determine its role in transcriptional regulation, we generated an inducible stable transfectant from HEK293 cells. RPF-1 showed nuclear localization, elevated stability, and transactivation of promoters featuring POU consensus sites, and led to reduced cell proliferation and in vivo tumor growth. By addressing the whole transcriptome regulated by its induction, we could detect a gross alteration of gene expression that is consistent with promoter occupancy predicted by genome-wide Chip-chip analysis. Comparison of bound regulatory regions with differentially expressed genes allowed identification of 217 candidate targets. Enrichment of divergent octamers in predicted regulatory regions revealed promiscuous binding to bipartite POUS and POUH consensus half-sites with intervening spacers. Gel-shift competition assay confirmed the specificity of RPF-1 binding to consensus motifs, and demonstrated that the Ser-rich region upstream of the POU domain is indispensable to achieve DNA-binding. Promoter-reporter activity addressing a few target genes indicated a dependence by RPF-1 on transcriptional response. In agreement with its expression in developing kidney and nervous system, the induced transcriptome appears to indicate a function for this protein in early renal differentiation and neuronal cell fate, providing a resource for understanding its role in the processes thereby regulated.

Abstract 5280: Identification of serum circulating non-coding RNAs as diagnostic markers by Next Generation Sequencing (NGS) and low-density array.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background. High incidence and mortality rate tumours present a more favourable prognosis if diagnosed in early stages (Jemal A. et al, 2011). Screening programs based on effective radiological techniques (e.g. mammography or low dose spiral CT) promise a mortality reduction, but their application on large scale suffers of disadvantages such as the use of ionizing radiation, overdiagnosis risk and high costs. Thus, the identification of molecular markers for cancer early diagnosis, especially if detectable through a simple non-invasive blood test, would provide powerful tools for the improvement of screening programs. Recently, a novel class of small non-coding RNAs, namely microRNAs, has been identified. MiRNA are extremely stable in biological fluids and their levels are different in malignant vs. control sera, making them a promising class of biological markers for early diagnosis. Indeed, studies from our and other labs led to the identification of “miRNA signatures” able to predict the presence of tumours even within a population of asymptomatic high-risk individuals (Bianchi F. et al., 2011; Boeri M. et al., 2011). However, methods used in these studies rely on QPCR approaches, which require previous knowledge on the molecules to be investigated. Indeed, the real complexity of circulating RNAs is still obscure, comprising other classes of molecules not yet investigated that could behave as novel biomarkers. The recent development of high-throughput sequencing technologies (Next Generation Sequencing - NGS) provided instruments to reveal the complexity of nucleic acids, but a complete protocol for the identification of circulating RNAs has not been established nor compared with current available approaches (i.e. QPCR). Aim: Our aim is to develop a simultaneous and comparative protocol for serum miRNA analysis, which encompasses both high-throughput QPCR and sequencing analysis in order to reveal the complexity of circulating small-RNAs. Methods: Total RNA has been isolated from the serum of healthy donors and analysed simultaneously by low-density QPCR (TaqMan Low Density Array, Life Technologies) and by NGS (Illumina). Results: A step-wise protocol to simultaneously analyze non-coding RNAs using NGS technology (Illumina) and high-throughput QPCR has been developed. The protocol has been optimized to allow both analyses using even limited amount of serum (up to 1mL). Both platforms resulted as highly efficient and quantitative, although NGS manages to score many molecules and RNA species that could not be analysed by current QPCR platforms, thus revealing a major complexity of circulating non-coding RNAs. Conclusion: We proposed that the combined use of NGS and QPCR platforms would allow a wider and more detailed analysis of circulating RNAs, expanding our ability to fish out robust and efficient molecular markers for early detection of cancer. Citation Format: Francesca Montani, Rose Mary Carletti, Matteo J. Marzi, Gabriele Bucci, Francesco Nicassio, Pier Paolo Di Fiore. Identification of serum circulating non-coding RNAs as diagnostic markers by Next Generation Sequencing (NGS) and low-density array. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5280. doi:10.1158/1538-7445.AM2013-5280

Abstract 1423: Transcription induced by the POU6F2-encoded RPF-1 and genome-wide screening of promoter occupancy unveil a potential regulatory role in kidney embryogenesis

Wilms Tumor (WT, or nephroblastoma), a tumor of embryonic origin, is the most frequent genitourinary tumor in childhood. Current view of this malignancy point out to a common origin with the fetal developing kidney. Undifferentiated mesenchymal cell types and differentiated immature structures are in fact present in WTs, whose gene expression tend to overlap with the early nephrogenesis transcriptome. Remarkably, several factors guiding the development of kidney are overexpressed in WT specimens. WT is also characterized by alterations at several genomic loci and 20% patients exhibit LOH at chr7p. We were able to identify the smallest overlapping region at 7p14 encoding the POU6F2 gene, of the POU family of transcription factors regulating developmental and biological processes. Moreover, expression of POU6F2 mouse homolog was shown during nephrogenesis. These finding may be indicative of a role for this gene in kidney development, and since perturbation of this process contribute to WT, we addressed whether the Retina-derived POU domain Factor-1 (RPF-1, product of POU6F2) is a bonafide transcription factor participating in the transcriptional program that drive nephrogenesis. By addressing RPF-1 protein-DNA interaction we could confirm functional interaction with consensus octamers for POU proteins. Following its induction in human embryo kidney (HEK293) stable transfectants, the pattern of transcriptional targets regulated was evaluated and about 750 genes showed significant modulation (p 80% genes, suggesting for RPF-1 a low specificity in its binding to DNA. Top biological processes emerging from functional annotation analysis of direct transcriptional targets included kidney, metanephron, tube and ureteric bud development, primary germ layer formation, cell fate commitment, and neurogenesis as well. Axonal guidance, ephrin signalling, and transcription were top canonical functions clustered by gene ontology. Accordingly, regulators of nephrogenesis, such as EYA1, PDGFD, SLIT2 and ROBO2, myogenic differentiator MEF2C, neurite regulator MAP2, and GATA2 stem cell differentiator, are all regulated through promoter featuring closely spaced octamers. Overall, our results suggest a direct implication of RPF-1 into the transcriptional program guiding kidney development, and perturbation of its function during embryogenesis may contribute to the development of nephroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1423. doi:1538-7445.AM2012-1423