Systematic identification of stem-loop containing sequence families in bacterial genomes

<p>Abstract</p> <p>Background</p> <p>Analysis of non-coding sequences in several bacterial genomes brought to the identification of families of repeated sequences, able to fold as secondary structures. These sequences have often been claimed to be transcribed and fulfill a functional role. A previous systematic analysis of a representative set of 40 bacterial genomes produced a large collection of sequences, potentially able to fold as stem-loop structures (SLS). Computational analysis of these sequences was carried out by searching for families of repetitive nucleic acid elements sharing a common secondary structure.</p> <p>Results</p> <p>The initial clustering procedure identified clusters of similar sequences in 29 genomes, corresponding to about 1% of the whole population. Sequences selected in this way have a substantially higher aptitude to fold into a stable secondary structure than the initial set. Removal of redundancies and regrouping of the selected sequences resulted in a final set of 92 families, defined by HMM analysis. 25 of them include all well-known SLS containing repeats and others reported in literature, but not analyzed in detail. The remaining 67 families have not been previously described. Two thirds of the families share a common predicted secondary structure and are located within intergenic regions.</p> <p>Conclusion</p> <p>Systematic analysis of 40 bacterial genomes revealed a large number of repeated sequence families, including known and novel ones. Their predicted structure and genomic location suggest that, even in compact bacterial genomes, a relatively large fraction of the genome consists of non-protein-coding sequences, possibly functioning at the RNA level.</p

Citizen-science reveals changes in the oral microbiome in Spain through age and lifestyle factors

The relevance of the human oral microbiome to our understanding of human health has grown in recent years as microbiome studies continue to develop. Given the links of the oral cavity with the digestive, respiratory and circulatory systems, the composition of the oral microbiome is relevant beyond just oral health, impacting systemic processes across the body. However, we still have a very limited understanding about intrinsic and extrinsic factors that shape the composition of the healthy oral microbiome. Here, we followed a citizen-science approach to assess the relative impact on the oral microbiome of selected biological, social, and lifestyle factors in 1648 Spanish individuals. We found that the oral microbiome changes across age, with middle ages showing a more homogeneous composition, and older ages showing more diverse microbiomes with increased representation of typically low abundance taxa. By measuring differences within and between groups of individuals sharing a given parameter, we were able to assess the relative impact of different factors in driving specific microbial compositions. Chronic health disorders present in the analyzed population were the most impactful factors, followed by smoking and the presence of yeasts in the oral cavity. Finally, we corroborate findings in the literature that relatives tend to have more similar oral microbiomes, and show for the first time a similar effect for classmates. Multiple intrinsic and extrinsic factors jointly shape the oral microbiome. Comparative analysis of metabarcoding data from a large sample set allows us to disentangle the individual effects.We are thankful to all citizens that participated in the second edition of the “Saca La Lengua” project by contributing samples and sharing ideas (see more details here www.sacalalengua.org). In particular, for the work described here we are extremely thankful to all the students and teachers of the schools that we have visited, for their enthusiasm and questions, which helped to expand our hypotheses, to those responsible for civic centers, libraries, museums, bars, for giving us their spaces and giving us the possibility of organizing all events, and to the following national associations: Spanish Federation of Cystic Fibrosis (www.fibrosisquistica.org), Down España (www.sindromedown.net), and Federación de Asociaciones de Celíacos de España (www.celiacos.org); and local associations: Asociación Madrileña de Fibrosis Quística, Associació Catalana de Fibrosi Quística, Cocemfe Cantabria, Down Lleida, Down Bilbao, Down Vigo, Down Málaga, Associació Celíacs de Catalunya, Celíacos Euskadi, Federación de Asociaciones Celíacos Andalucía, and Asojum Murcia. Only with their effort are studies like this possible. The authors acknowledge the CRG Genomics Core Facility, CRG Bioinformatics Core Facility, CRG Biomolecular Screening and Protein Technologies Unit, CRG Communication and Public Relationships Department, and UCT ICTS High Performance Computing unit for providing access to the computing facilities. CRG authors acknowledge the Spanish Ministry for Economy, Industry and Competitiveness (MEIC) for the EMBL partnership, and Centro de Excelencia Severo Ochoa. The following reagents were obtained through BEI Resources, NIAID, NIH as part of the Human Microbiome Project: (1) Genomic DNA from Microbial Mock Community B (Even, Low Concentration), v5.1 L, for 16 S rRNA Gene Sequencing, HM-782D, and (2) Genomic DNA from Microbial Mock Community B (Staggered, Low Concentration), v5.2 L, for 16 S rRNA Gene Sequencing, HM-783D. The project was financed by the CRG through Genomics and Bioinformatics Core Facilities funds, and by the EduCaixa program through funds from the Fundación Bancaria “La Caixa”, with the participation of the Center for Research into Environmental Epidemiology (CREAL), and the “Centre d’Excellència Severo Ochoa 2013–2017” program (SEV-2012-02-08) of the Ministry of Economy and Competitiveness. Eppendorf, Illumina, and ThermoFisher sponsored the research by donating some materials and reagents. TG group acknowledges support from the Spanish Ministry of Science and Innovation for grant PGC2018-099921-B-I00, cofounded by European Regional Development Fund (ERDF); from the CERCA Program / Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423; from the European Union’s Horizon 2020 research and innovation program under the grant agreement ERC-2016-724173; from Instituto de Salud Carlos III (INB Grant PT17/0009/0023 - ISCIII-SGEFI/ERDF).Peer Reviewed"Article signat per 15 autors/es: Jesse R. Willis, Ester Saus, Susana Iraola-Guzmán, Ewa Ksiezopolska, Luca Cozzuto, Luis A. Bejarano, Nuria Andreu-Somavilla, Miriam Alloza-Trabado, Andrea Blanco, Anna Puig-Sola, Elisabetta Broglio, Carlo Carolis, Julia Ponomarenko, Jochen Hecht & Toni Gabaldón"Postprint (published version

SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML

© 2023, The Author(s). The version of record of this article, first published in [Oncogene], is available online at Publisher’s website: http://dx.doi.org/10.1038/s41388-023-02840-1KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3β, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia. [Abstract copyright: © 2023. The Author(s).

Fine tuning the extracellular environment accelerates the derivation of kidney organoids from human pluripotent stem cells

The generation of organoids is one of the biggest scientific advances in regenerative medicine. Here, by lengthening the time that human pluripotent stem cells (hPSCs) were exposed to a three-dimensional microenvironment, and by applying defined renal inductive signals, we generated kidney organoids that transcriptomically matched second-trimester human fetal kidneys. We validated these results using ex vivo and in vitro assays that model renal development. Furthermore, we developed a transplantation method that utilizes the chick chorioallantoic membrane. This approach created a soft in vivo microenvironment that promoted the growth and differentiation of implanted kidney organoids, as well as providing a vascular component. The stiffness of the in ovo chorioallantoic membrane microenvironment was recapitulated in vitro by fabricating compliant hydrogels. These biomaterials promoted the efficient generation of renal vesicles and nephron structures, demonstrating that a soft environment accelerates the differentiation of hPSC-derived kidney organoids

SET-PP2A complex as a new therapeutic target in KMT2A (MLL) rearranged AML

KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3β, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia

SET-PP2A Complex as a New Therapeutic Target in KMT2A (MLL) Rearranged AML

KMT2A-rearranged (KMT2A-R) is an aggressive and chemo-refractory acute leukemia which mostly affects children. Transcriptomics-based characterization and chemical interrogation identified kinases as key drivers of survival and drug resistance in KMT2A-R leukemia. In contrast, the contribution and regulation of phosphatases is unknown. In this study we uncover the essential role and underlying mechanisms of SET, the endogenous inhibitor of Ser/Thr phosphatase PP2A, in KMT2A-R-leukemia. Investigation of SET expression in acute myeloid leukemia (AML) samples demonstrated that SET is overexpressed, and elevated expression of SET is correlated with poor prognosis and with the expression of MEIS and HOXA genes in AML patients. Silencing SET specifically abolished the clonogenic ability of KMT2A-R leukemic cells and the transcription of KMT2A targets genes HOXA9 and HOXA10. Subsequent mechanistic investigations showed that SET interacts with both KMT2A wild type and fusion proteins, and it is recruited to the HOXA10 promoter. Pharmacological inhibition of SET by FTY720 disrupted SET-PP2A interaction leading to cell cycle arrest and increased sensitivity to chemotherapy in KMT2A-R-leukemic models. Phospho-proteomic analyses revealed that FTY720 reduced the activity of kinases regulated by PP2A, including ERK1, GSK3β, AURB and PLK1 and led to suppression of MYC, supporting the hypothesis of a feedback loop among PP2A, AURB, PLK1, MYC, and SET. Our findings illustrate that SET is a novel player in KMT2A-R leukemia and they provide evidence that SET antagonism could serve as a novel strategy to treat this aggressive leukemia

Direct targets of Klf5 transcription factor contribute to the maintenance of mouse embryonic stem cell undifferentiated state

<p>Abstract</p> <p>Background</p> <p>A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed.</p> <p>Results</p> <p>By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes.</p> <p>Conclusions</p> <p>Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype.</p> <p>See: <url>http://www.biomedcental.com/1741-7007/8/125</url></p

Identification of novel regulatory elements in sequenced genomes by clustering and other data mining methods

In bacterial genomes a fraction of transcribed sequences do not code for proteins or structural RNAs, but have been shown to be involved in fundamental processes, such as regulation of gene expression, mRNA processing and stability or structural RNA maturation. In this thesis a systematic procedure to identify and classify families of repeated sequences sharing a common RNA secondary structure was applied to the study of 40 bacterial genomes. Sequences able to fold in a stable stem loop structure were clustered according to sequence similarity, and grouped within homogeneous families. The study led to the identification of 57 families of repeated sequences, sharing a common secondary structure and potentially coding for structured RNAs. All previously known such families have been detected by the used procedure, and are listed within the final set, together with 37 novel ones. Their location in relation to protein coding genes was evaluated, and a correlation was found between structure and positioning within intergenic regions. A new software tool is also described, Scaffolder, designed to help in high-throughput de novo genome sequencing by finding connections between contigs produced by random shotgun sequencing, and assisting the researcher in the whole process. The software, accessible both as a command line tool and as a web application, can guide all the final phases of genome assembly by storing the current assembly status, displaying networks of connected contigs and untangling multiply connected ones by a combination of computational and experimental procedures

Effects of a 8-oxoadenosine Incorporation on Quadruplex structures: thermal stabilities and structural studies

The effects of incorporation of 8-oxoadenosine in two different truncations of human telomeric sequence forming quadruplex structures are reported. In order to characterize their structures, a combination of NMR and UV spectroscopy and computational techniques were used. Both oligonucleotides have been found to form four-fold sym. quadruplex structures. As a tautomeric equil. between keto and enol forms of 8-oxoadenosine may establish in soln. and intrinsic stabilities effects, such as internal H-bonds, for example, may det. the predominance of some particular tautomer, mol. modeling studies were performed on quadruplex structures contg. both the tautomeric forms. Both mols. resulted to be thermally less stable than the natural