Biology subjects

Rinaldi, V. D.

Publications and source records attributed to Rinaldi, V. D..

4 recordsLinked to original sources

bioRxiv2022

Integrative genome-scale analyses reveal post-transcriptional signatures of early human smallintestinal development in a directed differentiation organoid model

MicroRNAs (miRNAs) are important post-transcriptional gene regulators in organ development. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Our analyses showed that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/499825v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ebf48eorg.highwire.dtl.DTLVardef@4a0d9org.highwire.dtl.DTLVardef@1dc3be9org.highwire.dtl.DTLVardef@1e14207_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2020

Single Cell Analysis Reveals Multi-faceted miR-375 Regulation of the Intestinal Crypt

The role of individual miRNAs in small intestinal (SI) epithelial homeostasis is under-explored. In this study, we discovered that miR-375 is among the most enriched miRNAs in intestinal crypts and stem cells (ISCs), especially facultative ISCs. We then showed by multiple manipulations, including CRISPR/Cas9 editing, that miR-375 is strongly suppressed by Wnt-signaling. Single-cell RNA-seq analysis of SI crypt-enriched cells from miR-375 knockout (375-KO) mice revealed elevated numbers of tuft cells and increased expression of pro-proliferative genes in ISCs. Accordingly, the genetic loss of miR-375 promoted resistance to helminth infection and enhanced the regenerative response to irradiation. The conserved effects of miR-375 were confirmed by gain-of-function studies in Drosophila midgut stem cells in vivo. Moreover, functional experiments in enteroids uncovered a regulatory relationship between miR-375 and Yap1 that controls cell survival. Finally, analysis of mouse model and clinical data revealed an inverse association between miR-375 levels and intestinal tumor development. HighlightsO_LImiR-375 is one of the most enriched miRNAs in ISCs, especially facultative ISCs. C_LIO_LImiR-375 modifies tuft cell abundance and pro-proliferative gene expression in ISCs. C_LIO_LILoss of miR-375 in mice enhances the host response to helminth infection and crypt regeneration. C_LIO_LIMouse and human intestinal cancer are associated with reduced miR-375 expression. C_LI eTOC BlurbSethupathy and colleagues show that miR-375 is a Wnt-responsive, ISC-enriched miRNA that serves as a break on intestinal crypt proliferation. They also show that miR-375 modulates tuft cell abundance and pro-proliferative gene expression in ISCs, that miR-375 loss enhances the host response to helminth infection as well as crypt regeneration post-irradiation, and its reduced expression is associated with intestinal cancer.

molecular biology↗

bioRxiv2017

Pharmacological inhibition of the DNA damage checkpoint prevents radiation-induced oocyte death

Ovarian function is directly correlated with survival of the primordial follicle reserve. Women diagnosed with cancer have a primary imperative of treating the cancer, but since the resting oocytes are hypersensitive to the DNA-damaging modalities of certain chemo- and radiotherapeutic regimens, such patients face the collateral outcome of premature loss of fertility and ovarian endocrine function. Current options for fertility preservation primarily include collection and cryopreservation of oocytes or in vitro fertilized oocytes, but this necessitates a delay in cancer treatment and additional assisted reproductive technology (ART) procedures. Here, we evaluated the potential of pharmacological preservation of ovarian function by inhibiting a key element of the oocyte DNA damage checkpoint response, checkpoint kinase 2 (CHK2; CHEK2). Whereas non-lethal doses of ionizing radiation (IR) eradicate immature oocytes in wild type mice, irradiated Chk2-/- mice retain their oocytes and thus, fertility. Using an ovarian culture system, we show that transient administration of the CHK2 inhibitor 2-(4-(4-Chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide-hydrate (\"CHK2iII\") blocked activation of the CHK2 targets TRP53 and TRP63 in response to sterilizing doses of IR, and preserved oocyte viability. After transfer into sterilized host females, these ovaries proved functional and readily yielded normal offspring. These results provide experimental evidence that chemical inhibition of CHK2 is a potentially valid treatment for preserving fertility and ovarian endocrine function of women exposed to DNA-damaging cancer therapies such as IR.\n\nSummary sentenceOocytes, which are highly sensitive to DNA damage caused by certain cancer treatments, can be protected from radiation-induced death by an inhibitor of the checkpoint protein CHK2.

cancer biology↗

bioRxiv2016

Mcmdc2, a minichromosome maintenance (MCM) paralog, is required for repair of meiotic DNA breaks and homolog pairing in mouse meiosis

The mammalian Mcm-domain containing 2 (Mcmdc2) gene encodes a protein of unknown function that is homologous to the mini-chromosome maintenance family of DNA replication licensing and helicase factors. Drosophila melanogaster contains two separate genes, the \"Mei-MCMs,\" that appear to have arisen from a single ancestral Mcmdc2 gene. The Mei-MCMs are involved in promoting meiotic crossovers by blocking the anti-crossover activity of BLM helicase, a function performed by MSH4 and MSH5 in metazoans. Here, we report that MCMDC2-deficient mice of both sexes are viable but sterile. Males fail to produce spermatozoa, and formation of primordial follicles is disrupted in females. Histology and immunocytological analyses of mutant testes revealed that meiosis is arrested in Prophase I, and is characterized by persistent meiotic double-stranded DNA breaks (DSBs), failure of homologous chromosome synapsis and XY body formation, and an absence of crossing over. These phenotypes essentially phenocopy those of MSH4/5 deficient meiocytes. The data indicate that MCMDC2 is essential for invasion of homologous sequences by RAD51- and DMC1-coated ssDNA filaments, or stabilization of recombination intermediates following strand invasion, both of which are needed to drive stable homolog pairing and DSB repair via recombination in mice.

genetics↗