NRL and CRX Define Photoreceptor Identity and Reveal Subgroup-Specific Dependencies in Medulloblastoma.
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ABSTRACT:
SUBMITTER: Garancher A
PROVIDER: S-EPMC6368680 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We used microarrays to compare gene expression between shRNA targeting NRL and control replicates in D458Med cell line.
2018-06-08 | GSE103902 | GEO
Project description:FIZ1 (Flt-3 Interacting Zinc-finger) interacts and co-purifies with the rod-specific transcription factor NRL (Neural Retina Leucine zipper). We hypothesize that FIZ1 is part of an interface between cell-specific factors, like NRL, and more ubiquitous regulatory networks that vary the absolute expression levels of some rod-specific genes (i.e. Rhodopsin). As part of an ongoing exploration of FIZ1's role in neural retina, in vivo, we have taken the first look at FIZ1 expression in the developing mouse retina during the retinal maturation period. Using the normal C57B6 mouse as a model, multiple approaches were used including: immunoblotting, immunohistochemistry, and quantitative real-time PCR. Functional implications of FIZ1/NRL interaction, on NRL- and CRX-mediated activation of the Rhodopsin (Rho) and cGMP-phosphodiesterase beta-subunit gene (PDE6B) promoters, were examined by co-transfection assays. Immunoblot analysis revealed that FIZ1 protein levels were lowest in immature mouse neural retina (P0). FIZ1 concentration increased at least ten-fold as the neural retina matured to the adult state (P21 and later). Immunohistochemical comparison of immature post-natal and mature adult retina revealed increasing FIZ1 protein in photoreceptors, the inner plexiform layer, and the ganglion cell layer. Total retinal Fiz1 mRNA content increased as the neural retina matured. The expected increase in Rho mRNA level was also monitored as a genetic marker of photoreceptor maturation. In transient co-transfection assays of CV1 cells, FIZ1 synergized with NRL to activate transcription from the Rho and PDE6B gene promoters with some differences. In the case of the Rho promoter, FIZ1 synergized when both NRL and CRX were present. With the PDE6B promoter, FIZ1 synergized with NRL alone, and the inclusion of CRX decreased this synergy. These findings support previous evidence that FIZ1 is present in rod-photoreceptors (co-immunoprecipitation from nuclear-protein extracts with rod-specific NRL). FIZ1 expression increases in the neural retina during the retinal maturation period. Additionally, in vitro experiments demonstrate that FIZ1 has the potential to significantly increase the NRL-mediated activation of photoreceptor-specific promoters. While CRX is not a strong activator of the PDE6B promoter, alone or with NRL, CRX decreased the synergy of NRL with FIZ1.
| S-EPMC5066392 | biostudies-literature
Project description:Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.
| S-EPMC5168934 | biostudies-literature
Project description:PurposeNRL is an influential transcription factor and central to animal modeling in ophthalmology. Disrupting NRL abrogates rod development and produces an excess of S-cones (also known as "UV cones" or "short-wavelength-sensitive1 [SWS1] cones"). Strikingly, mutations in zebrafish tbx2b produce the exact opposite phenotypes (excess rods and loss of SWS1 cones). We sought to define what genetic relationship exists, if any, between these transcription factors. We also infer whether these two phenotypes (altered rod abundance and altered SWS1 cone abundance) are independent versus inter-related.MethodsZebrafish mutants were bred to disrupt nrl and tbx2b in concert. Rods and SWS1 cones were quantified and characterized at ultrastructural and transcriptional levels.ResultsConsidering single mutant zebrafish, we confirmed previously established phenotypes and noted that the number of rods lost in nrl-/- mutants is reflected by a concomitant increase in SWS1 cone abundance. The tbx2b-/- mutants present the opposite phenotype(s) but exhibit a similar trade-off in cell abundances, with lots of rods and a concomitant decrease in SWS1 cones. Double mutant nrl-/-;tbx2b-/- zebrafish recapitulate the nrl-/- mutant phenotype(s).ConclusionsThe tbx2b is thought to be required for producing SWS1 cones in zebrafish, but this can be over-ridden when nrl is absent. Regarding the altered cell abundances observed in either tbx2b-/- or nrl-/- mutants, the alterations in rod and SWS1 cones appear to not be two separate phenotypes but are instead a single intertwined outcome. The tbx2b and nrl are in an epistatic relationship, with nrl phenotypes dominating, implying that tbx2b is upstream of nrl in photoreceptor cell fate determination.
| S-EPMC10810017 | biostudies-literature
Project description:BackgroundMedulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models.MethodsTo better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry, and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups.ResultsNeoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally differentiated subpopulation in the sonic hedgehog subgroup. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally related neuron-pruning and antigen-presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB.ConclusionsThese findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment.
| S-EPMC8804892 | biostudies-literature
Project description:Four distinct subgroups of cerebellar medulloblastomas (MBs) differ in their histopathology, molecular profiles, and prognosis. c-Myc (Myc) or MycN overexpression in granule neuron progenitors (GNPs) induces Group 3 (G3) or Sonic Hedgehog (SHH) MBs, respectively. Differences in Myc and MycN transcriptional profiles depend, in part, on their interaction with Miz1, which binds strongly to Myc but not MycN, to target sites on chromatin. Myc suppresses ciliogenesis and reprograms the transcriptome of SHH-dependent GNPs through Miz1-dependent gene repression to maintain stemness. Genetic disruption of the Myc/Miz1 interaction inhibited G3 MB development. Target genes of Myc/Miz1 are repressed in human G3 MBs but not in other subgroups. Therefore, the Myc/Miz1 interaction is a defining hallmark of G3 MB development.
| S-EPMC4714043 | biostudies-literature
Project description:Medulloblastoma comprises four distinct molecular variants: WNT, SHH, Group 3, and Group 4. We analyzed alternative splicing usage in 14 normal cerebellar samples and 103 medulloblastomas of known subgroup. Medulloblastoma samples have a statistically significant increase in alternative splicing as compared to normal fetal cerebella (2.3-times; P < 6.47E-8). Splicing patterns are distinct and specific between molecular subgroups. Unsupervised hierarchical clustering of alternative splicing events accurately assigns medulloblastomas to their correct subgroup. Subgroup-specific splicing and alternative promoter usage was most prevalent in Group 3 (19.4%) and SHH (16.2%) medulloblastomas, while observed less frequently in WNT (3.2%), and Group 4 (9.3%) tumors. Functional annotation of alternatively spliced genes reveals overrepresentation of genes important for neuronal development. Alternative splicing events in medulloblastoma may be regulated in part by the correlative expression of antisense transcripts, suggesting a possible mechanism affecting subgroup-specific alternative splicing. Our results identify additional candidate markers for medulloblastoma subgroup affiliation, further support the existence of distinct subgroups of the disease, and demonstrate an additional level of transcriptional heterogeneity between medulloblastoma subgroups.
| S-EPMC3984840 | biostudies-literature
Project description:Medulloblastoma is a heterogeneous disease with at least four distinct molecular subgroups: wingless (WNT), sonic hedgehog (SHH), Group 3, and Group 4. Recently there has been considerable progress defining the molecular drivers and prognostic factors of each subgroup. However, this information has only rarely been used to stratify risk or impact treatment. The purpose of this work is to provide an update on current clinical trials that provide molecularly stratified treatment paradigms. A search was conducted on ClinicalTrials.gov using the following search terms: "medulloblastoma and subgroup", "medulloblastoma and SHH", "medulloblastoma and WNT", and "medulloblastoma and Non-WNT/Non-SHH". This search resulted in nine distinct clinical trials, five for newly diagnosed medulloblastoma and four for recurrent medulloblastoma. Four trials for newly diagnosed medulloblastoma had a component of craniospinal irradiation reduction for patients with WNT medulloblastoma. Molecularly stratified trials for recurrent medulloblastoma largely focus on SHH. As these trials are ongoing, there are limited data available. A trial in which newly-diagnosed WNT patients received modest chemotherapy without radiation has been closed to accrual due to several early failures. Phase II trials evaluating vismodegib for SHH medulloblastoma in children and adults have been disappointing. In conclusion, although there is an expanding array of clinical trials which incorporate molecular data in prescribing treatment for newly-diagnosed and recurrent medulloblastoma, treatments for these diseases are fairly uniform, with craniospinal radiation dose being the main variable. As the drivers of the distinct subgroups and their associated prognoses are better elucidated, future clinical trials and novel targeted agents are needed to improve outcomes and reduce toxicity where feasible.
| S-EPMC7237968 | biostudies-literature
Project description:BackgroundRecent technological advances have delivered the genome-wide targets of many important transcription factors (TFs). However, increasing evidence suggests that not all target sites mediate regulatory function, raising the questions of how to determine which sites are active, what are the epigenetic consequences of TF binding at these sites, and how the specificity is coded. To address these questions, we focused on CRX, a disease-associated homeodomain TF required for photoreceptor gene expression and development. Since CRX binds more than 6000 sites across the genome in the retina, we profiled chromatin landscape changes at each binding site during normal development and in the absence of CRX and interpreted the results by thorough investigation of other epigenomic datasets and sequence features.ResultsCRX is required for chromatin remodeling at only a subset of its binding sites, which undergo retina or neuronal specific activation during photoreceptor differentiation. Genes near these "CRX Dependent" sites code for proteins important for photoreceptor physiology and function, and their transcription is significantly reduced in Crx deficient retinas. In addition, the nucleotide and motif content distinguish these CRX Dependent sites from other CRX-bound sites.ConclusionsTogether, our results suggest that CRX acts only at select, uniquely-coded binding sites to accelerate chromatin remodeling during photoreceptor differentiation. This study emphasizes the importance of connecting TF binding with its functional consequences and provides a framework for making such a connection using comparative analyses of available genomic datasets. Finally, this study prioritizes sets of non-coding DNA sites for future functional interrogation and identification of mutations associated with retinal disease.
| S-EPMC6069558 | biostudies-literature
Project description:Knowledge on immune and stromal cells in medulloblastoma microenvironment is still limited as previous work was frequently restricted by low sample size and the lack of molecular subgroup information. We characterized 10 microenvironment cell populations as well as PD-L1 from gene expression in 1422 brain tumors and 763 medulloblastomas. All in all, medulloblastomas showed low expression of immune markers. Still, there were substantial differences with a clustering of medulloblastoma subgroups according to their microenvironment profile. Specifically, SHH medulloblastomas displayed strong signatures of fibroblasts, T cells and macrophages, while markers of cytotoxic lymphocytes were enriched in Group 4 tumors. PD-L1 gene expression appeared to be relatively high in single SHH and WNT cases but was undetectable by immunohistochemistry. In addition, two diverse immuno-stromal patterns were identified, indicating distinct types of local tumor immunosuppression, which were primarily controlled by either macrophage and regulatory T cell-mediated mechanisms or immunosuppressive cytokines and checkpoints, respectively. None of the immune cell signatures had an independent prognostic value in the present dataset after multiple testing correction. These results suggest a mild, but subgroup-specific infiltration of immune cells in medulloblastoma.
| S-EPMC6140816 | biostudies-other