High-throughput RNA sequencing identifies the miRNA expression profile, target genes, and molecular pathways contributing to growth of sporadic vestibular schwannomas.

PURPOSE: To assess the differences in the miRNA expression profile between small (stage I Koos classification) and large solid vestibular schwannoma (VS) tumors, using the RNA-seq technique. METHODS: Twenty tumor samples (10 small and 10 large tumors) were collected from patients operated for VS in a Tertiary Academic Center. Tumor miRNA expression was analyzed using high-throughput RNA sequencing (RNA-seq) technique, with NovaSeq 6000 Illumina system. Bioinformatics analysis was done using statistical software R. Gene enrichment and functional analysis was performed using miRTargetLink 2.0 and DIANA miRpath 3.0 online tools. RESULTS: We identified 9 differentially expressed miRNAs in large VS samples: miR-7, miR-142 (-3p and -5p), miR-155, miR-342, miR-1269, miR-4664, and miR-6503 were upregulated, whereas miR-204 was significantly down-regulated in comparison to small VS samples. Gene enrichment analysis showed that the most enriched target genes were SCD, TMEM43, LMNB2, JARID2, and CCND1. The most enriched functional pathways were associated with lipid metabolism, along with signaling pathways such as Hippo and FOXO signaling pathway. CONCLUSION: We identified a set of 9 miRNAs that are significantly deregulated in large VS in comparison to small, intracanalicular tumors. The functional enrichment analysis of these miRNAs suggests novel mechanisms, such as that lipid metabolism, as well as Hippo and FOxO signaling pathways that may play an important role in VS growth regulation.

Targeted sequencing of cancer-related genes reveals a recurrent TOP2A variant which affects DNA binding and coincides with global transcriptional changes in glioblastoma.

High-grade gliomas are aggressive, deadly primary brain tumors. Median survival of patients with glioblastoma (GBM, WHO grade 4) is 14 months and <10% of patients survive 2 years. Despite improved surgical strategies and forceful radiotherapy and chemotherapy, the prognosis of GBM patients is poor and did not improve over decades. We performed targeted next-generation sequencing with a custom panel of 664 cancer- and epigenetics-related genes, and searched for somatic and germline variants in 180 gliomas of different WHO grades. Herein, we focus on 135 GBM IDH-wild type samples. In parallel, mRNA sequencing was accomplished to detect transcriptomic abnormalities. We present the genomic alterations in high-grade gliomas and the associated transcriptomic patterns. Computational analyses and biochemical assays showed the influence of TOP2A variants on enzyme activities. In 4/135 IDH-wild type GBMs we found a novel, recurrent mutation in the TOP2A gene encoding topoisomerase 2A (allele frequency [AF] = 0.03, 4/135 samples). Biochemical assays with recombinant, wild type (WT) and variant proteins demonstrated stronger DNA binding and relaxation activity of the variant protein. GBM patients carrying the altered TOP2A had shorter overall survival (median OS 150 vs 500 days, P = .0018). In the GBMs with the TOP2A variant we found transcriptomic alterations consistent with splicing dysregulation. luA novel, recurrent TOP2A mutation, which was found exclusively in four GBMs, results in the TOP2A E948Q variant with altered DNA binding and relaxation activities. The deleterious TOP2A mutation resulting in transcription deregulation in GBMs may contribute to disease pathology.

Genetic Risk Factors for Neurological Disorders in Children with Adverse Events Following Immunization: A Descriptive Study of a Polish Case Series.

Studies conducted on large populations show a lack of connection between vaccination and serious neurological symptoms. However, there are isolated cases that indicate such a relationship. These reports on adverse effects following immunization (AEFI) reduce social confidence in vaccination; however, their background may be rare genetic defects. The aim of the presented study was to examine if neurological AEFI in children may be associated with variants in genes related to neurodevelopment. To identify such possible associations, a descriptive study of the Polish case series was conducted. We performed next-generation sequencing in patients who, up to 4 weeks of injection of any vaccine, manifested neurological AEFI. We included 23 previously normally developing children with first seizures that occurred after vaccination. We identified pathogenic/likely pathogenic variants in genes engaged in neurodevelopment in nine patients and variants of uncertain significance in another nine patients. The mutated genes belonged to the group of genes related to epilepsy syndromes/epileptic encephalopathy. We showed that AEFI might have a genetic background. We hypothesized that in some AEFI patients, the vaccine might only trigger neurological symptoms that would have been manifested anyway as a result of a pathogenic variant in a gene engaged in neurodevelopment.

The Epidermal Transcriptome Analysis of a Novel c.639_642dup LORICRIN Variant-Delineation of the Loricrin Keratoderma Pathology.

Loricrin keratoderma (LK) is a rare autosomal dominant genodermatosis caused by LORICRIN gene mutations. The pathogenesis of the disease is not yet fully understood. So far, only 10 pathogenic variants in LORICRIN have been described, with all of them but one being deletions or insertions. The significance of rare nonsense variants remains unclear. Furthermore, no data regarding the RNA expression in affected patients are available. The aim of this study is to describe the two variants in the LORICRIN gene found in two distinct families: the novel pathogenic variant c.639_642dup and a rare c.10C > T (p.Gln4Ter) of unknown significance. We also present the results of the transcriptome analysis of the lesional loricrin keratoderma epidermis of a patient with c.639_642dup. We show that in the LK lesion, the genes associated with epidermis development and keratocyte differentiation are upregulated, while genes engaged in cell adhesion, differentiation developmental processes, ion homeostasis and transport, signaling and cell communication are downregulated. In the context of the p.Gln4Ter clinical significance evaluation, we provide data indicating that LORICRIN haploinsufficiency has no skin consequences. Our results give further insight into the pathogenesis of LK, which may have therapeutic implications in the future and important significance in the context of genetic counseling.

Defining molecular identity and fates of CNS-border associated macrophages after ischemic stroke in rodents and humans.

Central nervous system (CNS)-border associated macrophages (BAMs) maintain their steady-state population during adulthood and are not replaced by circulating monocytes under physiological conditions. Their roles in CNS integrity and functions under pathological conditions remain largely unknown. Until recently, BAMs and microglia could not be unequivocally distinguished due to expression of common macrophage markers. We investigated the transcriptional profiles of immunosorted BAMs from rat sham-operated and ischemic brains using RNA sequencing. We found that BAMs express the distinct transcriptional signature than microglia and infiltrating macrophages. The enrichment of functional groups associated with the cell cycle in CD163+ cells isolated 3 days after the ischemic injury indicates the proliferative capacity of these cells. The increased number of CD163+ cells 3 days post-ischemia was corroborated by flow cytometry and detecting the increased number of CD163+ cells positive for a proliferation marker Ki67 at perivascular spaces. CD163+ cells in the ischemic brains up-regulated many inflammatory genes and parenchymal CD163+ cells expressed iNOS, which indicates acquisition of a pro-inflammatory phenotype. In mice, BAMs typically express CD206 and we found a subset of these cells expressing CD169. Chimeric mice generated by transplanting bone marrow of donor Cx3cr1gfpCCR2rfp mice to wild type hosts showed an increased number of CX3CR1+CD169+ perivascular macrophages 3 days post-ischemia. Furthermore, these cells accumulated in the brain parenchyma and we detected replacement of perivascular macrophages by peripheral monocytic cells in the sub-acute phase of stroke. In line with the animal results, post-mortem brain samples from human ischemic stroke cases showed time-dependent accumulation of CD163+ cells in the ischemic parenchyma. Our findings indicate a unique transcriptional signature of BAMs, their local proliferation and migration of inflammatory BAMs to the brain parenchyma after stroke in animal models and humans.

Antifungal Agent 4-AN Changes the Genome-Wide Expression Profile, Downregulates Virulence-Associated Genes and Induces Necrosis in Candida albicans Cells.

In the light of the increasing occurrence of antifungal resistance, there is an urgent need to search for new therapeutic strategies to overcome this phenomenon. One of the applied approaches is the synthesis of small-molecule compounds showing antifungal properties. Here we present a continuation of the research on the recently discovered anti-Candida albicans agent 4-AN. Using next generation sequencing and transcriptional analysis, we revealed that the treatment of C. albicans with 4-AN can change the expression profile of a large number of genes. The highest upregulation was observed in the case of genes involved in cell stress, while the highest downregulation was shown for genes coding sugar transporters. Real-time PCR analysis revealed 4-AN mediated reduction of the relative expression of genes engaged in fungal virulence (ALS1, ALS3, BCR1, CPH1, ECE1, EFG1, HWP1, HYR1 and SAP1). The determination of the fractional inhibitory concentration index (FICI) showed that the combination of 4-AN with amphotericin B is synergistic. Finally, flow cytometry analysis revealed that the compound induces mainly necrosis in C. albicans cells.

Novel TG-FGFR1 and TRIM33-NTRK1 transcript fusions in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is most common among all thyroid cancers. Multiple genomic alterations occur in PTC, and gene rearrangements are one of them. Here we screened 14 tumors for novel fusion transcripts by RNA-Seq. Two samples harboring RET/PTC1 and RET/PTC3 rearrangements were positive controls whereas the remaining ones were negative regarding the common PTC alterations. We used Sanger sequencing to validate potential fusions. We detected 2 novel potentially oncogenic transcript fusions: TG-FGFR1 and TRIM33-NTRK1. We detected 4 novel fusion transcripts of unknown significance accompanying the TRIM33-NTRK1 fusion: ZSWIM5-TP53BP2, TAF4B-WDR1, ABI2-MTA3, and ARID1B-PSMA1. Apart from confirming the presence of RET/PTC1 and RET/PTC3 in positive control samples, we also detected known oncogenic fusion transcripts in remaining samples: TFG-NTRK1, ETV6-NTRK3, MKRN1-BRAF, EML4-ALK, and novel isoform of CCDC6-RET.

Dissecting functional phenotypes of microglia and macrophages in the rat brain after transient cerebral ischemia.

Ischemic brain injury causes local inflammation, which involves activation of resident microglia, leukocyte, and monocyte infiltration. Involvement of peripheral immune cells in ischemia-induced damage and repair is debatable. Using flow cytometry, gene expression profiling, and immunocytochemistry, we show that microglia predominate in the ischemic brain and express inflammation mediators at Day 1 after transient middle cerebral artery occlusion (MCAo) in rats. At Day 3, both resident microglia and bone marrow (BM)-derived macrophages are detected in the ischemic hemispheres and display unique transcriptomic profiles. Functional groups enriched in BM-macrophages are indicative of the pro-regenerative, immunosuppressive phenotype. Transient depletion of peripheral macrophages with clodronate-filled liposomes reduced the number of Arg1+ Iba1+ expressing cells in the ischemic brain. The analysis of microglia and macrophage signature genes shows that each cell type maintains the expression of their identity genes, even if gene expression is modified in a response to environmental clues. At Day 7, infiltrating BM-macrophages exhibit the reduced expression of Arg1, the elevated expression of iNos and many inflammatory genes, as shown by RNA sequencing. This is consistent with their switch toward a pro-inflammatory phenotype. We propose that BM-macrophages recruited to the injured brain early after ischemia could contribute to functional recovery after stroke, but they switch toward a pro-inflammatory phenotype in the ischemic parenchyma. Our results point to the detrimental role of microglia in an ischemic brain and the primarily pro-regenerative role of infiltrating BM-macrophages.

Open chromatin landscape of rat microglia upon proinvasive or inflammatory polarization.

Microglia are brain-resident, myeloid cells that play important roles in health and brain pathologies. Herein, we report a comprehensive, replicated, false discovery rate-controlled dataset of DNase-hypersensitive (DHS) open chromatin regions for rat microglia. We compared the open chromatin landscapes in untreated primary microglial cultures and cultures stimulated for 6 hr with either glioma-conditioned medium (GCM) or lipopolysaccharide (LPS). Glioma-secreted factors induce proinvasive and immunosuppressive activation of microglia, and these cells then promote tumor growth. The open chromatin landscape of the rat microglia consisted of 126,640 reproducible DHS regions, among which 2,303 and 12,357 showed a significant change in openness following stimulation with GCM or LPS, respectively. Active genes exhibited constitutively open promoters, but there was no direct dependence between the aggregated openness of DHS regions near a gene and its expression. Individual regions mapped to the same gene often presented different patterns of openness changes. GCM-regulated DHS regions were more frequent in areas away from gene bodies, while LPS-regulated regions were more frequent in introns. GCM and LPS differentially affected the openness of regions mapped to immune checkpoint genes. The two treatments differentially affected the aggregated openness of regions mapped to genes in the Toll-like receptor signaling and axon guidance pathways, suggesting that the molecular machinery used by migrating microglia is similar to that of growing axons and that modulation of these pathways is instrumental in the induction of proinvasive polarization of microglia by glioma. Our dataset of open chromatin regions paves the way for studies of gene regulation in rat microglia.

Consequences of IDH1/2 Mutations in Gliomas and an Assessment of Inhibitors Targeting Mutated IDH Proteins.

Isocitrate dehydrogenases (IDH) 1 and 2 are key metabolic enzymes that generate reduced nicotinamide adenine dinucleotide phosphate (NADPH) to maintain a pool of reduced glutathione and peroxiredoxin, and produce α-ketoglutarate, a co-factor of numerous enzymes. IDH1/2 is mutated in ~70⁻80% of lower-grade gliomas and the majority of secondary glioblastomas. The mutant IDH1 (R132H), in addition to losing its normal catalytic activity, gains the function of producing the d-(R)-2-hydroxyglutarate (2-HG). Overproduction of 2-HG in cancer cells interferes with cellular metabolism and inhibits histone and DNA demethylases, which results in histone and DNA hypermethylation and the blockade of cellular differentiation. We summarize recent findings characterizing molecular mechanisms underlying oncogenic alterations associated with mutated IDH1/2, and their impact on tumor microenvironment and antitumor immunity. Isoform-selective IDH inhibitors which suppress 2-HG production and induce antitumor responses in cells with IDH1 and IDH2 mutations were developed and validated in preclinical settings. Inhibitors of mutated IDH1/2 enzymes entered clinical trials and represent a novel drug class for targeted therapy of gliomas. We describe the development of small-molecule compounds and peptide vaccines targeting IDH-mutant gliomas and the results of their testing in preclinical and clinical studies. All those results support the translational potential of strategies targeting gliomas carrying IDH1 mutations.

RRAD, IL4I1, CDKN1A, and SERPINE1 genes are potentially co-regulated by NF-κB and p53 transcription factors in cells exposed to high doses of ionizing radiation.

BACKGROUND: The cellular response to ionizing radiation involves activation of p53-dependent pathways and activation of the atypical NF-κB pathway. The crosstalk between these two transcriptional networks include (co)regulation of common gene targets. Here we looked for novel genes potentially (co)regulated by p53 and NF-κB using integrative genomics screening in human osteosarcoma U2-OS cells irradiated with a high dose (4 and 10 Gy). Radiation-induced expression in cells with silenced TP53 or RELA (coding the p65 NF-κB subunit) genes was analyzed by RNA-Seq while radiation-enhanced binding of p53 and RelA in putative regulatory regions was analyzed by ChIP-Seq, then selected candidates were validated by qPCR. RESULTS: We identified a subset of radiation-modulated genes whose expression was affected by silencing of both TP53 and RELA, and a subset of radiation-upregulated genes where radiation stimulated binding of both p53 and RelA. For three genes, namely IL4I1, SERPINE1, and CDKN1A, an antagonistic effect of the TP53 and RELA silencing was consistent with radiation-enhanced binding of both p53 and RelA. This suggested the possibility of a direct antagonistic (co)regulation by both factors: activation by NF-κB and inhibition by p53 of IL4I1, and activation by p53 and inhibition by NF-κB of CDKN1A and SERPINE1. On the other hand, radiation-enhanced binding of both p53 and RelA was observed in a putative regulatory region of the RRAD gene whose expression was downregulated both by TP53 and RELA silencing, which suggested a possibility of direct (co)activation by both factors. CONCLUSIONS: Four new candidates for genes directly co-regulated by NF-κB and p53 were revealed.

SPEN protein expression and interactions with chromatin in mouse testicular cells.

SPEN (spen family transcription repressor) is a nucleic acid-binding protein putatively involved in repression of gene expression. We hypothesized that SPEN could be involved in general downregulation of the transcription during the heat shock response in mouse spermatogenic cells through its interactions with chromatin. We documented predominant nuclear localization of the SPEN protein in spermatocytes and round spermatids, which was retained after heat shock. Moreover, the protein was excluded from the highly condensed chromatin. Chromatin immunoprecipitation experiments clearly indicated interactions of SPEN with chromatin in vivo However, ChIP-Seq analyses did not reveal any strong specific peaks both in untreated and heat shocked cells, which might suggest dispersed localization of SPEN and/or its indirect binding to DNA. Using in situ proximity ligation assay we found close in vivo associations of SPEN with MTA1 (metastasis-associated 1), a member of the nucleosome remodeling complex with histone deacetylase activity, which might contribute to interactions of SPEN with chromatin.

Synthesis and biological evaluation of thiophosphate tricyclic coumarin derivatives as steroid sulfatase inhibitors.

Steroid sulfatase (STS) enzyme inhibition is an important approach to the management of hormone-dependent breast cancer. In this paper, we report convenient methods for the synthesis and biological evaluation of thiophosphate tricyclic coumarin analogs exhibiting STS activity. The described methods are based on the straightforward preparation of 7-hydroxy-2,3-dihydro-1H-cyclopenta[c]chromen-2-one, 3-hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one, and 3-hydroxy-8,9,10,11-tetrahydro-7H-cyclohepta[c]chromen-6-one and their further modification by the introduction of various thiophosphate moieties. The inhibition properties of the synthesized compounds were tested toward STS isolated from human placenta. Most of the new STS inhibitors possessed good to moderate activity toward STS. During the course of our investigation, the largest inhibitory effects in the STS enzyme assays were observed for the two compounds 3f and 4r, with IC50 values of 13.3 and 30.3 µM, respectively (the IC50 value of 1 µM for the 665-COUMATE was used as a reference). The structure-activity relationships of the synthesized coumarin derivatives toward STS enzymes are discussed.

Small RNA Deep Sequencing Uncovers microRNAs Associated with Hearing Loss in Vestibular Schwannoma.

OBJECTIVE: To analyze the correlation between the miRNA expression profile in vestibular schwannoma (VS) tumor tissue and preoperative patient's hearing status, using the RNA-seq technique. METHODS: Nineteen tumor samples were collected from patients operated for VS in a Tertiary Academic Center. Samples were classified into "good hearing" and "poor hearing" study group based on the results of audiometric studies. Tumor miRNA expression was analyzed using high-throughput RNA sequencing (RNA-seq) technique, using NovaSeq 6000 Illumina system. Functional analysis was performed with the use of DIANA miRpath v. 4.0 online tool. RESULTS: The most overexpressed miRNAs in VS samples derived from poor hearing patients belonged to miR 449a/b, miR 15/16-1, and hypoxamiR families. Functional analysis showed that the differentially expressed miRNAs regulate cellular pathways associated with hypoxia, adherence junction functions, and signaling pathways such as Hippo, FOXO, MAPK, and Wnt signaling pathway. CONCLUSION: Our study identified a specific miRNA expression profile in VS tumor tissues that correlates with hearing impairment. These results suggest potential new molecular mechanisms related to hearing loss in the course of VS. LEVEL OF EVIDENCE: 3 (cohort study) Laryngoscope, 134:3778-3785, 2024.

Comprehensive analysis of the REST transcription factor regulatory networks in IDH mutant and IDH wild-type glioma cell lines and tumors.

The RE1-silencing transcription factor (REST) acts either as a repressor or activator of transcription depending on the genomic and cellular context. REST is a key player in brain cell differentiation by inducing chromatin modifications, including DNA methylation, in a proximity of its binding sites. Its dysfunction may contribute to oncogenesis. Mutations in IDH1/2 significantly change the epigenome contributing to blockade of cell differentiation and glioma development. We aimed at defining how REST modulates gene activation and repression in the context of the IDH mutation-related phenotype in gliomas. We studied the effects of REST knockdown, genome wide occurrence of REST binding sites, and DNA methylation of REST motifs in IDH wild type and IDH mutant gliomas. We found that REST target genes, REST binding patterns, and TF motif occurrence proximal to REST binding sites differed in IDH wild-type and mutant gliomas. Among differentially expressed REST targets were genes involved in glial cell differentiation and extracellular matrix organization, some of which were differentially methylated at promoters or gene bodies. REST knockdown differently impacted invasion of the parental or IDH1 mutant glioma cells. The canonical REST-repressed gene targets showed significant correlation with the GBM NPC-like cellular state. Interestingly, results of REST or KAISO silencing suggested the interplay between these TFs in regulation of REST-activated and repressed targets. The identified gene regulatory networks and putative REST cooperativity with other TFs, such as KAISO, show distinct REST target regulatory networks in IDH-WT and IDH-MUT gliomas, without concomitant DNA methylation changes. We conclude that REST could be an important therapeutic target in gliomas.

H2A.Z histone variants facilitate HDACi-dependent removal of H3.3K27M mutant protein in pediatric high-grade glioma cells.

Diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain tumors, non-resectable due to brainstem localization and diffusive growth. Over 80% of DIPGs harbor a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine-to-methionine substitution (H3K27M). Patients with DIPG have a dismal prognosis with no effective therapy. We show that histone deacetylase (HDAC) inhibitors lead to a significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines. We discover that the SB939-mediated H3.3K27M loss is partially blocked by a lysosomal inhibitor, chloroquine. The H3.3K27M loss is facilitated by co-occurrence of H2A.Z, as evidenced by the knockdown of H2A.Z isoforms. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. We discover a mechanism showing that HDAC inhibition in DIPG leads to pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings show the possibility of directly targeting the H3.3K27M oncohistone.

HSF1 Can Prevent Inflammation following Heat Shock by Inhibiting the Excessive Activation of the ATF3 and JUN&FOS Genes.

Heat Shock Factor 1 (HSF1), a transcription factor frequently overexpressed in cancer, is activated by proteotoxic agents and participates in the regulation of cellular stress response. To investigate how HSF1 level affects the response to proteotoxic stress, we integrated data from functional genomics analyses performed in MCF7 breast adenocarcinoma cells. Although the general transcriptional response to heat shock was impaired due to HSF1 deficiency (mainly chaperone expression was inhibited), a set of genes was identified, including ATF3 and certain FOS and JUN family members, whose stress-induced activation was stronger and persisted longer than in cells with normal HSF1 levels. These genes were direct HSF1 targets, suggesting a dual (activatory/suppressory) role for HSF1. Moreover, we found that heat shock-induced inflammatory response could be stronger in HSF1-deficient cells. Analyses of The Cancer Genome Atlas data indicated that higher ATF3, FOS, and FOSB expression levels correlated with low HSF1 levels in estrogen receptor-positive breast cancer, reflecting higher heat shock-induced expression of these genes in HSF1-deficient MCF7 cells observed in vitro. However, differences between the analyzed cancer types were noted in the regulation of HSF1-dependent genes, indicating the presence of cell-type-specific mechanisms. Nevertheless, our data indicate the existence of the heat shock-induced network of transcription factors (associated with the activation of TNFα signaling) which includes HSF1. Independent of its chaperone-mediated cytoprotective function, HSF1 may be involved in the regulation of this network but prevents its overactivation in some cells during stress.

Identification of candidate enhancers controlling the transcriptome during the formation of interphalangeal joints.

The formation of the synovial joint begins with the visible emergence of a stripe of densely packed mesenchymal cells located between distal ends of the developing skeletal anlagen called the interzone. Recently the transcriptome of the early synovial joint was reported. Knowledge about enhancers would complement these data and lead to a better understanding of the control of gene transcription at the onset of joint development. Using ChIP-sequencing we have mapped the H3-signatures H3K27ac and H3K4me1 to locate regulatory elements specific for the interzone and adjacent phalange, respectively. This one-stage atlas of candidate enhancers (CEs) was used to map the association between these respective joint tissue specific CEs and biological processes. Subsequently, integrative analysis of transcriptomic data and CEs identified new putative regulatory elements of genes expressed in interzone (e.g., GDF5, BMP2 and DACT2) and phalange (e.g., MATN1, HAPLN1 and SNAI1). We also linked such CEs to genes known as crucial in synovial joint hypermobility and osteoarthritis, as well as phalange malformations. These analyses show that the CE atlas can serve as resource for identifying, and as starting point for experimentally validating, putative disease-causing genomic regulatory regions in patients with synovial joint dysfunctions and/or phalange disorders, and enhancer-controlled synovial joint and phalange formation.

Activation-induced chromatin reorganization in neurons depends on HDAC1 activity.

Spatial chromatin organization is crucial for transcriptional regulation and might be particularly important in neurons since they dramatically change their transcriptome in response to external stimuli. We show that stimulation of neurons causes condensation of large chromatin domains. This phenomenon can be observed in vitro in cultured rat hippocampal neurons as well as in vivo in the amygdala and hippocampal neurons. Activity-induced chromatin condensation is an active, rapid, energy-dependent, and reversible process. It involves calcium-dependent pathways but is independent of active transcription. It is accompanied by the redistribution of posttranslational histone modifications and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that the histone deacetylase HDAC1 is the key regulator of this process. Our results suggest that HDAC1-dependent chromatin reorganization constitutes an important level of transcriptional regulation in neurons.

Identification of the immune gene expression signature associated with recurrence of high-grade gliomas.

High-grade gliomas (HGGs), the most common and aggressive primary brain tumors in adults, inevitably recur due to incomplete surgery or resistance to therapy. Intratumoral genomic and cellular heterogeneity of HGGs contributes to therapeutic resistance, recurrence, and poor clinical outcomes. Transcriptomic profiles of HGGs at recurrence have not been investigated in detail. Using targeted sequencing of cancer-related genes and transcriptomics, we identified single nucleotide variations, small insertions and deletions, copy number aberrations (CNAs), as well as gene expression changes and pathway deregulation in 16 pairs of primary and recurrent HGGs. Most of the somatic mutations identified in primary HGGs were not detected after relapse, suggesting a subclone substitution during the tumor progression. We found a novel frameshift insertion in the ZNF384 gene which may contribute to extracellular matrix remodeling. An inverse correlation of focal CNAs in EGFR and PTEN genes was detected. Transcriptomic analysis revealed downregulation of genes involved in messenger RNA splicing, cell cycle, and DNA repair, while genes related to interferon signaling and phosphatidylinositol (PI) metabolism are upregulated in secondary HGGs when compared to primary HGGs. In silico analysis of the tumor microenvironment identified M2 macrophages and immature dendritic cells as enriched in recurrent HGGs, suggesting a prominent immunosuppressive signature. Accumulation of those cells in recurrent HGGs was validated by immunostaining. Our findings point to a substantial transcriptomic deregulation and a pronounced infiltration of immature dendritic cells in recurrent HGG, which may impact the effectiveness of frontline immunotherapies in the GBM management. KEY MESSAGES: Most of the somatic mutations identified in primary HGGs were not detected after relapse. Focal CNAs in EGFR and PTEN genes are inversely correlated in primary and recurrent HGGs. Transcriptomic changes and distinct immune-related signatures characterize HGG recurrence. Recurrent HGGs are characterized by a prominent infiltration of immature dendritic and M2 macrophages.