De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

Posterior fossa ependymoma in neurodevelopmental syndrome caused by a de novo germline pathogenic POLR2A variant.

Ependymoma is the third most common pediatric brain tumor. Predisposition to develop ependymomas has been reported in different hereditary diseases, but the pathogenic variants related to the familial syndromes have rarely been detected in sporadic ependymomas. De novo variants in POLR2A, the gene encoding the largest subunit of RNA polymerase II, cause a neurodevelopmental disorder with a wide range of clinical manifestations, characterized by severe infantile-onset hypotonia, developmental delay, feeding difficulties, palatal anomalies, and facial dysmorphisms. As somatic events, POLR2A mutations represent a recurrent somatic lesion in benign meningiomas. Here we describe a case of ependymoma in a 2-year-old male with a de novo pathogenic variant in POLR2A predicted to impair proper interaction of the subunit with transcription-elongation factor TFIIS, whose function is required for back-tracking of the enzyme due to elongation blocks or nucleotide misincorporation, and expected to result in an increased error and reduced elongation rates. To date, ependymoma has never been reported in patients harboring pathogenic POLR2A variants. Further information is required to explore the possibility of a differential clinical and functional impact of the pathogenic POLR2A variants and the eventual inclusion of the POLR2A neurodevelopmental disorder among the cancer predisposition syndromes with the possible development of ependymomas.

POLR2A blocks osteoclastic bone resorption and protects against osteoporosis by interacting with CREB1.

Bone-resorbing osteoclasts significantly contribute to osteoporosis, and understanding the mechanisms of osteoclastogenesis is crucial for developing new drugs to treat diseases associated with bone loss. Here, we report that POLR2A is upregulated during osteoclastogenesis. Functional analyses showed that the inhibition of POLR2A decreased osteoclastogenesis, whereas the overexpression of POLR2A had completely opposite effects in vitro. Notably, the osteoclast-specific deletion of POLR2A blocks bone resorption in vivo. Furthermore, POLR2A loss-of-function suppresses estrogen deficiency-induced bone resorption. Mechanistically, POLR2A regulates the assembly of CREB1 on the regulatory elements of its target genes. Collectively, using genetic, pharmacological, and disease mouse models, we have identified a previously undescribed protein that interacts with CREB1 to regulate osteoclastic bone resorption.

Comparison of Histone H3K4me3 between IVF and ICSI Technologies and between Boy and Girl Offspring.

Epigenetics play a vital role in early embryo development. Offspring conceived via assisted reproductive technologies (ARTs) have a three times higher risk of epigenetic diseases than naturally conceived children. However, investigations into ART-associated placental histone modifications or sex-stratified analyses of ART-associated histone modifications remain limited. In the current study, we carried out immunohistochemistry, chip-sequence analysis, and a series of in vitro experiments. Our results demonstrated that placentas from intra-cytoplasmic sperm injection (ICSI), but not in vitro fertilization (IVF), showed global tri-methylated-histone-H3-lysine-4 (H3K4me3) alteration compared to those from natural conception. However, for acetylated-histone-H3-lysine-9 (H3K9ac) and acetylated-histone-H3-lysine-27 (H3K27ac), no significant differences between groups could be found. Further, sex -stratified analysis found that, compared with the same-gender newborn cord blood mononuclear cell (CBMC) from natural conceptions, CBMC from ICSI-boys presented more genes with differentially enriched H3K4me3 (n = 198) than those from ICSI-girls (n = 79), IVF-girls (n = 5), and IVF-boys (n = 2). We also found that varying oxygen conditions, RNA polymerase II subunit A (Polr2A), and lysine demethylase 5A (KDM5A) regulated H3K4me3. These findings revealed a difference between IVF and ICSI and a difference between boys and girls in H3K4me3 modification, providing greater insight into ART-associated epigenetic alteration.

Aberrant activation of RPB1 is critical for cell overgrowth in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a group of highly aggressive malignancies with a 5-year overall survival of less than 40%. Cell overgrowth with defective apoptosis is a hallmark of AML, but little is known about how it occurs. Here, we show that aberrant activation of the largest subunit of RNA polymerase II (RPB1) encoded by POLR2A gene is critically involved in this hallmark. We retrospectively analyzed the expression profiles of POLR2A and RPB1 in a panel of AML cell lines, primary AML patients and peripheral blood samples. Meanwhile, correlation analysis was used to explore the correlation between the expression of RPB1 with tumor burden and overall survival time in untreated AML samples. RNA-Seq approach was performed to identify the differentially expressed genes between RPB1 silencing AML cells with control cells after knocking out RPB1. Furthermore, orthotopic AML models were established with RPB1 silencing and control cells to investigate the effects of RPB1 protein level on leukemia cell growth. In most AML patients, RPB1 was aberrantly activated and closely associated with poor prognosis, but not in normal hematopoietic cells. Global transcriptomic analysis revealed that POLR2A knockout strongly impaired growth of AML cells by selectively depleting a substantial set of AML-related oncogenic and anti-apoptosis genes such as MYC, RUNX2, MEIS1, CDC25A and BCL-2. Silencing RPB1 by genetic technology led to a potent regression of human refractory AML in mouse models. These findings reveal that dysregulated RPB1 is a central oncogenic hub that drives overgrowth by hijacking an array of oncogenic and anti-apoptosis factors. Targeting RPB1 is a potential therapeutic for treating AML.

Genetic characterization and mutational profiling of foramen magnum meningiomas: a multi-institutional study.

OBJECTIVE: Foramen magnum (FM) meningiomas pose significant surgical challenges and have high morbidity and mortality rates. This study aimed to investigate the distribution of clinically actionable mutations in FM meningiomas and identify clinical characteristics associated with specific mutational profiles. METHODS: The authors conducted targeted next-generation sequencing of 62 FM meningiomas from three international institutions, covering all relevant meningioma genes (AKT1, KLF4, NF2, POLR2A, PIK3CA, SMO, TERT promoter, and TRAF7). Patients with a radiation-induced meningioma or neurofibromatosis type 2 (NF2) were excluded from the study. Additionally, patient and tumor characteristics, including age, sex, radiological features, and tumor location, were retrospectively collected and evaluated. RESULTS: The study cohort consisted of 46 female and 16 male patients. Clinically significant driver mutations were detected in 58 patients (93.5%). The most commonly observed alteration was TRAF7 mutations (26, 41.9%), followed by AKT1E17K mutations (19, 30.6%). Both mutations were significantly associated with an anterolateral tumor location relative to the brainstem (p = 0.0078). NF2 mutations were present in 11 cases (17.7%) and were associated with posterior tumor location, in contrast to tumors with TRAF7 and AKT1E17K mutations. Other common mutations in FM meningiomas included POLR2A mutations (8, 12.9%; 6 POLR2AQ403K and 2 POLR2AH439_L440del), KLF4K409Q mutations (7, 11.3%), and PIK3CA mutations (4, 6.5%; 2 PIK3CAH1047R and 2 PIK3CAE545K). POLR2A and KLF4 mutations exclusively occurred in female patients and showed no significant association with specific tumor locations. All tumors harboring AKT1E17K and POLR2A mutations displayed meningothelial histology. Ten tumors exhibited intratumoral calcification, which was significantly more frequent in NF2-mutant compared with AKT1-mutant FM meningiomas (p = 0.047). CONCLUSIONS: These findings provide important insights into the molecular genetics and clinicopathological characteristics of FM meningiomas. The identification of specific genetic alterations associated with tumor location, volume, calcification, histology, and sex at diagnosis may have implications for personalized treatment strategies in the future.

De novo POLR2A p.(Ile457Thr) variant associated with early-onset encephalopathy and cerebellar atrophy: expanding the phenotypic spectrum.

BACKGROUND: Heterozygous POLR2A variants have been recently reported in patients with a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia. POLR2A encodes the highly conserved RBP1 protein, an essential subunit of the DNA-dependent RNA polymerase II. CASE PRESENTATION: We investigated a 12-year-old girl presenting with an early-onset encephalopathy characterized by psychomotor delay, facial dysmorphism, refractory epilepsy with variable seizure types, behavioural abnormalities, and sleep disorder. Brain MRI showed a slowly progressive cerebellar atrophy. Trio-exome sequencing (Trio-ES) revealed the de novo germline variant NM_000937.5:c.1370T>C; p.(Ile457Thr) in POLR2A. This variant was previously reported in a subject with profound generalized hypotonia and muscular atrophy by Haijes et al. Our patient displayed instead a severe epileptic phenotype with refractory hypotonic seizures with impaired consciousness, myoclonic jerks, and drop attacks. CONCLUSION: This case expands the clinical spectrum of POLR2A-related syndrome, highlighting its phenotypic variability and supporting the relevance of epilepsy as a core feature of this emerging condition.

Metformin Bicarbonate-Mediated Efficient RNAi for Precise Targeting of TP53 Deficiency in Colon and Rectal Cancers.

Colon and rectal cancers are the leading causes of cancer-related deaths in the United States and effective targeted therapies are in need for treating them. Our genomic analyses show hemizygous deletion of TP53, an important tumor suppressor gene, is highly frequent in both cancers, and the 5-year survival of patients with the more prevalent colon cancer is significantly reduced in the patients with the cancer harboring such deletion, although such reduction is not observed for rectal cancer. Unfortunately, direct targeting TP53 has been unsuccessful for cancer therapy. Interestingly, POLR2A, a gene essential for cell survival and proliferation, is almost always deleted together with TP53 in colon and rectal cancers. Therefore, RNA interference (RNAi) with small interfering RNAs (siRNAs) to precisely target/inhibit POLR2A may be an effective strategy for selectively killing cancer cells with TP53 deficiency. However, the difficulty of delivering siRNAs specifically into the cytosol where they perform their function, is a major barrier for siRNA-based therapies. Here, metformin bicarbonate (MetC) is synthesized to develop pH-responsive MetC-nanoparticles with a unique "bomb" for effective cytosolic delivery of POLR2A siRNA, which greatly facilitates its endo/lysosomal escape into the cytosol and augments its therapeutic efficacy of cancer harboring TP53 deficiency. Moreover, the MetC-based nanoparticles without functional siRNA show notable therapeutic effect with no evident toxicity or immunogenicity.

Micro-RNA378a-3p Induces Apoptosis in Sarcomatoid Renal Cell Carcinoma and Regulates POLR2A and RUNX2 Expression.

BACKGROUND/AIM: Sarcomatoid renal cell carcinoma (sRCC) is an aggressive subtype of RCC. In the current study, we investigated whether the POLR2A and RUNX2 genes are involved in RCC-related signaling pathway and associated with miR-378. MATERIALS AND METHODS: Thirteen formalin-fixed and paraffin-embedded RCC samples were collected. Three software programs were used to predict the potential gene regulation in RCC by miR-378 and immunohistochemistry analysis was applied to confirm the expression of targeted proteins in FFPE samples. MicroRNA-378 transfection, analysis of mRNA and protein expression via Western Blotting and cell apoptosis analysis via flow cytometry for POLR2A and RUNX2 were further studied in four renal cell carcinoma cell lines. RESULTS: Both the mRNA and protein expression levels of POLR2A and RUNX2 in sRCC cell lines and were significantly higher than those in other subtypes of RCC, and similar results were obtained in clinical samples (p<0.01). Second, overexpression of miR-378 significantly suppressed the expression of POLR2A and RUNX2 in sRCC cells (p<0.01) and enhanced apoptosis (p<0.05). CONCLUSION: miR-378 significantly inhibits the expression of POLR2A and RUNX2 in sRCC and further promotes apoptosis of sRCC cells. We speculated that the apoptosis mechanism in sRCC occurs via regulation of the ERK2 and PI3K/AKT pathways, which might distinguish it from other subtypes of RCC.

Complex Autism Spectrum Disorder with Epilepsy, Strabismus and Self-Injurious Behaviors in a Patient with a De Novo Heterozygous POLR2A Variant.

Autism spectrum disorder (ASD) describes a complex and heterogenous group of neurodevelopmental disorders. Whole genome sequencing continues to shed light on the multifactorial etiology of ASD. Dysregulated transcriptional pathways have been implicated in neurodevelopmental disorders. Emerging evidence suggests that de novo POLR2A variants cause a newly described phenotype called 'Neurodevelopmental Disorder with Hypotonia and Variable Intellectual and Behavioral Abnormalities' (NEDHIB). The variable phenotype manifests with a spectrum of features; primarily early onset hypotonia and delay in developmental milestones. In this study, we investigate a patient with complex ASD involving epilepsy and strabismus. Whole genome sequencing of the proband−parent trio uncovered a novel de novo POLR2A variant (c.1367T>C, p. Val456Ala) in the proband. The variant appears deleterious according to in silico tools. We describe the phenotype in our patient, who is now 31 years old, draw connections between the previously reported phenotypes and further delineate this emerging neurodevelopmental phenotype. This study sheds new insights into this neurodevelopmental disorder, and more broadly, the genetic etiology of ASD.

POLR2A Promotes the Proliferation of Gastric Cancer Cells by Advancing the Overall Cell Cycle Progression.

RNA polymerase II subunit A (POLR2A) is the largest subunit encoding RNA polymerase II and closely related to cancer progression. However, the biological role and underlying molecular mechanism of POLR2A in gastric cancer (GC) are still unclear. Our study demonstrated that POLR2A was highly expressed in GC tissue and promoted the proliferation of GC in vitro and in vivo. We also found that POLR2A participated in the transcriptional regulation of cyclins and cyclin-dependent kinases (CDKs) at each stage and promoted their expression, indicated POLR2A's overall promotion of cell cycle progression. Moreover, POLR2A inhibited GC cell apoptosis and promoted GC cell migration. Our results indicate that POLR2A play an oncogene role in GC, which may be an important factor involved in the occurrence and development of GC.

BCAR1 promotes proliferation and cell growth in lung adenocarcinoma via upregulation of POLR2A.

BACKGROUND: This study was designed to investigate the effects of a novel carcinogenetic molecule, p130cas (breast cancer antiestrogen resistance protein 1 or BCAR1) on proliferation and cell growth in lung adenocarcinoma. The study also aimed to identify the possible underlying signal networks of BCAR1. METHODS: First, we evaluated proliferation, cell colony formation, apoptosis, and cell cycle after BCAR1 was knocked out (KO) using CRISPR-Cas9 technology in H1975 and H1299 human lung adenocarcinoma cells. Subsequently, BCAR1 was upregulated in 293T cells and immunoprecipitation-mass spectrometry (IP-MS) was used with bioinformatics analysis to screen for potential networks of BCAR1 interacting proteins. Ultimately, we validated the correlated expressions of BCAR1 and a selected hub gene, RNA polymerase II subunit A (POLR2A), in 54 lung adenocarcinoma tissues, as well as in H1975 and H1299 cells. RESULTS: Cell proliferation of H1975 and H1299 was significantly inhibited following BCAR1-KO. Colony formation of H1975 cells was also significantly decreased following BCAR1-KO. IP-MS demonstrated 419 potential proteins that may interact with BCAR1. Among them, 68 genes were significantly positively correlated to BCAR1 expression, as verified by TCGA. Six hub genes were revealed by PPI String. High expression of POLR2A, MAPK3, MOV10, and XAB2 predicted poor prognosis in lung adenocarcinoma, as verified by the K-M plotter database. POLR2A and MAPK3 are involved in both catalytic activity and transferase activity. POLR2A and BCAR1 were significantly increased in lung cancer tissues as compared with matched normal tissues. High expression of POLR2A was significantly positively correlated to BCAR1 overexpression and predicted poor prognosis in 54 lung cancer cases. POLR2A expression was significantly decreased following BCAR1-KO in H1975 and H1299 cells. CONCLUSIONS: BCAR1 promotes proliferation and cell growth, probably via upregulation of POLR2A and subsequent enhancement of catalytic and transferase activities. However, additional robust studies are required to elucidate the mechanisms involved.

Distinct DNA Sequence Preference for Histone Occupancy in Primary and Transformed Cells.

Genome-wide occupancy of several histone modifications in various cell types has been studied using chromatin immunoprecipitation (ChIP) sequencing. Histone occupancy depends on DNA sequence features like inter-strand symmetry of base composition and periodic occurrence of TT/AT. However, whether DNA sequence motifs act as an additional effector of histone occupancy is not known. We have analyzed the presence of DNA sequence motifs in publicly available ChIP-sequence datasets for different histone modifications. Our results show that DNA sequence motifs are associated with histone occupancy, some of which are different between primary and transformed cells. The motifs for primary and transformed cells showed different levels of GC-richness and proximity to transcription start sites (TSSs). The TSSs associated with transformed or primary cell-specific motifs showed different levels of TSS flank transcription in primary and transformed cells. Interestingly, TSSs with a motif-linked occupancy of H2AFZ, a component of positioned nucleosomes, showed a distinct pattern of RNA Polymerase II (POLR2A) occupancy and TSS flank transcription in primary and transformed cells. These results indicate that DNA sequence features dictate differential histone occupancy in primary and transformed cells, and the DNA sequence motifs affect transcription through regulation of histone occupancy.

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer.

Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer.

The nuclear receptors pregnane X receptor and constitutive androstane receptor contribute to the impact of fipronil on hepatic gene expression linked to thyroid hormone metabolism.

Fipronil is described as a thyroid disruptor in rat. Based on the hypothesis that this results from a perturbation of hepatic thyroid hormone metabolism, our goal was to investigate the pathways involved in fipronil-induced liver gene expression regulations. First, we performed a microarray screening in the liver of rats treated with fipronil or vehicle. Fipronil treatment led to the upregulation of several genes involved in the metabolism of xenobiotics, including the cytochrome P450 Cyp2b1, Cyp2b2 and Cyp3a1, the carboxylesterases Ces2 and Ces6, the phase II enzymes Ugt1a1, Sult1b1 and Gsta2, and the membrane transporters Abcc2, Abcc3, Abcg5, Abcg8, Slco1a1 and Slco1a4. Based on a large overlap with the target genes of constitutive androstane receptor (CAR) and pregnane X receptor (PXR), we postulated that these two nuclear receptors are involved in mediating the effects of fipronil on liver gene expression in rodents. We controlled that liver gene expression changes induced by fipronil were generally reproduced in mice, and then studied the effects of fipronil in wild-type, CAR- and PXR-deficient mice. For most of the genes studied, the gene expression modulations were abolished in the liver of PXR-deficient mice and were reduced in the liver of CAR-deficient mice. However, CAR and PXR activation in mouse liver was not associated with a marked increase of thyroid hormone clearance, as observed in rat. Nevertheless, our data clearly indicate that PXR and CAR are key modulators of the hepatic gene expression profile following fipronil treatment which, in rats, may contribute to increase thyroid hormone clearance.