Human mediator subunit MED26 functions as a docking site for transcription elongation factors.

Promoter-proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of super-elongation complexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is an unanswered question. Here, we present evidence for a role of human Mediator subunit MED26 in this process. We identify in the conserved N-terminal domain of MED26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that MED26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription.

PAR3 restricts the expansion of neural precursor cells by regulating hedgehog signaling.

During brain development, neural precursor cells (NPCs) expand initially, and then switch to generating stage-specific neurons while maintaining self-renewal ability. Because the NPC pool at the onset of neurogenesis crucially affects the final number of each type of neuron, tight regulation is necessary for the transitional timing from the expansion to the neurogenic phase in these cells. However, the molecular mechanisms underlying this transition are poorly understood. Here, we report that the telencephalon-specific loss of PAR3 before the start of neurogenesis leads to increased NPC proliferation at the expense of neurogenesis, resulting in disorganized tissue architecture. These NPCs demonstrate hyperactivation of hedgehog signaling in a smoothened-dependent manner, as well as defects in primary cilia. Furthermore, loss of PAR3 enhanced ligand-independent ciliary accumulation of smoothened and an inhibitor of smoothened ameliorated the hyperproliferation of NPCs in the telencephalon. Thus, these findings support the idea that PAR3 has a crucial role in the transition of NPCs from the expansion phase to the neurogenic phase by restricting hedgehog signaling through the establishment of ciliary integrity.

Chimera RNA transcribed from integrated HPV18 genome with adjacent host genomic region promotes oncogenic gene expression through condensate formation.

Most cervical cancers are caused by human papillomavirus (HPV) infection. In HeLa cells, the HPV18 viral genome is integrated at chromosome 8q24.21 and activates transcription of the proto-oncogene c-Myc. However, the mechanism of how the integrated HPV genome and its transcribed RNAs exhibit transcription activation function has not been fully elucidated. In this study, we found that HPV18 transcripts contain an enhancer RNA-like function to activate proximal genes including CCAT1-5L and c-Myc. We showed that the human genome-integrated HPV18 genes are activated by transcription coregulators including BRD4 and Mediator. The transcribed HPV18 RNAs form a liquid-like condensate at chromosome 8q24.21 locus, which in turn accumulates RNA polymerase II. Moreover, we focused on a relatively uncharacterized transcript from the upstream region of CCAT1, named URC. The URC RNA is transcribed as a chimera RNA with HPV18 and is composed of the 3'-untranslated region of the HPV18 transcript. We experimentally showed that the URC contributes to stabilization of HPV18 RNAs by supplying a polyadenylation site for the HPV18 transcript. Our findings suggest that integrated HPV18 at 8q24.21 locus produces HPV18-URC chimera RNA and promotes tumorigenesis through RNA-based condensate formation.

RNA Foci in Two bi-Allelic RFC1 Expansion Carriers.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is a late-onset, autosomal recessive neurodegenerative disorder caused by biallelic AAGGG/ACAGG repeat expansion (AAGGG-exp/ACAGG-exp) in RFC1. The recent identification of patients with CANVAS exhibiting compound heterozygosity for AAGGG-exp and truncating variants supports the loss-of-function of RFC1 in CANVAS patients. We investigated the pathological changes in 2 autopsied patients with CANVAS harboring biallelic ACAGG-exp and AAGGG-exp. RNA fluorescence in situ hybridization of the 2 patients revealed CCTGT- and CCCTT-containing RNA foci, respectively, in neuronal nuclei of tissues with neuronal loss. Our findings suggest that RNA toxicity may be involved in the pathogenesis of CANVAS. ANN NEUROL 2024;95:607-613.

MED26-containing Mediator may orchestrate multiple transcription processes through organization of nuclear bodies.

Mediator is a coregulatory complex that plays essential roles in multiple processes of transcription regulation. One of the human Mediator subunits, MED26, has a role in recruitment of the super elongation complex (SEC) to polyadenylated genes and little elongation complex (LEC) to non-polyadenylated genes, including small nuclear RNAs (snRNAs) and replication-dependent histone (RDH) genes. MED26-containing Mediator plays a role in 3' Pol II pausing at the proximal region of transcript end sites in RDH genes through recruitment of Cajal bodies (CBs) to histone locus bodies (HLBs). This finding suggests that Mediator is involved in the association of CBs with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing by supplying 3'-end processing factors from CBs. Thus, we argue the possibility that Mediator is involved in the organization of nuclear bodies to orchestrate multiple processes of gene transcription.

De novo ARF3 variants cause neurodevelopmental disorder with brain abnormality.

An optimal Golgi transport system is important for mammalian cells. The adenosine diphosphate (ADP) ribosylation factors (ARF) are key proteins for regulating cargo sorting at the Golgi network. In this family, ARF3 mainly works at the trans-Golgi network (TGN), and no ARF3-related phenotypes have yet been described in humans. We here report the clinical and genetic evaluations of two unrelated children with de novo pathogenic variants in the ARF3 gene: c.200A > T (p.Asp67Val) and c.296G > T (p.Arg99Leu). Although the affected individuals presented commonly with developmental delay, epilepsy and brain abnormalities, there were differences in severity, clinical course and brain lesions. In vitro subcellular localization assays revealed that the p.Arg99Leu mutant localized to Golgi apparatus, similar to the wild-type, whereas the p.Asp67Val mutant tended to show a disperse cytosolic pattern together with abnormally dispersed Golgi localization, similar to that observed in a known dominant negative variant (p.Thr31Asn). Pull-down assays revealed that the p.Asp67Val had a loss-of-function effect and the p.Arg99Leu variant had increased binding of the adaptor protein, Golgi-localized, γ-adaptin ear-containing, ARF-binding protein 1 (GGA1), supporting the gain of function. Furthermore, in vivo studies revealed that p.Asp67Val transfection led to lethality in flies. In contrast, flies expressing p.Arg99Leu had abnormal rough eye, as observed in the gain-of-function variant p.Gln71Leu. These data indicate that two ARF3 variants, the possibly loss-of-function p.Asp67Val and the gain-of-function p.Arg99Leu, both impair the Golgi transport system. Therefore, it may not be unreasonable that they showed different clinical features like diffuse brain atrophy (p.Asp67Val) and cerebellar hypoplasia (p.Arg99Leu).

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities.

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy.

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1-4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA-binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole-exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense-mediated mRNA decay (NMD), and one canonical splice site variant, c.272-1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272-1G>C, were clustered within 20 amino acid residues of the C-terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.

AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia.

Chromosomal translocations involving the MLL gene are associated with infant acute lymphoblastic and mixed lineage leukemia. There are a large number of translocation partners of MLL that share very little sequence or seemingly functional similarities; however, their translocations into MLL result in the pathogenesis of leukemia. To define the molecular reason why these translocations result in the pathogenesis of leukemia, we purified several of the commonly occurring MLL chimeras. We have identified super elongation complex (SEC) associated with all chimeras purified. SEC includes ELL, P-TEFb, AFF4, and several other factors. AFF4 is required for SEC stability and proper transcription by poised RNA polymerase II in metazoans. Knockdown of AFF4 in leukemic cells shows reduction in MLL chimera target gene expression, suggesting that AFF4/SEC could be a key regulator in the pathogenesis of leukemia through many of the MLL partners.

p53 represses the transcription of snRNA genes by preventing the formation of little elongation complex.

The regulation of transcription by RNA polymerase II (Pol II) is important for a variety of cellular functions. ELL/EAF-containing little elongation complex (LEC) was found to be required for transcription of Pol II-dependent small nuclear RNA (snRNA) genes. It was shown that the tumor suppressor p53 interacts with ELL and inhibits transcription elongation activity of ELL. Here, we show that p53 inhibits interaction between ELL/EAF and ICE1 in LEC and thereby p53 represses transcription of Pol II-dependent snRNA genes through inhibiting LEC function. Furthermore, induction of p53 expression by ultraviolet (UV) irradiation decreases the occupancy of ICE1 at Pol II-dependent snRNA genes. Consistent with the results, knockdown of p53 increased both the expression of snRNA genes and the occupancy of Pol II and components of LEC at snRNA genes. Our results indicate that p53 interferes with the interaction between ELL/EAF and ICE1 and represses transcription of snRNA genes by Pol II.

Sez6l2 regulates phosphorylation of ADD and neuritogenesis.

Increasing evidence shows that immune-mediated mechanisms may contribute to the pathogenesis of central nervous system disorders including cerebellar ataxias, as indicated by the aberrant production of neuronal surface antibodies. We previously reported a patient with cerebellar ataxia associated with production of a new anti-neuronal antibody, anti-seizure-related 6 homolog like 2 (Sez6l2). Sez6l2 is a type 1 membrane protein that is highly expressed in the hippocampus and cerebellar cortex and mice lacking Sez6l2 protein family members develop ataxia. Here we used a proteomics-based approach to show that serum derived from this patient recognizes the extracellular domain of Sez6l2 and that Sez6l2 protein binds to both adducin (ADD) and glutamate receptor 1 (GluR1). Our results indicate that Sez6l2 is one of the auxiliary subunits of the AMPA receptor and acts as a scaffolding protein to link GluR1 to ADD. Furthermore, Sez6l2 overexpression upregulates ADD phosphorylation, whereas siRNA-mediated downregulation of Sez612 prevents ADD phosphorylation, suggesting that Sez6l2 modulates AMPA-ADD signal transduction.

TRIM39 negatively regulates the NFκB-mediated signaling pathway through stabilization of Cactin.

NFκB is one of the central regulators of cell survival, immunity, inflammation, carcinogenesis and organogenesis. The activation of NFκB is strictly regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. Several types of ubiquitination play important roles in multi-step regulations of the NFκB pathway. Some of the tripartite motif-containing (TRIM) proteins functioning as E3 ubiquitin ligases are known to regulate various biological processes such as inflammatory signaling pathways. One of the TRIM family proteins, TRIM39, for which the gene has single nucleotide polymorphisms, has been identified as one of the genetic factors in Behcet's disease. However, the role of TRIM39 in inflammatory signaling had not been fully elucidated. In this study, to elucidate the function of TRIM39 in inflammatory signaling, we performed yeast two-hybrid screening using TRIM39 as a bait and identified Cactin, which has been reported to inhibit NFκB- and TLR-mediated transcriptions. We show that TRIM39 stabilizes Cactin protein and that Cactin is upregulated after TNFα stimulation. TRIM39 knockdown also causes activation of the NFκB signal. These findings suggest that TRIM39 negatively regulates the NFκB signal in collaboration with Cactin induced by inflammatory stimulants such as TNFα.

The novel heart-specific RING finger protein 207 is involved in energy metabolism in cardiomyocytes.

A failing heart shows severe energy insufficiency, and it is presumed that this energy shortage plays a critical role in the development of cardiac dysfunction. However, little is known about the mechanisms that cause energy metabolic alterations in the failing heart. Here, we show that the novel RING-finger protein 207 (RNF207), which is specifically expressed in the heart, plays a role in cardiac energy metabolism. Depletion of RNF207 in neonatal rat cardiomyocytes (NRCs) leads to a reduced cellular concentration of adenosine triphosphate (ATP) and mitochondrial dysfunction. Consistent with this result, we observed here that the expression of RNF207 was significantly reduced in mice with common cardiac diseases including heart failure. Intriguingly, proteomic approaches revealed that RNF207 interacts with the voltage-dependent anion channel (VDAC), which is considered to be a key regulator of mitochondria function, as an RNF207-interacting protein. Our findings indicate that RNF207 is involved in ATP production by cardiomyocytes, suggesting that RNF207 plays an important role in the development of heart failure.

TRIM29 regulates the p63-mediated pathway in cervical cancer cells.

Cell invasion and adhesion play an important role in cancer metastasis and are orchestrated by a complicated network of transcription factors including p63. Here, we show that a member of the tripartite motif protein family, TRIM29, is required for regulation of the p63-mediated pathway in cervical cancer cells. TRIM29 knockdown alters the adhesion and invasion activities of cervical cancer cells. TRIM29 knockdown and overexpression cause a significant decrease and increase of TAp63α expression, respectively. TRIM29 knockdown alters the expression pattern of integrins and increases ZEB1 expression. TRIM29 is required for suppression of an increase in the adhesion activity of cells by TAp63α. These findings suggest that TRIM29 regulates the p63-mediated pathway and the behavior of cervical cancer cells.

Chd5 Regulates MuERV-L/MERVL Expression in Mouse Embryonic Stem Cells Via H3K27me3 Modification and Histone H3.1/H3.2.

Chd5 is an essential factor for neuronal differentiation and spermatogenesis and is a known tumor suppressor. H3K27me3 and H3K4un are modifications recognized by Chd5; however, it remains unclear how Chd5 remodels chromatin structure. We completely disrupted the Chd5 locus using the CRISPR-Cas9 system to generate a 52 kbp long deletion and analyzed Chd5 function in mouse embryonic stem cells. Our findings show that Chd5 represses murine endogenous retrovirus-L (MuERV-L/MERVL), an endogenous retrovirus-derived retrotransposon, by regulating H3K27me3 and H3.1/H3.2 function.

Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes.

OBJECTIVE: Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphatidylserine-prothrombin complex, which is associated with APS. We have previously reported that aPS-PT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression. METHODS: RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography-tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR). RESULTS: RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT. CONCLUSION: We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS.

Distinct modes of regulation of the Uch37 deubiquitinating enzyme in the proteasome and in the Ino80 chromatin-remodeling complex.

Deubiquitinating enzymes (DUBs) are proteases that can antagonize ubiquitin-mediated signaling by disassembling ubiquitin-protein conjugates. How DUBs are regulated in vivo and how their substrate specificities are achieved are largely unknown. The conserved DUB Uch37 is found on proteasomes in organisms ranging from fission yeast to humans. Deubiquitination by Uch37 is activated by proteasomal binding, which enables Uch37 to process polyubiquitin chains. Here we show that in the nucleus Uch37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80). In hINO80, Uch37 is held in an inactive state; however, it can be activated by transient interaction of the Ino80 complex with the proteasome. Thus, DUB activities can be modulated both positively and negatively via dynamic interactions with partner proteins. In addition, our findings suggest that the proteasome and the hINO80 chromatin-remodeling complex may cooperate to regulate transcription or DNA repair, processes in which both complexes have been implicated.

Peripartum cardiomyopathy in patients with psychiatric disorders successfully treated with bromocriptine: Two case reports.

Peripartum cardiomyopathy (PPCM) is a rare disorder in which left ventricular systolic dysfunction and heart failure symptoms occur during the peripartum period. Inhibition of prolactin secretion by bromocriptine mediates beneficial effects on cardiac function in PPCM. Mental disorders are also associated with the onset of PPCM. Psychiatric medications for mental disorders would affect serotonin production and tryptophan and dopamine metabolism, and they are associated with PPCM. Conversely, bromocriptine affects psychiatric symptoms; therefore, the treatment of PPCM complicated by mental disorders using bromocriptine may be difficult. Herein, we report cases of two patients with PPCM and mental disorders successfully treated with bromocriptine therapy. The first case involved a 33-year-old woman with a history of atypical depression and anxiety disorder, who developed PPCM with a left ventricular ejection fraction (LVEF) of 19 %. The second case was that of a 42-year-old woman with a history of bipolar and panic disorders who developed PPCM with an LVEF of 18 %. Both patients were administered bromocriptine; however, psychiatric symptoms did not worsen and cardiac function improved. We also review the literature on the relationship between PPCM and mental disorders. Learning objective: Mental disorders and psychiatric medications may be associated with the onset of peripartum cardiomyopathy (PPCM). Although bromocriptine has beneficial effects on PPCM, it has also been reported to increase the risk of worsening psychiatric symptoms; therefore, the efficacy and safety of bromocriptine in PPCM patients with mental disorders is controversial. Our cases showed that bromocriptine can be used safely without worsening psychiatric symptoms in PPCM with mental disorders.