Mechanistic insights into nucleosomal H2B monoubiquitylation mediated by yeast Bre1-Rad6 and its human homolog RNF20/RNF40-hRAD6A.

Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.

The Histone Acetyltransferase Mst2 Protects Active Chromatin from Epigenetic Silencing by Acetylating the Ubiquitin Ligase Brl1.

Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we report a positive feedback loop that preserves the transcription-competent state of RNA polymerase II-transcribed genes. We found that Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3-marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription and prevents ectopic heterochromatin assembly. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Directly Stimulates H2B Ubiquitylation through an Interaction with Rad6.

The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.

Clinical value of BRE-AS1 in myocardial infarction and its role in myocardial infarction-induced cardiac muscle cell apoptosis.

Objectives. The aim of this study was to investigate the expression of long non-coding RNA (lncRNA) brain and reproductive organ-expressed protein (BRE) antisense RNA 1 (BRE-AS1) in patients with acute myocardial infarction (AMI) and its effect on ischemia/reperfusion (I/R)-induced oxidative stress and apoptosis of cardiomyocytes. Methods. Serum BRE-AS1 levels in patients with AMI was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic and prognostic values of BRE-AS1 were evaluated. H9c2 cells were treated with hypoxia/reoxygenation to establish an in vitro myocardial infarction cell model. The levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). Levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were determined by commercial kits. Cell counting kit-8 (CCK-8) and flow cytometry were used to evaluate the cell viability and cell apoptosis. Results. The expression of BRE-AS1 in serum of patients with AMI is upregulated, which shows the clinical diagnostic value for AMI. In the I/R injury cell model, the knockout of BRE-AS1 can significantly alleviate the increase in TNF-α, IL-1ß, and IL-6 levels, inhibit the production of LDH and MDA, increase the activities of SOD and GSH-Px, promote the cell viability and suppress cell apoptosis. Conclusions. Abnormally elevated BRE-AS1 has a high diagnostic value for AMI as well as a prognostic value for major adverse cardiovascular events (MACEs). The elevation of BRE-AS1 promoted oxidative stress injury and cell apoptosis in vitro.

Biochemical insights into Paf1 complex-induced stimulation of Rad6/Bre1-mediated H2B monoubiquitination.

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HMD stimulates H2Bub, probably through accelerating ubiquitin discharge from Rad6 alone or in cooperation with Cdc73 and Bre1, and that the Paf1/Leo1 heterodimer in PAF1C specifically recognizes the histone H3 tail of nucleosomal substrates, stimulating H2Bub. Collectively, our biochemical results indicate that intact PAF1C is required to efficiently stimulate Rad6/Bre1-mediated H2Bub.

RPA-mediated recruitment of Bre1 couples histone H2B ubiquitination to DNA replication and repair.

The ubiquitin E3 ligase Bre1-mediated H2B monoubiquitination (H2Bub) is essential for proper DNA replication and repair in eukaryotes. Deficiency in H2Bub causes genome instability and cancer. How the Bre1-H2Bub pathway is evoked in response to DNA replication or repair remains unknown. Here, we identify that the single-stranded DNA (ssDNA) binding factor RPA acts as a key mediator that couples Bre1-mediated H2Bub to DNA replication and repair in yeast. We found that RPA interacts with Bre1 in vitro and in vivo, and this interaction is stimulated by ssDNA. This association ensures the recruitment of Bre1 to replication forks or DNA breaks but does not affect its E3 ligase activity. Disruption of the interaction abolishes the local enrichment of H2Bub, resulting in impaired DNA replication, response to replication stress, and repair by homologous recombination, accompanied by increased genome instability and DNA damage sensitivity. Notably, we found that RNF20, the human homolog of Bre1, interacts with RPA70 in a conserved mode. Thus, RPA functions as a master regulator for the spatial-temporal control of H2Bub chromatin landscape during DNA replication and recombination, extending the versatile roles of RPA in guarding genome stability.

The Ubp3/Bre5 deubiquitylation complex modulates COPII vesicle formation.

The appropriate delivery of secretory proteins to the correct subcellular destination is an essential cellular process. In the endoplasmic reticulum (ER), secretory proteins are captured into COPII vesicles that generally exclude ER resident proteins and misfolded proteins. We previously characterized a collection of yeast mutants that fail to enforce this sorting stringency and improperly secrete the ER chaperone, Kar2 (Copic et al., Genetics 2009). Here, we used the emp24Δ mutant strain that secretes Kar2 to identify candidate proteins that might regulate ER export, reasoning that loss of regulatory proteins would restore sorting stringency. We find that loss of the deubiquitylation complex Ubp3/Bre5 reverses all of the known phenotypes of the emp24Δ mutant, and similarly reverses Kar2 secretion of many other ER retention mutants. Based on a combination of genetic interactions and live cell imaging, we conclude that Ubp3 and Bre5 modulate COPII coat assembly at ER exit sites. Therefore, we propose that Ubp3/Bre5 influences the rate of vesicle formation from the ER that in turn can impact ER quality control events.

Bre1 and Ubp8 regulate H2B mono-ubiquitination and the reversible yeast-hyphae transition in Candida albicans.

The reversible yeast-hyphae transition of the human fungal pathogen Candida albicans is tightly linked to its pathogenicity. In this study, we show that histone H2B mono-ubiquitination (H2Bub) at lysine 123 was maintained at a low level in the yeast state, whereas it increased significantly during yeast-to-hyphae transition and decreased when hyphae converted to yeast. The increased H2Bub level is correlated with activation of the hyphal program. H2B ubiquitination and deubiquitination are dynamically regulated by the E3 ligase Bre1 and the deubiquitinase Ubp8 during the reversible yeast-hyphae transition. The functions of Bre1 and Ubp8 in hypha-specific gene (HSG) regulation appears to be direct because both are recruited to the coding regions of HSGs during hyphal induction. The sequential recruitment of Bre1 and Ubp8 to HSGs coding regions is important for the initiation and maintenance of HSG expression. Additionally, Ubp8 contributes to the pathogenicity of C. albicans during early infection in a mouse model. Our study is the first to link H2B ubiquitination to the morphological plasticity and pathogenicity of the human fungal pathogen C. albicans and shed light on potential antifungal treatments.

Development and validation of a novel luciferase reporter gene assay to detect pyrogen.

To develop and validate a novel reporter gene assay (RGA) to detect pyrogen, HL60 cells were transfected with an NF-κB-RE plasmid containing the luciferase gene to generate stably transfected cells. Through stimulation with pyrogens, a signal was obtained that was dose-dependent with the concentration of pyrogen. Using the cells, we selected and optimized the parameters and found that the optimal conditions may be with 5 × 105/ml cells that were seeded and incubated with pyrogen for 3-6 h in IMDM medium with 2% FBS. Based on the optimized parameters, a novel RGA was developed. Then, the RGA was validated and the results showed that the linearity was greater than 0.95 between the signals and the concentrations of pyrogen, the recoveries of pyrogen were all between 50% and 200%, and the precision was less than 35%. There was no difference in the sensitivity, specificity or reproducibility between RGA and BET, and the results from RGA and MAT and RPT were consistent. Furthermore, the RGA can be applied to the pyrogen detection of monoclonal antibodies. Due to its advantages including a fast detection speed, high sensitivity, convenient mode of operation and wide-pyrogen spectrum detection, RGA is promising as a supplementary method to detect pyrogen.

Bmp4 Synexpression Gene, Sizzled, Transcription Is Collectively Modulated by Smad1 and Ventx1.1/Ventx2.1 in Early Xenopus Embryos.

Sizzled (Szl) is a secreted frizzled protein, having a sequence homology with the extracellular cysteine-rich domain (CRD) of the Wnt receptor, 'Frizzled'. Contrary to the other secreted frizzled like proteins (Sfrps), szl belongs to the bone morphogenetic protein 4 (Bmp4) synexpression group and is tightly coexpressed with Bmp4. What is not known is how the szl transcription achieves its Bmp4 synexpression pattern. To address the molecular details of szl transcription control, we cloned a promoter of size 1566 base pairs for szl (bps) from the Xenopus laevis genomic DNA. Luciferase and eGFP reporter gene results of this szl promoter (-1566 bp) in its activation and repression patterns by Bmp4/Smad1 and a dominant negative Bmp4 receptor (DNBR) were similar to those of the endogenous szl expression. Reporter gene assays and site-directed mutagenesis of the szl promoter mapped an active Bmp4/Smad1 response element (BRE) and a cis-acting element, which competitively share a direct binding site for Ventx1.1 and Ventx2.1 (a Ventx response element, VRE). Smad1 and ventx2.1 alone increased szl promoter activity; in addition, the binding of each protein component was enhanced with their coexpression. Interestingly, Ventx1.1 repressed this reporter gene activity; however, Ventx1.1 and Ventx2.1 together positively regulated the szl promoter activity. From our analysis, Ventx2.1 binding was enhanced by Ventx1.1, but Ventx1.1 inhibitory binding was inhibited by co-injection of Ventx2.1 for the VRE site. The inhibitory Ventx1.1 co-injection decreased Smad1 binding on the szl promoter. In a triple combination of overexpressed Smad1/Ventx1.1/Ventx2.1, the reduced binding of Smad1 from Ventx1.1 was recovered to that of the Smad1/Ventx2 combination. Collectively, this study provides evidence of Bmp4/Smad1 signaling for a primary immediate early response and its two oppositely behaving target transcription factors, Ventx1.1 and Ventx2.1, for a secondary response, as they together upregulate the szl promoter's activity to achieve szl expression in a Bmp4 synexpression manner.

Genome Sequence, Proteome Profile, and Identification of a Multiprotein Reductive Dehalogenase Complex in Dehalogenimonas alkenigignens Strain BRE15M.

Bacteria of the genus Dehalogenimonas respire with vicinally halogenated alkanes via dihaloelimination. We aimed to describe involved proteins and their supermolecular organization. Metagenomic sequencing of a Dehalogenimonas-containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained 31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for membrane-anchoring proteins. Shotgun proteomics of cells grown with 1,2-dichloropropane as an electron acceptor identified 1152 proteins representing more than 60% of the total proteome. Ten RdhA proteins were detected, including a DcpA ortholog, which was the strongest expressed RdhA. Blue native gel electrophoresis (BNE) demonstrating maximum activity was localized in a protein complex of 146-242 kDa. Protein mass spectrometry revealed the presence of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake hydrogenase subunits (HupL and HupS), an iron-sulfur protein (HupX), and subunits of a redox protein with a molybdopterin-binding motif (OmeA and OmeB) in the complex. BNE after protein solubilization with different detergent concentrations revealed no evidence for an interaction between the putative respiratory electron input module (HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated with the organohalide respiration complex. Based on genomic and proteomic analysis, we propose quinone-independent respiration in Dehalogenimonas.

New Endohyphal Relationships between Mucoromycota and Burkholderiaceae Representatives.

Mucoromycota representatives are known to harbor two types of endohyphal bacteria (EHB)-Burkholderia-related endobacteria (BRE) and Mycoplasma-related endobacteria (MRE). While both BRE and MRE occur in fungi representing all subphyla of Mucoromycota, their distribution is not well studied. Therefore, it is difficult to resolve the evolutionary history of these associations in favor of one of the following two alternative hypotheses explaining their origin: "early invasion" and "late invasion." Our main goal was to fill this knowledge gap by surveying Mucoromycota fungi for the presence of EHB. We screened 196 fungal strains from 16 genera using a PCR-based approach to detect bacterial 16S rRNA genes, complemented with fluorescence in situ hybridization (FISH) imaging to confirm the presence of bacteria within the hyphae. We detected Burkholderiaceae in ca. 20% of fungal strains. Some of these bacteria clustered phylogenetically with previously described BRE clades, whereas others grouped with free-living Paraburkholderia Importantly, the latter were detected in Umbelopsidales, which previously were not known to harbor endobacteria. Our results suggest that this group of EHB is recruited from the environment, supporting the late invasion scenario. This pattern complements the early invasion scenario apparent in the BRE clade of EHB.IMPORTANCE Bacteria living within fungal hyphae present an example of one of the most intimate relationships between fungi and bacteria. Even though there are several well-described examples of such partnerships, their prevalence within the fungal kingdom remains unknown. Our study focused on early divergent terrestrial fungi in the phylum Mucoromycota. We found that ca. 20% of the strains tested harbored bacteria from the family Burkholderiaceae Not only did we confirm the presence of bacteria from previously described endosymbiont clades, we also identified a new group of endohyphal Burkholderiaceae representing the genus Paraburkholderia We established that more than half of the screened Umbelopsis strains were positive for bacteria from this new group. We also determined that, while previously described BRE codiverged with their fungal hosts, Paraburkholderia symbionts did not.

Long non-coding RNA BRE-AS1 inhibits the proliferation, migration, and invasion of cancer cells in triple-negative breast cancer and predicts patients' survival by downregulating miR-21.

BACKGROUND: BRE-AS1 is a recently identified tumor suppressor in non-small cell lung cancer. It role in other human diseases remains elusive. METHODS: Differential expression of BRE-AS1 in with triple-negative breast cancer (TNBC) patients (n = 74, patient group) and healthy volunteers (n = 58, control group) was studied with RT-qPCR. The direct interaction between BRE-AS1 and premature microRNA-21 (miR-21) was assessed by RNA pull-down assay. The interactions among BRE-AS1, miR-21 and PTEN were evaluated by overexpression assays. CCK-8 assay and Transwell assay were used to evaluate cell behaviors. RESULTS: BRE-AS1 was downregulated in TNBC, while miR-21 was highly expressed in TNBC. Low expression levels of lncRNA BRE-AS1 and high expression levels of miR-21 were significantly correlated with unfavorable survival outcomes. BRE-AS1 and miRNA-21 were inversely correlated across TNBC samples, not control samples. BRE-AS1 decreased miR-21 expression and increased PTEN expression while miR-21showed no role in BRE-AS1 expression. RNA pull-down assay illustrated that BRE-AS1 may sponge premature miR-21 to suppress it maturation. Overexpression of BRE-AS1 decreased cell behaviors, while overexpression of miR-21 promoted cell behaviors. MiR-21 suppressed the role of BRE-AS1 in cancer cell behaviors. CONCLUSION: Therefore, BRE-AS1 may inhibit TNBC by downregulating miR-21.

Refolding, purification, and characterization of constitutive-active human-Smad8 produced as inclusion bodies in ClearColi® BL21 (DE3).

Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-ß (TGF-ß) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and ß-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.

The Bre1/Rad6 machinery: writing the central histone ubiquitin mark on H2B and beyond.

Mono-ubiquitination on H2B (H2Bub1) is an evolutionarily conserved histone post-translational modification implicated in various important physiological processes including DNA replication, transcription activation, and DNA damage repair. The Bre1/Rad6 ubiquitination machinery is currently considered to be the sole writer of H2Bub1, but the mechanistic basis by which it operates is unclear. Recently, the RING-type E3 ligase Bre1 was proposed to associate with the E2 enzyme Rad6 through a novel interaction between Bre1 RBD (Rad6 binding domain) and Rad6; and the RING domain of Bre1 that is responsible for the nucleosomal acidic patch binding also interacts with Rad6 to stimulate its catalytic activity. Recent discoveries have yielded evidence for the phenomenon of liquid-liquid phase separation in the context of H2Bub1, and its regulation by other histone post-translational modifications. This review summarizes current knowledge about Bre1/Rad6-mediated H2B ubiquitination, including the physiological functions and the molecular basis for writing and regulation of this central histone ubiquitin mark. Possible models for the Bre1/Rad6 machinery bound to nucleosomes bearing different modifications in the writing step are also disclosed.

Expression of ß-1,4-galactosyltransferases during Aging in Caenorhabditis elegans.

BACKGROUND: Altered plasma activity of ß-1,4-galac-tosyl-transferases (B4GALTs) is a novel candidate biomarker of human aging. B4GALT1 is assumed to be largely responsible for this activity increase, but how it modulates the aging process is unclear at present. OBJECTIVES: To determine how expression of B4GALT1 and other B4GALT enzymes changes during aging of an experimentally tractable model organism, Caenorhabditis elegans. METHODS: Targeted analysis of mRNA levels of all 3 C. elegans B4GALT family members was performed by qPCR in wild-type and in long-lived daf-2 (insulin/IGF1-like receptor)-deficient or germline-deficient animals. RESULTS: bre-4 (B4GALT1/2/3/4) is the only B4GALT whose expression increases during aging in wild-type worms. In addition, bre-4 levels also rise during aging in long-lived daf-2-deficient worms, but not in animals that are long-lived due to the lack of germline stem cells. On the other hand, expression of sqv-3 (B4GALT7) and of W02B12.11 (B4GALT5/6) appears decreased or constant, respectively, in all backgrounds during aging. CONCLUSIONS: The age-dependent bre-4 mRNA increase in C. elegans parallels the age-dependent B4GALT activity increase in humans and is consistent with C. elegans being a suitable experimental organism to define potentially conserved roles of B4GALT1 during aging.

Monoubiquitylation of histone H2B contributes to the bypass of DNA damage during and after DNA replication.

DNA lesion bypass is mediated by DNA damage tolerance (DDT) pathways and homologous recombination (HR). The DDT pathways, which involve translesion synthesis and template switching (TS), are activated by the ubiquitylation (ub) of PCNA through components of the RAD6-RAD18 pathway, whereas the HR pathway is independent of RAD18 However, it is unclear how these processes are coordinated within the context of chromatin. Here we show that Bre1, an ubiquitin ligase specific for histone H2B, is recruited to chromatin in a manner coupled to replication of damaged DNA. In the absence of Bre1 or H2Bub, cells exhibit accumulation of unrepaired DNA lesions. Consequently, the damaged forks become unstable and resistant to repair. We provide physical, genetic, and cytological evidence that H2Bub contributes toward both Rad18-dependent TS and replication fork repair by HR. Using an inducible system of DNA damage bypass, we further show that H2Bub is required for the regulation of DDT after genome duplication. We propose that Bre1-H2Bub facilitates fork recovery and gap-filling repair by controlling chromatin dynamics in response to replicative DNA damage.

Changes in background electroencephalographic activity in benign childhood epilepsy with centrotemporal spikes after oxcarbazepine treatment: a standardized low-resolution brain electromagnetic tomography (sLORETA) study.

BACKGROUND: Several neuroimaging studies have reported neurophysiological alterations in patients with benign childhood epilepsy with centrotemporal spikes (BCECTS). However, reported outcomes have been inconsistent, and the progression of these changes in the brain remains unresolved. Moreover, background electroencephalography (EEG) in cases of BCECTS has not been performed often. METHODS: We investigated background EEG activity changes after six months of oxcarbazepine treatment to better understand the neurophysiological alterations and progression that occur in BCECTS. In 18 children with BCECTS, non-parametric statistical analyses using standardized low resolution brain electromagnetic tomography (sLORETA) were performed to compare the current density distribution of four frequency bands (delta, theta, alpha, and beta) between untreated and treated conditions. RESULTS: Background EEG activity for the delta frequency band was significantly decreased in the fronto-temporal and limbic regions of the left hemisphere after oxcarbazepine treatment (threshold log-F-ratio = ±2.729, P < 0.01). The maximum current density difference was found in the parahippocampal gyrus of the left limbic lobe (Montreal Neurological Institute coordinate [x, y, z = 25, - 20, - 10], Brodmann area 28) (log-F-ratio = 3.081, P < 0.01). CONCLUSIONS: Our results indicate the involvement of the fronto-temporal and limbic cortices in BCECTS, and limbic lobe involvement, including the parahippocampal gyrus, was noted. In addition to evidence of the involvement of the fronto-temporal and limbic cortices in BCECTS, this study also found that an antiepileptic drug could reduce the delta frequency activity of the background EEG in these regions.

Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1.

Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.

Bre Enhances Osteoblastic Differentiation by Promoting the Mdm2-Mediated Degradation of p53.

Bre is a conserved cellular protein expressed in various tissues. Its major function includes DNA damage repair and anti-apoptosis. Recent studies indicate that Bre is potentially involved in stem cell differentiation although pathophysiological significance along with the molecular mechanisms is still unclear. Here, we report that Bre protein was substantially expressed in the bone tissue and its expression was highly upregulated during the osteogenic differentiation. To test a hypothesis that Bre plays functional roles in the process of osteogenic differentiation, we examined the expression of Bre in an osteoporosis mouse model. Compared with the normal bone tissue, Bre expression in osteoporotic bone was also significantly reduced. Moreover, knockdown of Bre in the mouse bone marrow mesenchymal cells significantly reduced the expression of osteogenic marker genes, the alkaline phosphatase activity, and the mineralization capacity, while overexpression of Bre greatly promoted the osteogenesis both in vitro and in vivo. Interestingly, we founded that knockdown of Bre led to activation of the p53 signaling pathways exhibited by increased p53, p21, and Mdm2. However, when we inhibited the p53 by siRNA silencing or pifithrin-α, the impaired osteogenesis caused by Bre knockdown was greatly restored. Finally, we found that Bre promoted the Mdm2-mediated p53 ubiquitination and degradation by physically interacting with p53. Taken together, our results revealed a novel function of Bre in osteoblast differentiation through modulating the stability of p53. Stem Cells 2017;35:1760-1772.