Integrated single-cell and bulk RNA sequencing reveals CREM is involved in the pathogenesis of ulcerative colitis.

Background: Ulcerative colitis (UC) is an inflammatory bowel disease characterized by persistent colonic inflammation. Here, we performed a systematic analysis to gain better insights into UC pathogenesis. Methods: We analyzed two UC-related datasets extracted from the gene expression omnibus database using several bioinformatics tools. The primary cell types and key subgroups of primary cells associated with UC and differentially expressed genes (DEGs) between UC and control samples were identified. The molecular regulation of the key genes was also predicted. The gene ontology and Kyoto encyclopedia of genes and genomes enrichment analyses of marker genes of key cell subgroups and model genes were performed. The expression of key enriched genes was validated in 10 clinical samples using real-time quantitative polymerase chain reaction (RT-qPCR). Results: Monocytes were identified as the major cell type. Ten differentially expressed marker genes were obtained by intersecting the 3121 DEGs, 38 marker genes in major cell types, and 104 marker genes in key cell subgroups. Four essential genes, associated with immune response, were obtained using support vector machine recursive feature elimination and least absolute shrinkage and selection operator analyses. The four essential genes were highly expressed in Cluster 0 during differentiation. Validation of the four key genes in colonic mucosal biopsy specimens from 10 normal and 10 UC patients revealed that CREM was highly expressed in both the lesion-free sites and lesion sites colonic mucosa of UC patients compared with normal adults. Conclusions: We identified CREM involved in UC pathogenesis, which is expected to provide a new therapeutic target for UC.

Repetitive transcranial magnetic stimulation and fluoxetine attenuate astroglial activation and benefit behaviours in a chronic unpredictable mild stress mouse model of depression.

Objectives: Repetitive transcranial magnetic stimulation (rTMS) has been considered as an effective antidepressant treatment; however, the mechanism of its antidepressant effect is still unclear. Fluoxetine, a selective serotonin reuptake inhibitor antidepressant, may be neuroprotective. The objective of the present study was to evaluate the effect and underlying possible neuroprotective mechanism of rTMS and fluoxetine on abnormal behaviours in a depressive mouse model induced by chronic unpredictable mild stress (CUMS).Methods: After 28 days of CUMS exposure, mice were chronically treated with rTMS (10 Hz for 5 s per train, total 20 trains per day) and (or) fluoxetine (5 mg/kg/day, intraperitoneally) for 28 days targeting on the frontal cortex. After the behavioural tests, the protein expressions of glial fibrillary acidic protein (GFAP), brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) were measured by immunohistochemistry and (or) Western Blot.Results: The results showed rTMS and (or) fluoxetine attenuated the locomotion decrease, anxiety and depressive like behaviours in the CUMS-exposed mice.Conclusion: Our results suggest that both rTMS and fluoxetine could benefit the CUMS-induced abnormal behaviours including depressive-like behaviours, and the beneficial effects of rTMS as well as fluoxetine on depression might be partly related to their neuroprotective effect on attenuating astroglial activation and BDNF decrease.

Role of MAPK and PI3K-Akt signaling pathways in cuprizone-induced demyelination and cognitive impairment in mice.

This study aimed to find the genes and signaling pathways underlying cuprizone-induced demyelination and cognitive impairments in mice. We used the cuprizone-exposed mice as an animal model of schizophrenia and assessed cognitive function in mice. Total RNA was extracted from mouse brain tissues for RNA sequencing. The DESeq2 R package was utilized to analyze the differentially expressed genes (DEGs). Functional and pathway enrichment analyses were performed simultaneously. We also constructed a protein-protein interaction (PPI) network to screen potential hub genes, and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to validate the screened genes. After 6 weeks of cuprizone treatment, the cognitive function of mice was impaired. Compared to the controls, the cuprizone-exposed mice contained 351 DEGs, including 167 upregulated and 184 downregulated genes. Enrichment analysis showed that the DEGs were enriched in some biological processes involved in demyelination, including the MAPK pathway. Functional pathway analysis revealed that the DEGs were significantly enriched in the PI3K-Akt signaling pathway, which may be associated with cognitive impairments. MBP, IGF1, GFAP, PTPRC, CD14, CD68, ITGB2, LYN, TLR2, TLR4, VAV1, and PLEK were considered as potential hub genes. Except for MBP, all genes were upregulated in the cuprizone models, as verified by qRT-PCR. We suggest that the MAPK and PI3K-Akt signaling pathways may be associated with demyelination and cognitive impairments, respectively. GFAP and IGF-1 expression levels increased in cuprizone-exposed mice, suggesting that astrocytes may play a role in protecting the myelin sheath following treatment with cuprizone.

Effects of Glycogen Synthase Kinase-3 Beta Gene Polymorphisms on the Plasma Concentration of Aripiprazole in Chinese Patients with Schizophrenia: A Preliminary Study.

This study explored the differences in glycogen synthase kinase-3 beta (GSK3ß) gene polymorphisms between patients with schizophrenia and healthy controls and investigated the association between gene polymorphisms and plasma concentration of aripiprazole. We enrolled 127 patients with schizophrenia and 125 healthy controls from southern Fujian. The genotypes of the rs6438552, rs12630592, and rs3732361 loci of GSK3ß were evaluated by sequencing with amplified polymerase chain reaction, and the plasma concentration of aripiprazole was determined by high-performance liquid chromatography-tandem mass spectrometry. All three loci of GSK3ß had three genotypes each. The genotype distribution in each locus was not significantly different, but there was a significant difference in the allele frequency between the schizophrenia and control groups within each locus. Linkage disequilibrium analyses of the three single-nucleotide polymorphisms (SNPs) revealed strong linkage. The haplotype analysis results showed two haplotypes in the three SNPs of GSK3ß. The plasma concentrations, dose-corrected concentrations, and normalized concentrations of aripiprazole were significantly different among the different genotypes of the three SNPs. In conclusion, the rs6438552, rs12630592, and rs3732361 loci of GSK3ß may be involved in schizophrenia, and GSK3ß gene polymorphism may be correlated with the plasma concentration of aripiprazole.

Huangkui lianchang decoction attenuates experimental colitis by inhibiting the NF-κB pathway and autophagy.

Ulcerative colitis (UC) is a chronic inflammatory colorectal disease characterized by excessive mucosal immune response activation and dysfunction of autophagy in intestinal epithelial cells. Traditional herbal preparations, including the Huangkui lianchang decoction (HLD), are effective in UC clinical treatment in East Asia, but the underlying mechanism is unclear. This study evaluated the therapeutic effects and associated molecular mechanisms of HLD in UC in vivo and in vitro. A C57BL/6 UC mouse model was established using 2.5% dextran sulfate sodium. The effects of HLD on the colonic structure and inflammation in mice were evaluated using mesalazine as the control. The anti-inflammatory effects of HLD were assessed using disease activity index (DAI) scores, histological scores, enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, and western blotting. HLD displayed a protective effect in UC mice by reducing the DAI and colonic histological scores, as well as levels of inflammatory cytokines and NF-κB p65 in colonic tissues. NCM460 lipopolysaccharide-induced cells were administered drug serum-containing HLD (HLD-DS) to evaluate the protective effect against UC and the effect on autophagy. HLD-DS exhibited anti-inflammatory effects in NCM460 cells by reducing the levels of inflammatory cytokines and increasing interleukin 10 levels. HLD-DS reduced p-NF-κB p65, LC3II/I, and Beclin 1 expression, which suggested that HLD alleviated colitis by inhibiting the NF-κB pathway and autophagy. However, there was no crosstalk between the NF-κB pathway and autophagy. These findings confirmed that HLD was an effective herbal preparation for the treatment of UC.

NCAPD3 enhances Warburg effect through c-myc and E2F1 and promotes the occurrence and progression of colorectal cancer.

BACKGROUND: NCAPD3 is one of the three non-SMC subunits of condensin II complex, which plays an important role in the chromosome condensation and segregation during mitosis. Notably, elevated levels of NCAPD3 are found in many somatic cancers. However, the clinical role, biological functions of NCAPD3 in cancers especially in colorectal cancer (CRC) and the underlying molecular mechanisms remain poorly elucidated. METHODS: Clinical CRC and adjacent normal tissues were used to confirm the expression of NCAPD3. The association of NCAPD3 expression with clinicopathological characteristics and patient outcomes were analyzed by using online database. In vivo subcutaneous tumor xenograft model, NCAPD3 gene knockout following azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced tumor mouse model, Co-IP, western blot, qRT-PCR, IHC, ChIP assays and cell functional assays were used to investigate the biological functions of NCAPD3 in CRC and the underlying molecular mechanisms. RESULTS: NCAPD3 was overexpressed in CRC tissues and positively correlated with poor prognosis of CRC patients. NCAPD3 knockout suppressed CRC development in AOM/DSS induced and xenograft mice models. Moreover, we found that NCAPD3 promoted aerobic glycolysis in CRC. Mechanistically, NCAPD3 up-regulated the level of c-Myc and interacted with c-Myc to recruit more c-Myc to the gene promoter of its downstream glycolytic regulators GLUT1, HK2, ENO1, PKM2 and LDHA, and finally enhanced cellular aerobic glycolysis. Also, NCAPD3 increased the level of E2F1 and interacted with E2F1 to recruit more E2F1 to the promoter regions of PDK1 and PDK3 genes, which resulted in the inhibition of PDH activity and TCA cycle. CONCLUSIONS: Our data demonstrated that NCAPD3 promoted glucose metabolism reprogramming and enhanced Warburg effect in colorectal tumorigenesis and CRC progression. These findings reveal a novel mechanism underlying NCAPD3 mediated CRC cell growth and provide new targets for CRC treatment.

Sphincter-Preserving Fistulectomy Is an Effective Minimally Invasive Technique for Complex Anal Fistulas.

Background: The optimal treatment of complex anal fistulas remains unclear, though many different sphincter-preserving procedures have been described. A minimally invasive technique with a better outcome is desired. The purpose of this study was to present a new technique-sphincter-preserving fistulectomy (SPF) and its clinical outcomes. Materials and Methods: A retrospective study was performed to compare the efficacy and outcomes of SPF with ligation of the intersphincteric fistula tract (LIFT) in the management of complex anal fistulas in regards to postoperative pain, complications, wound healing time, recurrence, overall success rate, fecal continence function, and quality of life. Continence function was evaluated using the Wexner incontinence scale and anal manometry. The fecal incontinence quality of life (FIQL) scale was used to assess patients' quality of life. Results: From June 2020 to July 2021, 41 patients with 43 SPF procedures and 35 patients with 35 LIFT procedures were included. Postoperative pain was comparable between two groups. The morbidity rate and the mean wound healing time in the SPF group were lower than those in the LIFT group (2.3% vs. 48.6%, p < 0.001; 1.4 ± 0.3 vs. 1.7 ± 0.4 months, p = 0.001). At a mean follow-up duration of 11.4 ± 3.5 months in the SPF group and 10.7 ± 4.3 months in the LIFT group, SPF achieved a better overall success rate than LIFT (97.7% vs. 77.1%, p = 0.014). Three patients in the SPF group and 4 patients in the LIFT group who all underwent a simultaneous fistulotomy procedure complained new incontinence of flatus. There was no statistical difference between the two groups in regards to the Wexner scores (p = 0.790), the maximum resting anal canal pressure (p = 0.641), the maximum squeeze pressure (p = 0.289), and the FIQL scores including lifestyle (p = 0.188), coping (p = 0.188), depression (p = 0.850), and embarrassment (p = 0.910). Conclusions: SPF is a novel, safe, and effective minimally invasive technique for the management of complex anal fistulas, with a promising success rate and negligible impairment on continence. Future prospective studies are needed to evaluate the long-term outcomes of SPF.

Proteomics identifies a novel role of fibrinogen-like protein 1 in Crohn's disease.

BACKGROUND: Crohn's disease (CD) is an incurable intestinal disorder with unclear etiology and pathogenesis. Currently, there is a lack of specific biomarkers and drug targets for CD in clinical practice. It is essential to identify the precise pathophysiological mechanism of CD and investigate new therapeutic targets. AIM: To explore a new biomarker and therapeutic target for CD and verify its role in the CD pathological mechanism. METHODS: Proteomics was performed to quantify the protein profile in the plasma of 20 CD patients and 20 matched healthy controls. Hub genes among the selected differentially expressed proteins (DEPs) were detected via the MCODE plugin in Cytoscape software. The expression level of one hub gene with an immunoregulatory role that interested us was verified in the inflamed intestinal tissues of 20 CD patients by immunohistochemical analysis. After that, the effects of the selected hub gene on the intestinal inflammation of CD were identified in a CD cell model by examining the levels of proinflammatory cytokines by enzyme-linked immunosorbent assays and the expression of the NF-κB signalling pathway by quantitative real-time PCR analysis and Western blot assays. RESULTS: Thirty-five DEPs were selected from 393 credible proteins identified by proteomic analysis. Among the DEPs, fibrinogen-like protein 1 (FGL1), which attracted our attention due to its function in the regulation of the immune response, had 1.722-fold higher expression in the plasma of CD patients and was identified as a hub gene by MCODE. Furthermore, the expression of FGL1 in the intestinal mucosal and epithelial tissues of CD patients was also upregulated (P < 0.05). In vitro, the mRNA levels of FGL1 and NF-κB; the protein expression levels of FGL1, IKKα, IKKß, p-IKKα/ß, p-IκBα, and p-p65; and the concentrations of the proinflammatory cytokines IL-1ß, IL-6, IL-17, and TNF-α were increased (P < 0.05) after stimulation with lipopolysaccharide, which were reversed by knockdown of FGL1 with siRNA transfection (P < 0.05). Conversely, FGL1 overexpression enhanced the abovementioned results (P < 0.05). CONCLUSION: FGL1 can induce intestinal inflammation by activating the canonical NF-κB signalling pathway, and it may be considered a potential biomarker and therapeutic target for CD.

Formation rate of secondary anal fistula after incision and drainage of perianal Sepsis and analysis of risk factors.

BACKGROUND: The choice of surgery for perianal sepsis is currently controversial. Some people advocate one-time radical surgery for perianal sepsis, while others advocate incision and drainage. The objective of this study is to observe the formation probability of secondary anal fistula after incision and drainage in patients with perianal sepsis and determine factors that contribute to secondary anal fistula after incision and drainage. METHODS: A retrospective descriptive analysis was conducted in 288 patients with perianal sepsis who were treated with anorectal surgery in the Suzhou Hospital of Traditional Chinese Medicine from January 2016 to June 2018. The patients were followed by telephone, physical examination, and pelvic MRI examination for at least 1 year after surgery. RESULTS: Three patients were not followed, 98 patients did not receive surgical treatment or one-time radical surgery for perianal sepsis, and 187 patients were ultimately identified for the study. Anal fistula was present in 105 patients, and the rate of formation of secondary anal fistula was 56.15%. There was no statistically significant difference in the fistula formation rate between different types of sepsis (P>0.05). And, in patients with secondary anal fistula, there was no significant correlation between the location of sepsis and the type of secondary anal fistula (P>0.05). CONCLUSIONS: The incidence of secondary anal fistula after incision and drainage of perianal sepsis is 56.15%, which is lower than the incidence found in previous study. Young is a risk factor for secondary anal fistula after incision and drainage of perianal sepsis. There is no significant correlation between the location of sepsis and the type of secondary anal fistula. Simple incision and drainage is a suitable choice for patients with acute perianal sepsis.

Effect of Loureirin B on Crohn's disease rat model induced by TNBS via IL-6/STAT3/NF-κB signaling pathway.

BACKGROUND: Crohn's disease (CD) is a chronic relapsing form of inflammatory bowel disease, seriously threatening human beings health. However, the pathogenesis of CD is still unclear and there is no specific effective drug for treatment of CD. Resina Donis (RD) obtained from Dracaena cochinchinensis (Lour.) S. C. Chen (Liliaceae), has been used for the treatment of CD clinically. Loureirin B (LB) is one of the most important chemical compositions and physiologically active ingredients of resina draconis. It has the molecular structure propan-1-one, 1-(4-hydroxyphenyl)-3-(2,4,6-trimethoxyphenyl)-1-(4-hydroxyphenyl)-3-(2,4,6-trimethoxyphenyl) propan-1-one. The aim of this study was to investigate the effect of LB on CD and explore the underlying mechanisms. METHODS AND RESULTS: In this study, the result demonstrated that LB prolonged the survival time of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rats and alleviated colonic damage in a dose dependent manner. Besides, LB remarkably ameliorated TNBS-induced inflammatory response via regulation of cytokines in the colonic tissues. Moreover, LB could reverse the established fibrosis and impede the accumulation infiltration, and improve the apoptosis induced by TNBS in a dose dependent manner. Further, LB dramatically suppressed TNBS-induced the activation of IL-6/STAT3/NF-κB signaling pathway. CONCLUSIONS: These findings suggested that LB could be beneficial regarding ameliorating TNBS-induced CD, which may represent a novel approach to treat CD and provide an alternative choice for disorders associated with CD.

Arecoline attenuates memory impairment and demyelination in a cuprizone-induced mouse model of schizophrenia.

Cerebral demyelination is possibly one of the main pathological factors involved in the development of schizophrenia. Our previous studies have showed that Areca catechu nut extract could ameliorate cognitive decline by facilitating myelination processes in the frontal cortex in a cuprizone (CPZ)-induced mouse model of schizophrenia. The aim of the present study was to evaluate the effects of arecoline, one of the alkaloids in A. catechu nut extract, on memory impairment and cerebral demyelination in CPZ-treated mice. Mice were treated with CPZ (0 or 0.2%) in chow food and arecoline hydrobromide (0, 2.5, or 5 mg/kg/day) in drinking water for 12 weeks before Y-maze behavioral test. After the behavioral test, the mice were sacrificed for the measurement of myelin basic protein in the frontal cortex. We showed that arecoline-attenuated spatial working memory impairment, concurrent with attenuated demyelination related to vehicle-treated CPZ mice for the first time. Arecoline is one of the primary active ingredients in A. catechu nut responsible for attenuating memory impairment and demyelination in CPZ mice, cerebral demyelination may have a role in memory impairment, and modulation of cerebral demyelination could be a useful strategy in schizophrenia treatment.

Chronic administration of quetiapine attenuates the phencyclidine-induced recognition memory impairment and hippocampal oxidative stress in rats.

The underlying mechanism of atypical antipsychotics in treating cognitive impairment in schizophrenia is unclear. The aim of the present study was to evaluate the effects of quetiapine, an atypical antipsychotic drug, on object recognition memory and hippocampal oxidative stress in a phencyclidine (PCP) rat model of schizophrenia. Rats were treated with chronic quetiapine (10 mg/kg/day, intraperitoneally) for 16 days or acute quetiapine (10 mg/kg/day, intraperitoneally) on day 16. On day 16, 1 h after the administration of quetiapine, the rats were administered PCP (50 mg/kg, subcutaneously). After the last object recognition behavioral test on day 18, the rats were killed for the measurement of hippocampal protein expression of nitrotyrosine, a protein marker of oxidative stress. The results showed that chronic quetiapine significantly attenuated object recognition memory impairment and hippocampal oxidative stress in the PCP-injected rats. These suggest that the attenuating effect of chronic quetiapine on hippocampal oxidative stress may be related to quetiapine's beneficial effects on object recognition memory in PCP rats, and further suggest that neuroprotective mechanisms are involved in chronic quetiapine treatment.

Magnetic resonance imaging findings for differential diagnosis of perianal plexiform schwannoma: Case report and review of the literature.

Plexiform schwannoma is an extremely rare variant of schwannoma, accounting for approximately 5% of cases. Due to the rarity and lack of typical symptoms, signs and radiological images, a definite diagnosis of plexiform schwannoma may not be made by clinicians prior to biopsy. In the present study, we report the first case (to our knowledge) of perianal plexiform schwannoma arising from the overlapped skin of the ischioanal fossa, and we propose an intratumorally nonenhanced circumferential capsule dividing the tumour into multiple homogeneously enhanced nodules as a magnetic resonance imaging feature to aid in the differential diagnosis of plexiform schwannoma from ancient schwannoma, cavernous haemangioma, liposarcoma and plexiform neurofibroma.

The Aqueous Fraction of Areca catechu Nut Ameliorates Demyelination in Prefrontal Cortex-Induced Depressive Symptoms and Cognitive Decline through Brain-Derived Neurotrophic Factor-Cyclic Adenosine Monophosphate Response Element-Binding Activation.

OBJECTIVE: To investigate if Areca catechu L. treatment could ameliorate depressive symptoms and cognitive decline by facilitating myelination processes in prefrontal cortex. METHODS: A mouse model of cuprizoneinduced demyelination was used to mimic demyelinating disease. Two concentrations of A. catechu nut extract (ANE; 1% and 2%) were administered orally in the diet for 8 weeks. Depressive symptoms and cognition-associated behaviors were evaluated in tests of locomotor activity, tail suspension, and forced swimming; spatial memory was tested with the Y-maze. Expression of myelin basic protein (MBP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), glutathione S-transferases pi (GSTpi), brain-derived neurotrophic factor (BDNF), and the transcription factor cyclic adenosine monophosphate (cAMP) response element-binding (CREB) were evaluated by western blot. RESULTS: Animals subjected to demyelination showed hyperactivity (P<0.01), impaired spatial memory (P<0.01), and depressive behaviors (P<0.05). Internally, they displayed signifificant myelin damage in the cortex, lower expression of CNPase and GSTpi, slightly decreased BDNF (P>0.05), and signifificantly reduced p-CREB (P<0.05). Nevertheless, ANE treatment demonstrated signifificant anti-depressant activity and enhancement of working memory (P<0.05 or 0.01). In addition, ANE treatment increased MBP, CNPase and GSTpi protein expression in prefrontal cortex (P<0.05). Concomitant with increased BDNF production (P<0.05), ANE treatment up-regulated phosphorylated CREB, but without statistical signifificance (P>0.05). CONCLUSION: ANE treatment might ameliorate depressive symptoms and cognitive decline by facilitating myelination processes in prefrontal cortex via induction of BDNF-CREB activation.

The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice.

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.

MicroRNA miR-92a-1 biogenesis and mRNA targeting is modulated by a tertiary contact within the miR-17~92 microRNA cluster.

While functional mature microRNAs (miRNAs) are small ∼22 base oligonucleotides that target specific mRNAs, miRNAs are initially expressed as long transcripts (pri-miRNAs) that undergo sequential processing to yield the mature miRNAs. We have previously reported that the pri-miR-17∼92 cluster adopts a compact globular folded structure that internalizes a 3' core domain resulting in reduced miRNA maturation and subsequent mRNA targeting. Using a site-specific photo-cross-linker we have identified a tertiary contact within the 3' core domain of the pri-miRNA between a non-miRNA stem-loop and the pre-miR-19b hairpin. This tertiary contact is involved in the formation of the compact globular fold of the cluster while its disruption enhances miR-92a expression and mRNA targeting. We propose that this tertiary contact serves as a molecular scaffold to restrict expression of the proposed antiangiogenic miR-92a, allowing for the overall pro-angiogenic effect of miR-17∼92 expression.

Probing the leucyl/phenylalanyl tRNA protein transferase active site with tRNA substrate analogues.

Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.

A small molecule inhibitor of polycomb repressive complex 1 inhibits ubiquitin signaling at DNA double-strand breaks.

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.

CBX4-mediated SUMO modification regulates BMI1 recruitment at sites of DNA damage.

Polycomb group (PcG) proteins are involved in epigenetic silencing where they function as major determinants of cell identity, stem cell pluripotency and the epigenetic gene silencing involved in cancer development. Recently numerous PcG proteins, including CBX4, have been shown to accumulate at sites of DNA damage. However, it remains unclear whether or not CBX4 or its E3 sumo ligase activity is directly involved in the DNA damage response (DDR). Here we define a novel role for CBX4 as an early DDR protein that mediates SUMO conjugation at sites of DNA lesions. DNA damage stimulates sumoylation of BMI1 by CBX4 at lysine 88, which is required for the accumulation of BMI1 at DNA damage sites. Moreover, we establish that CBX4 recruitment to the sites of laser micro-irradiation-induced DNA damage requires PARP activity but does not require H2AX, RNF8, BMI1 nor PI-3-related kinases. The importance of CBX4 in the DDR was confirmed by the depletion of CBX4, which resulted in decreased cellular resistance to ionizing radiation. Our results reveal a direct role for CBX4 in the DDR pathway.