RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

BACKGROUND: Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. METHODS: The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. RESULTS: RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. CONCLUSION: This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

Background Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication origin binding protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. Methods The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. Results RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. Conclusion This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production.

The differentially expressed gene signatures of the Cullin 3-RING ubiquitin ligases in neuroendocrine cancer.

Cullin-RING ubiquitin E3 ligase (CRLs) composed of four components including cullin scaffolds, adaptors, substrate receptors, and RING proteins mediates the ubiquitination of approximately 20% of cellular proteins that are involved in numerous biological processes. While CRLs deregulation contributes to the pathogenesis of many diseases, including cancer, how CRLs deregulation occurs is yet to be fully investigated. Here, we demonstrate that components of CRL3 and its transcriptional regulators are possible prognosis marker of neuroendocrine (NE) cancer. Analysis of Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal revealed that expression of CRL3 scaffold Cullin 3 (CUL3) highly correlates with NE signature, and CUL3 silencing inhibited NE cancer proliferation. Moreover, subset of 151 BTB (Bric-a-brac, Tramtrack, Broad complex) domain-containing proteins that have dual roles as substrate receptors and adaptor subunits in CRL3, as well as the expression of transcription factors (TFs) that control the transcription of BTB genes were upregulated in NE cancer. Analysis using published ChIP-sequencing data in small cell lung cancer (SCLC), including NE or non-NE SCLC verified that gene promoter of candidates which show high correlation with NE signature enriched H3K27Ac. These observations suggest that CRL3 is a master regulator of NE cancer and knowledge of specifically regulated CRL3 genes in NE cancer may accelerate new therapeutic approaches.

Safety evaluation and consideration of 4 Pin Multi-needle for meso-therapy.

This study was conducted according to the method presented in the Republic of Korea Pharmacopoeia 11th Revision, aseptic test method to evaluate the suitability of sterilization for a sterile needle (4 Pin Multi-needle). In this study, four tests were conducted: sterility test, cytotoxicity test, acute toxicity test, skin sensitization test. First, in the aseptic test, the microorganism was not proliferated in the aseptic test of the medium. As a result of the performance test of the medium, it was confirmed that the microorganism developed within 3 days and the fungus was evident within 5 days. Based on this, it was confirmed that the medium was suitable, and as a result of the aseptic test, the development of microorganisms was not observed during the total culture period. Based on these results, tests were conducted which were confirmed to be suitable for aseptic testing because the development of bacteria on the provided samples was not recognized. For cytotoxicity tests ISO10993-5; 2009 (Biological Evaluation of Medical Devices, Part 5: Test for in vitro Cytotoxicity). As a result, the MEM eluate of the test substance caused very slight cytotoxicity to the fibroblasts of the mouse and was judged to be Grade 1 (Slightly cytotoxic) according to the judgment standard of ISO 10993-5. On the other hand, solvent control, negative control and positive control showed the expected results on the test. Acute Toxicity Test Results: It was judged that there was no systemic toxicity change when ICR mice were treated with 50 mL/kg B.W. of the eluate of sterile injectable needle for 72 hours. Skin sensitization test result: The Hartley guinea pig was evaluated as a substance which is evaluated as a substance which does not induce any skin reaction when skin sensitization is applied to the dissected material of the sterile injectable needle and is weak in skin sensitivity. Based on the above tests, we will study the stability and efficacy of more reliable medical devices based on the verification and performance of medical devices.

A case of medullary thyroid carcinoma in which the skin metastasis was concurrently present and response occurred to chemotherapy.

Medullary thyroid carcinoma accounts for 3% of all thyroid gland malignancies. It commonly metastasizes to liver, lung, and bone. It rarely metastasizes to skin, and only a few such cases have been documented. Cutaneous metastasis suggests a poor prognosis, with a mean survival of 7.5-19 months. The most effective treatment for skin metastasis is complete surgical removal of all local and regional lesions. The response to systemic chemotherapy is typically poor. We report a case of medullary thyroid carcinoma with cutaneous metastases, which responded to chemotherapy.

Early modification of initial HAART regimen associated with poor clinical outcome in HIV patients.

Although the importance of the initial regimen in highly active antiretroviral therapy (HAART) has been emphasized, the effect on clinical outcome of early modification of the initial HAART regimen has not been well-defined. The analysis included antiretroviral-naive HIV patients who started HAART between 1999 and 2005 at a university hospital. Early modification was defined as any drug change within 2 months of starting HAART. The effect of early regimen modification on the occurrence of new AIDS-defining illness or death was assessed using Kaplan-Meier survival estimates, and hazard ratios were estimated using Cox's proportional hazards regression model. Of 398 patients beginning HAART, 21% experienced early modification of their regimen, the most common reason being gastrointestinal toxicity (49%). After adjusting for risk factors for occurrence of a new AIDS event or death, identified by univariate analysis, the hazards ratio contrasting early modification with maintenance of initial HAART regimen was 3.06 (95% confidence interval, 1.36-6.90; p = 0.007). There were significant differences between the AIDS event-free survival curves of patients in clinical categories B or C with or without early modification of initial HAART regimen (p = 0.0002), but no significant difference in patients in clinical category A (p = 0.706). A considerable proportion of patients who started HAART changed treatment regimen mainly due to intolerance early after start of HAART. Early modification of an initial HAART regimen was associated with poor clinical outcome in HIV patients in the advanced clinical categories.

Tuberculosis manifested by immune reconstitution inflammatory syndrome during HAART.

The proportion of tuberculosis manifested by immune reconstitution inflammatory syndrome (IRIS) after HAART has not been established. We describe the incidence and clinical features of tuberculosis manifested by IRIS after HAART in an intermediate tuberculosis burden area. The findings suggest that a significant proportion of the tuberculosis occurring early after starting HAART is manifested by IRIS.

Immune reconstitution inflammatory syndrome in the first year after HAART: influence on long-term clinical outcome.

Little is known about the effect of immune reconstitution inflammatory syndrome (IRIS) on the long-term clinical outcome. Of 52 opportunistic infections (OI) occurring within one year after the start of HAART in 387 HIV patients, 33 (63%) were classified as having IRIS. The patients with IRIS showed no significant difference in the AIDS event-free survival curve compared with the matched control group without OI and in contrast to non-IRIS OI.