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Introduction: According to the statistics, vascular injury occurs during the onset of diabetic changes after the production of several byproducts. Many authorities have focused to find an alternative therapy for diabetic patients. In this study, we investigated the therapeutic effects of natural polyphenol like resveratrol on human endothelial cells exposed to malondialdehyde for 48 hours. Methods: Human Umbilical Vein Endothelial Cells were randomly classified into four groups;control, malondialdehyde (2.5 mM), resveratrol (100 mu M), and cells received the combined regime for 48 hours. Cell viability was determined by 3-(4, 5-dimethyl thiazol-2-yl) 2, 5-diphenyl-tetrazoliumbromide (MTT) assay. Griess reaction was performed to measure the content of Nitric oxide (NO). Apoptosis was studied by using real-time polymerase chain reaction (RT-PCR) and western blotting assays. Levels of receptor tyrosine kinases like VEGFR-1, -2, Tie-1, and -2 were analyzed by enzyme-linked immunosorbent assay(ELISA). The affinity of resveratrol and malondialdehyde to serum albumin was measured by Surface Plasmon Resonance Assay. Any changes in chromatin remodeling were detected by PCR array analysis. Results: Resveratrol reduced cytotoxicityand NO contentinside cells induced by malondialdehyde(MDA) (P < 0.05). Endothelial cell apoptosis was decreased by the reduction of pro-apoptotic factor Bax and increase of Bcl-2 following the incubation with resveratrol (P < 0.05). MDA-induced receptor tyrosine kinases increase was inhibited by resveratrol and reached near-to-normal levels (P < 0.05). Surface Plasmon Resonance revealed a higher affinity of resveratrol to albumin compared to the malondialdehyde-albumin complex. Polymerase chain reaction (PCR) array revealed the potency of resveratrol in chromatin remodeling following the treatment with malondialdehyde (P < 0.05). Conclusion: Based on our findings, resveratrol has the potential to decrease diabetic vascular injury induced by lipid byproducts such as MDA., Tabriz University of Medical Sciences, This study was supported by a grant from Tabriz University of Medical Sciences.

Les protéines du groupe Polycomb (PcG) sont des protéines essentielles et conservées, qui forment deux complexes principaux, PRC1 et PRC2, qui sont recrutés au niveau de la chromatine et qui répriment stablement l’expression génique. Chez Drosophila melanogaster, les complexes Polycomb sont recrutés à des éléments d’ADN appelés éléments de réponse Polycomb (PREs) pour réprimer la transcription. PREs sont des éléments mémoires permutables qui peuvent maintenir la répression ou l’expression génique. Malgré des dizaines d’années d’étude, des questions fondamentales sur le fonctionnement du système PcG subsistent. 1) Comment les protéines PcG sont recrutées aux PREs uniquement lors du contexte développemental approprié, et comment les PREs peuvent conduire à la fois à l’activation et à la répression stable. 2) Comment les complexes PcG répriment la transcription, et si cela implique de nouvelles activités biochimiques et interactions. 3) Comment la répression dépendante des PcG peut-elle être propagé à travers le cycle cellulaire. La recherche de nouvelles activités biochimiques pour les complexes PcG pouvant répondre à ces questions fait l’objet de cette thèse. Les PREs sont transcrits en ARN qui pourraient donner la spécificité de contexte pour recruter les protéines PcG. Nous avons supposé que des R-loops puissent se former aux PREs, et être reconnues par les complexes Polycomb, ce que vous avons testé dans le chapitre 2. Nous avons identifié les séquences formant des R-loops dans des embryons et une lignée cellulaire de Drosophila melanogaster, et nous avons trouvé que ~30% des PREs forment des R-loops. Nous avons découvert que les PREs ayant formé des R-loops ont une plus forte probabilité d’être liés par les protéines PcG in vivo et in vitro. PRC2 lie des milliers d’ARN in vivo, mais aucune fonction claire n’y a été associée. En utilisant des expériences in vitro, nous avons identifié une activité d’invasion de brins pour PRC2 qui induit la formation d’hybride ARN-ADN, la partie principale d’une R-loop. Dans ce chapitre, nous avons trouvé que les PREs forment des R-loops, et sont impliquées dans le recrutement des protéines PcG qui induisent la répression génique stable. Nous avons découvert une activité d’invasion de brins pour PRC2 qui pourrait impliquer ce complexe Polycomb dans la formation de R-loops in vivo. Dans le chapitre 3, nous avons identifié une activité similaire à celle de la topoisomérase I associée avec PRC1 et la région C-terminale de sa sous-unité PSC (PSC-CTR). PRC1 et PSC-CTR peuvent relaxer un plasmide surenroulé négativement et ajouter des supertours négatifs à un plasmide relaxé en absence de topoisomérase. Cette activité suggère que la régulation de la topologie de l’ADN puisse être un nouveau mécanisme utilisé par les complexes PcG. PRC1 peut résoudre les R-loops formées sur un ADN négativement surenroulé in vitro. Une fonction possible pour cette activité de topoisomérase peut être la régulation des R-loops, dont la stabilité dépend à la fois de la séquence d’ADN et de la topologie de l’ADN environnant, in vivo. Dans cette thèse, nous avons identifié de nouvelles activités chez les complexes PcG : une activité d’invasion de brins pour PRC2 et une activité similaire à celle des topoisomérases pour PRC1. Ces deux activités impliquent les complexes PcG dans la formation et la résolution de R-loops. De plus, ces deux complexes peuvent reconnaitre les R-loops et sont recrutés aux PREs ayant formé ces structures. En conclusion, nous avons identifié de nouvelles activités pour les complexes Polycomb PRC1 et PRC2 qui les lient à la formation, la reconnaissance et la résolution de R-loops., Polycomb group (PcG) proteins are conserved, essential proteins, which assemble in two main complexes, PRC1 and PRC2, which are targeted to chromatin and stably repress gene expression. In Drosophila melanogaster, Polycomb complexes are targeted to DNA elements called Polycomb response elements (PREs) to repress transcription. PREs are switchable memory elements that can maintain either gene repression or gene activation. Despite decades of study, fundamental questions about how the PcG system functions remain. These include: 1) how PcG proteins are targeted to PREs only in the appropriate developmental context, and how PREs can mediate both stable activation and repression; 2) how PcG complexes repress transcription, and whether it involves novel biochemical mechanisms and interactions; 3) how PcG repression can be propagated through cell cycles. The search for new biochemical activities for PcG complexes that may answer these questions is the topic of this thesis. PREs are transcribed into RNAs which may give the context specificity to recruit PcG proteins. We hypothesized that R-loops may form at PREs, and be recognized by PcG complexes, which we tested in Chapter 2. We identified R-loop forming sequences in Drosophila melanogaster embryos and tissue culture cells, and found that ~30% of the PREs form R-loops. We found that PREs which have formed R-loops are more likely to be bound by PcG proteins both in vivo and in vitro. PRC2 binds to thousand RNA in vivo but no clear activity has been associated with it. Using in vitro assays, we identified a strand exchange activity for PRC2 which induces the formation of RNA-DNA hybrid, the main part of an R-loop. In this chapter, we have found that PREs form R-loops and are involved in the targeting of PcG proteins which induce stable gene repression. We have discovered an RNA strand exchange activity for PRC2 which may involve this Polycomb complex in the formation of R-loops in vivo. In Chapter 3, we identified a type I topoisomerase-like activity associated with PRC1 and the C-terminal region of its subunit PSC (PSC-CTR). PRC1 and PSC-CTR can relax a negatively supercoiled plasmid and add negative coils to a relaxed plasmid in absence of topoisomerase. This activity suggests regulation of DNA topology may be a novel mechanism used by PcG complexes. PRC1 can resolve R-loops formed on negatively supercoiled DNA in vitro. One role for the topoisomerase-like activity may be to regulate R-loops, whose stability of depends on both the DNA sequence and the topology of the surrounding DNA, in vivo. In this thesis, we identified new activities for Polycomb group complexes: an RNA strand exchange activity for PRC2 and a topoisomerase-like activity for PRC1. Both activities link PcG complexes to the formation and resolution of R-loops. In addition, both complexes can recognize R-loops and are recruited to PREs which have formed these structures. In conclusion, we have identified new nucleic acid-based activities for the Polycomb complexes PRC1 and PRC2, which link them to the formation, recognition and resolution of R-loops.

The Polycomb group genes are a general class of regulators that are responsible for maintaining homeotic gene expression throughout cell division. Polycomb group expression plays an important role in oncogenesis of several types of human cancer. Melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 are key Polycomb group proteins. Studies have shown that melanoma nuclear protein 18 is a potential tumor suppression, and B-cell-specific Moloney leukemia virus insert site 1 is overexpressed in several human malignancies. However, the roles of melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 in esophageal squamous cell carcinoma are still unclear. In this study, we analyzed the expression levels of melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 in 89 esophageal cancer tissues and paired normal mucosal tissues using immunohistochemistry, Western blotting, and quantitative real-time polymerase chain reaction analyses. We found that the expression of melanoma nuclear protein 18 in the carcinoma tissues was significantly lower than that in the noncancerous mucosal tissues (P < .05), and B-cell-specific Moloney leukemia virus insert site 1 expression in the carcinoma tissues was significantly higher than that in the noncancerous mucosal tissues (P < .05). In addition, the expression of melanoma nuclear protein 18 was correlated with clinical stage, depth of invasion, and lymph node metastasis (P < .05) but was not correlated with gender, age, degree of differentiation, or disease-free survival (P > .05). B-cell-specific Moloney leukemia virus insert site 1 expression was strongly correlated with the degree of differentiation, clinical stage, and lymph node metastasis (P .05). Moreover, there was a negative correlation between melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 expressions in esophageal squamous cell carcinoma (P < .05). Our study suggests that melanoma nuclear protein 18 and B-cell-specific Moloney leukemia virus insert site 1 may play a crucial role in esophageal squamous cell carcinoma. Melanoma nuclear protein 18 or B-cell-specific Moloney leukemia virus insert site 1 may be a potential biomarker for diagnosis and prognosis of esophageal squamous cell carcinoma.

Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/β-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/β-catenin signaling pathway activities.

How multicellular organisms modulate transcription in response to internal and external cues is a fundamental question in plant and animal development and growth. In Arabidopsis, TOPLESS(TPL), a Groucho/Tup1 like co-repressor, is involved in the transcriptional response to multiple plant hormones and plays a crucial role in a range of major developmental processes, such as embryonic apical-basal patterning and post-embryonic floral patterning. This dissertation describes the advances made toward the elucidation of the molecular mechanisms underlying transcriptional regulation by TPL. As a co-repressor, TPL lacks intrinsic DNA binding activity and has been shown to be recruited by transcription factors with repressive motifs. To date, a range of transcription factors have been reported to interact with TPL, yet the functionality of most of these interactions is not known. We found that the members of the Class III Homedomain-Leucine Zipper (HD- ZIPIII) transcription factor family co-localize with TPL to form complexes at the promoters of thousands of genes in the genome. HD-ZIPIIIs both antagonistically and cooperatively regulate entire pathways at multiple steps in a cell or tissue specific manner. The five members in the family form homo- and hetero-dimers and act both as activators and repressors. The association with TPL confers the repressive function of HD-ZIPIIIs. Just like its counterparts in animals and fungi, TPL also associates with histone deacetylases(HDACs) to repress transcription. We characterized the binding profiles of TPL and HDA19, a class I histone deacetylase that interacts with TPL, and found they tend to bind to the promoters of dynamically expressed genes. Almost all HDA19 peaks have an overlapping TPL peak, while about half of the TPL peaks are in common with HDA19, suggesting HDA19 is involved only at a subset of TPL targets. Specifically, we found the three members of the evolutionarily conserved NAM/CUC (for NO APICAL MERISTEM and CUP-SHAPED COTYLEDON) transcription factors, CUC1, CUC2 and CUC3, are regulated by TPL and HDA19 to control boundary specification during floral development. Finally, we showed that TPL interacts with lysine 27-trimethylated histone H3(H3K27me3) and binds to a significant number of genomic regions with this modification, suggesting a novel layer of regulation which has not been reported in animals. A decrease in TPL binding was observed in plants that have a mutation in the Polycomb Repressive Complexes 2 (PRC2), which mediates the deposition of H3K27me3. TPL itself affects H3K27me3 levels and interacts with a component of the PRC1 complex, which recognizes H3K27me3 and further contributes to chromatin compaction. Genes potentially regulated by both TPL and PRCs are involved in multiple developmental processes and stress responses. These data suggest that upon the recruitment of TPL, binding affinity is enhanced by H3K27me3 marked nucleosomes and TPL may further contributes to the repressive transcriptional state via the association with Polycomb group (PcG) proteins. Overall, we propose a model of transcriptional regulation by TPL that consists of three steps: TPL first is recruited by specific transcription factors; the association of TPL at some target genes is enhanced by the presence of H3K27me3; and TPL forms co-repressor complexes with HDA19 and possibly PcGs to repress gene expression.

Enhancer of zeste homolog 2 (EZH2), the critical component of polycomb group protein family, has been demonstrated to be overexpressed in various types of human cancer, including hepatocellular carcinoma, breast, bladder and lung cancer. The mechanism of how EZH2 promotes oncogenesis has also been well studied. However, little is known about the role of EZH2 in colorectal cancer (CRC). The main purpose of the present study was to analyze the association between EZH2 expression and the clinicopathological features of CRC. Therefore, the mRNA and protein expression levels were analyzed in tumor tissues and adjacent non-cancerous tissues by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The expression of EZH2 was demonstrated to be significantly increased in tumor tissues compared with adjacent noncancerous tissues, according to the results of western blot analysis and RT-qPCR in the majority of cases. Patients with low EZH2 expression had a longer overall survival rate compared with those with high EZH2 expression. An analysis of the association between clinicopathological features and EZH2 expression indicated that high EZH2 expression was significantly associated with tumor stage, tumor size, histological differentiation and lymph node metastasis. Multivariate analysis demonstrated that high EZH2 expression was an independent predictor of overall survival. In conclusion, to the best of our knowledge, the data presented in the present study is the first to indicate that EZH2 is upregulated in CRC and may serve as a predictor of poor outcome for patients with CRC.

// Jianjun Tang 1, * , Gang Wang 1, * , Meifang Zhang 1, * , Feng-yan Li 1 , Yi Sang 1 , Boqing Wang 1, 2 , Kaishun Hu 1 , Yuanzhong Wu 1 , Rongzhen Luo 1 , Dan Liao 1 , Jingying Cao 1 , Xin Wang 1 , Li Wang 1 , Ruhua Zhang 1 , Xiaoshi Zhang 1 , Wu-Guo Deng 1 , Dan Xie 1 , Rui-hua Xu 1 , Tiebang Kang 1 1 State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou 510060, China 2 Department of Hepatobiliarypancreatic Surgery, Affiliated Tumor Hospital, Xinjiang Medical University, Urumqi 830000, China * These authors contributed equally to this work Correspondence to: Tiebang Kang, e-mail: kangtb@mail.sysu.edu.cn Keywords: CBX8, Polycomb Group protein, colorectal cancer, metastasis, proliferation, p53, integrin Received: July 23, 2014 Accepted: September 13, 2014 Published: October 24, 2014 ABSTRACT The effect of polycomb chromobox (Cbx) proteins in cancer is context-dependent. The Chromobox homolog 8 (CBX8) was originally characterized as a transcriptional repressor, which inhibits cell proliferation in Ink4a-Arf-dependent and -independent manner. However, the role of CBX8 in colorectal cancer remains unknown. Here, we found that high CBX8 expression was associated with a low rate of distant metastasis and good prognosis in CRC patients, even though CBX8 was up-regulated in CRC cell lines and clinical samples. Knockdown of CBX8 inhibited CRC proliferation in vitro and in vivo, mostly by increasing p53 and its downstream effectors. However, knockdown of CBX8 enhanced CRC migration, invasion and metastasis in vitro and in vivo, in part through direct up-regulation of integrin β4 (ITGB4) that in turn decreased RhoA activity. Collectively, the knockdown of CBX8 inhibited CRC proliferation, while promoting its metastasis, thus exerting paradoxical effects in CRC progression.

Summary Yin yang 1 (YY1) is a ubiquitous transcription factor and mammalian polycomb group protein (PcG) with important functions to regulate embryonic development, lineage differentiation, and cell proliferation. YY1 mediates stable PcG-dependent transcriptional repression via recruitment of PcG proteins that catalyze histone modifications. Many questions remain unanswered regarding how cell- and tissue-specificity is achieved by PcG proteins. Here, we demonstrate that a conditional knockout of Yy1 in hematopoietic stem cells (HSCs) decreases long-term repopulating activity and ectopic YY1 expression expands HSCs. Although the YY1 PcG domain is required for Igκ chain rearrangement in B cells, the YY1 mutant lacking the PcG domain retained the capacity to stimulate HSC self-renewal. YY1 deficiency deregulated the genetic network governing HSC cell proliferation and impaired stem cell factor/c-Kit signaling, disrupting mechanisms conferring HSC quiescence. These results reveal a mechanism for how a ubiquitously expressed transcriptional repressor mediates lineage-specific functions to control adult hematopoiesis.

Background MicroRNAs (miRNAs) are short non-coding RNAs that are emerging as important post-transcriptional regulators of neuronal and synaptic development. The precise impact of miRNAs on presynaptic function and neurotransmission remains, however, poorly understood. Results Here, we identify miR-27b—an abundant neuronal miRNA implicated in neurological disorders—as a global regulator of the presynaptic transcriptome. miR-27b influences the expression of three quarters of genes associated with presynaptic function in cortical neurons. Contrary to expectation, a large majority of these genes are up-regulated by miR-27b. This stimulatory effect is mediated by miR-27b-directed silencing of several transcriptional repressors that cooperate to suppress the presynaptic transcriptome. The strongest repressive activity appears to be mediated by Bmi1, a component of the polycomb repressive complex implicated in self-renewal of neural stem cells. miR-27b knockdown leads to reduced synaptogenesis and to a marked decrease in neural network activity, which is fully restored by RNAi-mediated silencing of Bmi1. Conclusions We conclude that silencing of Bmi1 by miR-27b relieves repression of the presynaptic transcriptome and supports neurotransmission in cortical networks. These results expand the repressive activity of Bmi1 to genes involved in synaptic function and identify a unique post-transcriptional circuitry that stimulates expression of synaptic genes and promotes synapse differentiation. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3139-7) contains supplementary material, which is available to authorized users.

Polycomb group (PcG) proteins regulate major developmental processes in Arabidopsis. EMBRYONIC FLOWER 2 (EMF2), the VEFS domain-containing PcG gene, regulates diverse genetic pathways and is required for vegetative development and plant survival. Despite widespread EMF2-like sequences in plants, little is known about their function other than in Arabidopsis and rice. To study the role of EMF2 in broccoli (Brassica oleracea var. italica cv. Elegance) development, we identified two broccoli EMF2 (BoEMF2) genes with sequence homology to and a similar gene expression pattern to that in Arabidopsis (AtEMF2). Reducing their expression in broccoli resulted in aberrant phenotypes and gene expression patterns. BoEMF2 regulates genes involved in diverse developmental and stress programs similar to AtEMF2 in Arabidopsis. However, BoEMF2 differs from AtEMF2 in the regulation of flower organ identity, cell proliferation and elongation, and death-related genes, which may explain the distinct phenotypes. The expression of BoEMF2.1 in the Arabidopsis emf2 mutant (Rescued emf2) partially rescued the mutant phenotype and restored the gene expression pattern to that of the wild type. Many EMF2-mediated molecular and developmental functions are conserved in broccoli and Arabidopsis. Furthermore, the restored gene expression pattern in Rescued emf2 provides insights into the molecular basis of PcG-mediated growth and development.

Enhancer of zeste homologue 2 (EZH2), a member of the polycomb group protein family, plays a crucial role in the regulation of embryonic development and has been associated with the regulation of the cell cycle. Recently, several studies have shown that EZH2 is highly expressed in aggressive tumours, including human breast cancer, prostate cancer, and lymphomas. We thus analysed EZH2 expression using real-time reverse transcription-polymerase chain reaction, and correlated its expression status with various clinicopathological parameters in 66 patients with hepatocellular carcinoma (HCC). We found high expression of EZH2 in human liver cancer cell lines. Furthermore, EZH2 gene-expression levels in tumour tissue specimens (0.34+/-0.52) were significantly higher (P0.0001) than those in the corresponding nontumour tissue specimens (0.07+/-0.09). The incidence of cancer cell invasion into the portal vein was significantly higher (P0.001) in the high EZH2 expression group (26 of the 33, 79%) than in the low expression group (13 of the 33, 39%). However, there was no significant difference in the disease-free survival rate between the two groups. The findings of this study indicate that EZH2 mRNA expression was upregulated in human HCC and may play an important role in tumour progression, especially by facilitating portal vein invasion.

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27. 18 1592 1605 Cited By :364

During last decades studies on embryonic stem cell (ESC) differentiation have led to a better understanding of tissue homeostasis, including hematopoietic cell development. It is also generally accepted that ESC-derived progenitors or somatic cells hold great potential as novel cell sources for cell or tissue replacement therapy. This thesis focuses on establishing ESC culture and ESC differentiation into hematopoietic cells. Hematopoietic cells were derived from ESC following established protocols by OP9 stroma cell co-culture and embryoid body (EB) formation. To enhance hematopoietic cell generation from ESC, we assessed the impact of Bmi1 (B lymphoma Mo-MLV insertion region 1 homolog). Bmi1 is a Polycomb group (PcG) protein and a key epigenetic regulator during development. It plays an essential role in maintaining adult stem cell self-renewal as revealed by loss of function and over-expression studies. However, the role of Bmi1 on pluripotent stem cells has not been explored so far. Here we found that Bmi1 is not expressed in pluripotent ESC. To examine the impact of Bmi1 on self-renewal and differentiation of pluripotent stem cells, we introduced Bmi1 into ESC by lentivirus vector. Bmi1-transduced ESC (Bmi1-ESC) could be maintained in an undifferentiated state during culture similar to control ESC. Upon differentiation, Bmi1-ESC showed similar pattern of mesoderm hemangioblasts formation, yielding Flk1+ cells at frequencies similar as controls. However, primitive hematopoietic cell generation from mesoderm was highly enhanced, as determined by colony forming assay. Global transcriptional profiling by DNA microarrays identified a panel of genes that were distinctly regulated by Bmi1 during differentiation. Several homeotic genes were repressed, but GATA-2 expression was induced. When we studied EB-derived Bmi1-ESC in serum-free medium with hematopoietic cytokines, Bmi1 led to more than 100-fold expansion of hematopoietic stem/progenitor cells within 2-3 weeks of culture versus control ESC. Additionally, Ink4a/Arf locus appeared being silenced in Bmi1-ESC derived hematopoietic progenitor cells, which relates to their high proliferation capacity. Taken together, these data demonstrate distinct activities of Bmi1 on ESC and ESC-derived hematopoietic progenitor cells. The activity of Bmi1 described here suggests how aberrant Bmi1 expression might contribute to leukemia formation in adult hematopoietic cells. Finally, Bmi1 might be a candidate gene for facilitating adult stem cell derivation from ESC. The better understanding of the transcriptional circuities that maintain ESC identity has allowed novel approaches of generating pluripotent stem cells from somatic cells. For a variety of reasons, hematopoietic stem/progenitor cells (HSC/HPC) represent an advantage cell source for reprogramming. For example, they are easily accessible and can be readily isolated. They can be rapidly expanded to a large cell number with hematopoietic cytokines. Therefore, the second part of this thesis focuses on reprogramming of ex vivo expanded Flt3+ HSC into pluripotent ESC-like cells. Flt3+ HSC were reprogrammed into pluripotent state by different approaches, including ESC fusion and pluripotent factor transduction. Initially, we found that reprogrammed Flt3 ESC hybrids obtained by cell fusion of Flt3+ HSC with ESC, contained donor cell memory, both in epigenetic information and in differentiation behavior. To examine whether somatic memory is also retained in Flt3 iPSC, we compared gene expression profiles of reprogrammed Flt3 ESC hybrids, Flt3 iPSC, ESC and Flt3+ HSC by DNA microarray. We found that expression profiles of pluripotent stem cells from different origins clustered together. This indicates that pluripotent stem cells from different origins maintain their specific epigenetic and gene expression profiles. Further analysis of Flt3+ HSC-derived pluripotent stem cells is ongoing in our lab. These studies will be helpful for understanding the molecular mechanisms underlying reprogramming, also will shed light on the understanding of the ground state of pluripotency.

Both Caspase-3 and Caspase-9 play critical roles in the execution of mitochondria-mediated apoptosis. Caspase-9 binds to Apaf-1 in the presence of cytochrome c and dATP/ATP, and is activated by self-cleavage. Caspase-3 is activated by cleavage of caspase-8 and caspase-9. Over hundred direct caspase-3 substrates are identified whereas only few direct caspase-9 substrates are known. Here, we demonstrate that Ring1B, a component of polycomb protein complex that plays important roles in modulating chromatin structures, is a direct substrate of active caspase-3 and caspase-9 both in vitro and in vivo. The specific cleavage sites for caspase-3 and caspase-9 were mapped to Asp175 and Asp208, respectively. Importantly, cleavage of Ring1B by active caspases-3 and caspase-9 triggers the redistribution of Ring1B, from exclusive nuclear localization to even distribution throughout the entire cell. The transcriptional repression activity of Ring1B was also disrupted by caspase cleavage. Our data suggest that caspases-3 and caspase-9 play novel roles in transcription by regulating polycomb protein function through direct cleaving of Ring1B.

Polycomb group proteins are epigenetic gene silencers that have been implicated in cancer development and stem cell maintenance.1 Biological and genetic studies indicate that PcG proteins exist in at least two separate protein complexes, polycomb-repressive complex 2 (PRC2) and polycomb-repressive complex 1 (PRC1), which act in concert to promote and maintain gene repression.1 EZH2, the catalytically active component of PRC2, plays an important role in many biological processes through its ability to trimethylate lysine 27 in histone H3. The core enzymatic activity of PRC1 is an E3 ubiquitin ligase activity contributed by RING1B (and enhanced by BMI1), which ubiquitinates histone H2A at lysine 119.1 PcG proteins are displaced from the promoters of one set of target genes while being recruited to the promoters of another set of target genes during lineage specification.1 How extracellular signaling regulates this process is not well-understood. In 2005, Cha and colleagues found Akt mediated phosphorylation of EZH2 results in a decrease of lysine 27 trimethylation and derepression of silenced genes.2 Recently, we reported that phosphorylation of BMI1 at serine 316 by Akt impairs its function by triggering its dissociation from the Ink4a-Arf locus, which results in decreased histone H2A ubiquitination and the inability of BMI1 to promote cell growth and tumorigenesis.3 It is very likely that other PcG proteins, including EZH1 and MEL18, are downstream targets of Akt. Thus, Akt appears to regulate the activities of both the PRC2 and PRC1 complexes in the cell (Fig. 1). Figure 1. PI3K-Akt signaling regulates polycomb group protein and hematopoietic stem cell self-renewal. PcG proteins are required for the maintenance of embryonic as well as a broad range of adult stem cells, including hematopoietic stem cells (HSCs).1 HSCs have both the capacity to self-renew and to differentiate into many different mature blood cell types. Understanding the molecular mechanisms governing stem cell self-renewal remains the holy grail of stem cell biology and holds great promise for the development of stem cell-based therapies aimed at treating debilitating and life-threatening diseases such as cancer. Self-renewal requires the integration of survival signals and proliferation controls with the maintenance of an undifferentiated state. This demands a complex crosstalk between extrinsic signals from the microenvironment and the cell-intrinsic regulators of HSCs to maintain an undifferentiated state.4 However, how the crosstalk is coordinated has not been well-defined at the molecular level. Activation of the PI3K-Akt signaling impairs HSC self-renewal,5 and we demonstrated that BMI1 is a critical downstream target of PI3K-Akt signaling in HSCs and that Akt-mediated phophorylation of BMI1 inhibits its ability to promote hematopoietic stem/progenitor cell (HSPC) self-renewal.3 At virtually the same time, the van Lohuizen lab reported that Akt-mediated BMI1 phosphorylation enhances its oncogenic potential in an Ink4a and Arf-independent mouse model of human prostate cancer.6 They identified three sites of serine phosphorylation [Ser251, Ser253 and Ser255 (SDSGS)], none of which were consensus Akt phosphorylation sites (RXRXXS or T).6 Nonetheless, it appears that the inhibition of cell proliferation and oncogenesis by Akt-mediated phosphorylation of BMI1, occurs in a site-specific and possibly context-dependent manner. Similar to BMI1, a component of Polycomb-repressive complex 1 (PRC1), PRC2 and its components play important roles in hematopoiesis. Ezh2-deficient embryos died of anemia because of insufficient expansion of HSCs/progenitor cells. Deletion of Ezh2 in adult BM, however, did not significantly compromise hematopoiesis.7 On the contrary, Ezh1 maintains repopulating HSCs in a slow-cycling, undifferentiated state, protecting them from senescence. Ezh1 ablation induces significant loss of adult HSCs, with concomitant impairment of their self-renewal capacity due to a potent senescence response, suggesting that Ezh1 is an important histone methyltransferase for adult HSC maintenance.8 Therefore, it is possible that Akt-mediated phosphorylation of EZH2 or EZH1 may limit HSC function as well. Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of diseases. The ability to increase the number of HSCs either in vivo or in vitro would provide new treatment options, but the amplification of HSCs has been difficult to achieve. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Most hematopoietic cell growth factors activate Akt in ex vivo cultures, promoting HSC differentiation. Therefore, treating HSCs with Akt inhibitors may expand HSCs ex vivo. Our understanding of the crosstalk between PI3K-Akt signaling and polycomb group proteins may facilitate the development of innovative clinical strategies that can enhance ex vivo HSC expansion.

2020年度