Ectopic Viral Integration Site Research Articles

Ectopic Viral Integration Site-1 Promotes NRAS/ERK Pathway through Epigenetic Silencing of microRNA-124 in AML

BACKGROUNDː Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by genetic mutations that promote proliferation of myeloid progenitors and prevent their differentiation. Over-expression of Ectopic Viral Integration site-1(EVI-1) is related to the poor prognosis in myeloid leukemia, but the underlying mechanism remains unclear. METHODSː Using qRT-PCR and western blotting, we quantified expressions of EVI-1, NRAS and ERK/p-ERK in leukemia cell lines and PBMCs. Using WTS-8 and cell cycle analysis, we further investigated whether downregulation of EVI-1 by siRNA can inhibit cell proliferation. Microscopic observation of peripheral blood cells from EVI-1 transgenic zebrafish and WT control were analyzed by Wright Giemsa staining. Using miR-seq, qPCR, dual-luciferase reporter and coimmunoprecipitation assays, we revealed the relationship between EVI-1, miR-124 and NRAS. RESULTSː EVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). In a transgenic zebrafish model, EVI-1 mediated higher mortality and induced immature hematopoietic cells in the blood circulation, suggesting its oncogenic role. Furthermore, our results suggested that EVI-1 upregulated NRAS expression, thereby activating the RAS/ERK pathway through epigenetic silencing of a potent NRAS suppressor, miR-124. In this study, we found that EVI1 physically interacts with Dnmt3a to form a protein complex that targets and binds to regulatory elements of miR-124. CONCLUSIONSː Overall, the current findings demonstrate that EVI-1 overexpression converges on the regulation of miR-124 promoter methylation and activation of the RAS/ERK pathway in AML carcinogenesis, and suggest EVI-1 and/or miR-124 as therapeutic targets for this dismal disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analysis of somatic mutations in 131 human brains reveals aging-associated hypermutability.

We analyzed 131 human brains (44 neurotypical, 19 with Tourette syndrome, 9 with schizophrenia, and 59 with autism) for somatic mutations after whole genome sequencing to a depth of more than 200×. Typically, brains had 20 to 60 detectable single-nucleotide mutations, but ~6% of brains harbored hundreds of somatic mutations. Hypermutability was associated with age and damaging mutations in genes implicated in cancers and, in some brains, reflected in vivo clonal expansions. Somatic duplications, likely arising during development, were found in ~5% of normal and diseased brains, reflecting background mutagenesis. Brains with autism were associated with mutations creating putative transcription factor binding motifs in enhancer-like regions in the developing brain. The top-ranked affected motifs corresponded to MEIS (myeloid ectopic viral integration site) transcription factors, suggesting a potential link between their involvement in gene regulation and autism.

Ectopic viral integration Site-1 oncogene promotes NRAS pathway through epigenetic silencing of microRNA-124 in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by genetic mutations that promote proliferation of myeloid progenitors and prevent their differentiation. Over-expression of Ectopic Viral Integration site-1(EVI-1) is related to the poor prognosis in myeloid leukemia, but the underlying mechanism remains unclear. MethodsUsing qRT-PCR and western blotting, we quantified expressions of EVI-1, NRAS and ERK/p-ERK in leukemia cell lines and PBMCs. Using WTS-8 and cell cycle analysis, we further investigated whether downregulation of EVI-1 by siRNA can inhibit cell proliferation. Microscopic observation of peripheral blood cells from EVI-1 transgenic zebrafish and WT control were analyzed by Wright Giemsa staining. Using miR-seq, qPCR, dual-luciferase reporter and coimmunoprecipitation assays, we revealed the relationship between EVI-1, miR-124 and NRAS. ResultsEVI-1 was highly expressed in both primary AML and leukemia cell lines (THP-1 and K562). In a transgenic zebrafish model, EVI-1 mediated higher mortality and induced immature hematopoietic cells in the blood circulation, suggesting its oncogenic role. Furthermore, our results suggested that EVI-1 upregulated NRAS expression, thereby activating the RAS/ERK pathway through epigenetic silencing of a potent NRAS suppressor, miR-124. In this study, we found that EVI1 physically interacts with Dnmt3a to form a protein complex that targets and binds to regulatory elements of miR-124. ConclusionsOverall, the current findings demonstrate that EVI-1 overexpression converges on the regulation of miR-124 promoter methylation and activation of the RAS/ERK pathway in AML carcinogenesis, and suggest EVI-1 and/or miR-124 as therapeutic targets for this dismal disease.

Acute Myeloid Leukemia Leading to Central Diabetes Insipidus

Background: Central diabetes insipidus (CDI) as a complication of acute myeloid leukemia (AML) is rare, occurring in less than 0.6% of AML cases. The mechanism is thought to involve leukemic infiltration in or around the pituitary gland, not always seen on imaging. In one study, as many as 61.4% of patients with CDI due to AML had no abnormalities on MRI, and at autopsy 46% of AML patients had perihypohyseal leukemic infiltration in the absence of overt CDI. CDI is also associated with AML in cases that involve monosomy 7 and inversion 3q21q26, both of which result in ectopic viral integration site 1 (EVI-1) overexpression. It is postulated that EVI-1 overexpression interferes with hypothalamic secretion of antidiuretic hormone (ADH) or may lead to its inactivation. We present a case of adipsic CDI due to AML in a patient with monosomy 7. Case: A 70-year-old female presented for routine follow-up and was found to have a white blood cell count of 2.6 K/µL with 29% blasts, anemia (Hgb 10.3 g/dL) and normal platelets (300 K/µL). She was diagnosed with AML and molecular evaluation showed del (3)(q21),-7,add(17(p13) consistent with monosomy 7. She was admitted for induction chemotherapy with cytarabine, daunorubicin and intrathecal methotrexate. She denied thirst. On physical exam she was euvolemic and visual fields were full on confrontation. Her admission sodium was 146 mmol/L, urine osmolality was 149 mOsm/kg H2O, urine sodium 14 mmol/L. Urine output was 5.1 L over the first 24 hours. She underwent a 6 hour water deprivation test, during which her urine output averaged 250 cc/hr. Her sodium increased to 158 mmol/L, serum osmolality 331 mOsm/kg H2O, urine osmolality 146 mOsm/kg H2O. She was then administered 100 µg of DDAVP PO and her serum sodium and osmolality decreased to 155 mmol/L and 326 mOsm/kg H2O, respectively, while her urine osmolality nearly doubled to 292 mOsm/kg H2O. Urine output decreased to 50-100 cc/hr. At no point during her testing did she report thirst. The patient’s pituitary laboratory profile did not show any other abnormalities. Her pituitary MRI revealed subtle thickening of the proximal infundibulum and hypothalamus but no definitive intra-sellar pathology. She was discharged on twice daily DDAVP with a sodium of 142. Unfortunately, her AML was refractory to treatment. She was transitioned to comfort care and died peacefully. Conclusion: CDI as a complication of AML is very rare and is a poor prognostic marker. Based on her MRI findings, the most likely mechanism in this case was infundibular/hypothalamic infiltration. Her adypsia is interesting and may point to more generalized hypothalamic involvement including thirst center. Her monosomy 7 mutation may have also played a role. We present this case to bring awareness to this etiology of DI and its proposed mechanisms.

Molecular characterization of ABHD5 gene promoter in intramuscular preadipocytes of Qinchuan cattle: Roles of Evi1 and C/EBPα

The genetic regulation of lipolytic enzyme is closely related to carcass quality traits through deposition of intramuscular fat (marbling) in beef cattle breeds. The α/β hydrolase domain containing 5 (ABHD5) is an accelerating factor of adipose triglyceride lipase (ATGL), which plays a key role in triglyceride metabolism. In this study, we determined that bovine ABHD5 gene was highly expressed in adult bovine adipose tissue. To elucidate the molecular mechanisms involved in bovine ABHD5 regulation, we cloned and characterized the promoter region of ABHD5. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified transcriptional start site (TSS) found in the predicted CpG island within promoter region of ABHD5 gene. Using the recombinant dual fluorescent reporter vectors, the fragment of –109/+307 was identified as proximal minimum core promoter region of ABHD5 in bovine intramuscular adipocytes. Site directed mutagenesis and electrophoretic mobility shift assay (EMSA) confirmed the role of two transcription factors, namely Ectopic viral integration site-1 (Evi1) and CCAAT/enhancer binding protein alpha (C/EBPα), in the regulation of ABHD5 gene. Taken together these findings we can conclude that ABHD5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits.

MEIS2 regulates endothelial to hematopoietic transition of human embryonic stem cells by targeting TAL1

BackgroundDespite considerable progress in the development of methods for hematopoietic differentiation, efficient generation of transplantable hematopoietic stem cells (HSCs) and other genuine functional blood cells from human embryonic stem cells (hESCs) is still unsuccessful. Therefore, a better understanding of the molecular mechanism underlying hematopoietic differentiation of hESCs is highly demanded.MethodsIn this study, by using whole-genome gene profiling, we identified Myeloid Ectopic Viral Integration Site 2 homolog (MEIS2) as a potential regulator of hESC early hematopoietic differentiation. We deleted MEIS2 gene in hESCs using the CRISPR/CAS9 technology and induced them to hematopoietic differentiation, megakaryocytic differentiation.ResultsIn this study, we found that MEIS2 deletion impairs early hematopoietic differentiation from hESCs. Furthermore, MEIS2 deletion suppresses hemogenic endothelial specification and endothelial to hematopoietic transition (EHT), leading to the impairment of hematopoietic differentiation. Mechanistically, TAL1 acts as a downstream gene mediating the function of MEIS2 during early hematopoiesis. Interestingly, unlike MEIS1, MEIS2 deletion exerts minimal effects on megakaryocytic differentiation and platelet generation from hESCs.ConclusionsOur findings advance the understanding of human hematopoietic development and may provide new insights for large-scale generation of functional blood cells for clinical applications.

MEIS1 Regulates Hemogenic Endothelial Generation, Megakaryopoiesis, and Thrombopoiesis in Human Pluripotent Stem Cells by Targeting TAL1 and FLI1.

SummaryHuman pluripotent stem cells (hPSCs) provide an unlimited source for generating various kinds of functional blood cells. However, efficient strategies for generating large-scale functional blood cells from hPSCs are still lacking, and the mechanism underlying human hematopoiesis remains largely unknown. In this study, we identified myeloid ectopic viral integration site 1 homolog (MEIS1) as a crucial regulator of hPSC early hematopoietic differentiation. MEIS1 is vital for specification of APLNR+ mesoderm progenitors to functional hemogenic endothelial progenitors (HEPs), thereby controlling formation of hematopoietic progenitor cells (HPCs). TAL1 mediates the function of MEIS1 in HEP specification. In addition, MEIS1 is vital for megakaryopoiesis and thrombopoiesis from hPSCs. Mechanistically, FLI1 acts as a downstream gene necessary for the function of MEIS1 during megakaryopoiesis. Thus, MEIS1 controls human hematopoiesis in a stage-specific manner and can be potentially manipulated for large-scale generation of HPCs or platelets from hPSCs for therapeutic applications in regenerative medicine.

Meis1 Coordinates Cerebellar Granule Cell Development by Regulating Pax6 Transcription, BMP Signaling and Atoh1 Degradation.

Cerebellar granule cell precursors (GCPs) and granule cells (GCs) represent good models to study neuronal development. Here, we report that the transcription factor myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse GC development. We found that Meis1 is expressed in GC lineage cells and astrocytes in the cerebellum during development. Targeted disruption of the Meis1 gene specifically in the GC lineage resulted in smaller cerebella with disorganized lobules. Knock-down/knock-out (KO) experiments for Meis1 and in vitro assays showed that Meis1 binds to an upstream sequence of Pax6 to enhance its transcription in GCPs/GCs and also suggested that the Meis1-Pax6 cascade regulates morphology of GCPs/GCs during development. In the conditional KO (cKO) cerebella, many Atoh1-positive GCPs were observed ectopically in the inner external granule layer (EGL) and a similar phenomenon was observed in cultured cerebellar slices treated with a bone morphogenic protein (BMP) inhibitor. Furthermore, expression of Smad proteins and Smad phosphorylation were severely reduced in the cKO cerebella and Meis1-knock-down GCPs cerebella. Reduction of phosphorylated Smad was also observed in cerebellar slices electroporated with a Pax6 knock-down vector. Because it is known that BMP signaling induces Atoh1 degradation in GCPs, these findings suggest that the Meis1-Pax6 pathway increases the expression of Smad proteins to upregulate BMP signaling, leading to degradation of Atoh1 in the inner EGL, which contributes to differentiation from GCPs to GCs. Therefore, this work reveals crucial functions of Meis1 in GC development and gives insights into the general understanding of the molecular machinery underlying neural differentiation from neural progenitors.SIGNIFICANCE STATEMENT We report that myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse granule cell (GC) development. Here, we show Meis1 is expressed in GC precursors (GCPs) and GCs during development. Our knock-down and conditional knock-out (cKO) experiments and in vitro assays revealed that Meis1 is required for proper cerebellar structure formation and for Pax6 transcription in GCPs and GCs. The Meis1-Pax6 cascade regulates the morphology of GCs. In the cKO cerebella, Smad proteins and bone morphogenic protein (BMP) signaling are severely reduced and Atoh1-expressing GCPs are ectopically detected in the inner external granule layer. These findings suggest that Meis1 regulates degradation of Atoh1 via BMP signaling, contributing to GC differentiation in the inner EGL, and should provide understanding into GC development.

EVI and MDS/EVI are required for adult intestinal stem cell formation during postembryonic vertebrate development.

The gene ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI encode zinc-finger proteins that have been recognized as important oncogenes in various types of cancer. In contrast to the established role of EVI and MDS/EVI in cancer development, their potential function during vertebrate postembryonic development, especially in organ-specific adult stem cells, is unclear. Amphibian metamorphosis is strikingly similar to postembryonic development around birth in mammals, with both processes taking place when plasma thyroid hormone (T3) levels are high. Using the T3-dependent metamorphosis in Xenopus tropicalis as a model, we show here that high levels of EVI and MDS/EVI are expressed in the intestine at the climax of metamorphosis and are induced by T3. By using the transcription activator-like effector nuclease gene editing technology, we have knocked out both EVI and MDS/EVI and have shown that EVI and MDS/EVI are not essential for embryogenesis and premetamorphosis in X. tropicalis On the other hand, knocking out EVI and MDS/EVI causes severe retardation in the growth and development of the tadpoles during metamorphosis and leads to tadpole lethality at the climax of metamorphosis. Furthermore, the homozygous-knockout animals have reduced adult intestinal epithelial stem cell proliferation at the end of metamorphosis (for the few that survive through metamorphosis) or during T3-induced metamorphosis. These findings reveal a novel role of EVI and/or MDS/EVI in regulating the formation and/or proliferation of adult intestinal adult stem cells during postembryonic development in vertebrates.-Okada, M., Shi, Y.-B. EVI and MDS/EVI are required for adult intestinal stem cell formation during postembryonic vertebrate development.

RPL5 on 1p22.1 is recurrently deleted in multiple myeloma and its expression is linked to bortezomib response.

Chromosomal region 1p22 is deleted in ≥20% of multiple myeloma (MM) patients, suggesting the presence of an unidentified tumor suppressor. Using high-resolution genomic profiling, we delimit a 58 kb minimal deleted region (MDR) on 1p22.1 encompassing two genes: ectopic viral integration site 5 (EVI5) and ribosomal protein L5 (RPL5). Low mRNA expression of EVI5 and RPL5 was associated with worse survival in diagnostic cases. Patients with 1p22 deletion had lower mRNA expression of EVI5 and RPL5, however, 1p22 deletion status is a bad predictor of RPL5 expression in some cases, suggesting that other mechanisms downregulate RPL5 expression. Interestingly, RPL5 but not EVI5 mRNA levels were significantly lower in relapsed patients responding to bortezomib and; both in newly diagnosed and relapsed patients, bortezomib treatment could overcome their bad prognosis by raising their progression-free survival to equal that of patients with high RPL5 expression. In conclusion, our genetic data restrict the MDR on 1p22 to EVI5 and RPL5 and although the role of these genes in promoting MM progression remains to be determined, we identify RPL5 mRNA expression as a biomarker for initial response to bortezomib in relapsed patients and subsequent survival benefit after long-term treatment in newly diagnosed and relapsed patients.

RPL5 Is a Candidate Tumor Suppressor on 1p22.1 in Multiple Myeloma of Which the Expression Is Linked to Bortezomib Response

Chromosomal region 1p22 is deleted in ~20% of multiple myeloma patients, suggesting the presence of an unidentified tumor suppressor gene in this region. Using high resolution copy number arrays, we delimit a 58 kb minimal deleted region on 1p22.1 encompassing two genes: ectopic viral integration site 5 (EVI5) and ribosomal protein L5 (RPL5). Although mutations in 1p22 genes are rare in multiple myeloma, the tumor suppressor role of EVI5 and RPL5 may be supported by the fact that these genes show the highest frequency of mutations predicted to impair protein function on 1p22. Interestingly, inactivation of RPL5 was also recently described in T-cell acute lymphoblastic leukemia (T-ALL) and glioblastoma.We found that 1p22 deleted patients have significantly lower levels of EVI5 and RPL5 mRNA expression (59% and 71% residual expression respectively; p<0.0001 for each gene). Whereas 1p22 deletion status correlates well with EVI5 expression in all patients, it is a bad predictor of RPL5 expression level in some cases, suggesting that other mechanisms besides 1p22 deletion can downregulate RPL5 expression in multiple myeloma. Deletion of 1p22 has been associated with decreased progression free (PFS) and overall survival (OS) in newly diagnosed multiple myeloma (Hebraud et al., Leukemia, 2013). We saw that low mRNA expression of EVI5 and RPL5 also correlates with worse survival in diagnosis but not in relapse cases.Interestingly, RPL5 but not EVI5 mRNA expression levels were significantly lower in multiple myeloma patients responding to bortezomib compared to patients that are not responding (68% residual RPL5 expression in responders versus non-responders). The clinical relevance of these findings is illustrated by the observation that PFS of newly diagnosed patients with low RPL5 expression was significantly longer when they were treated on a bortezomib containing protocol (median PFS bortezomib protocol 29.8 months versus 18.7 months for non-bortezomib, p=0.03, data phase III HOVON-65/ GMMG-HD4 trial). In contrast, PFS of RPL5 high patients was not influenced by bortezomib. These associations between RPL5 expression level and bortezomib response were confirmed when performing the same analyses on the PFS data from relapse patients in the APEX trial. For low EVI5 expressing patients, there was a non-significant trend towards benefit from bortezomib which was not present in the EVI5 high cases. Taken together, these data suggest that RPL5 expression levels are a better biomarker for bortezomib response than 1p22 deletion.In conclusion, we provide genetic data that narrow down the list of candidate tumor suppressors on 1p22 to EVI5 and RPL5. Although the exact role of these genes in suppressing multiple myeloma disease progression remains to be determined, we identify low RPL5 expression levels in multiple myeloma as a novel, clinically relevant biomarker associated with initial response and survival benefit upon treatment with bortezomib. DisclosuresMulligan:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Delforge:Amgen: Honoraria; Novartis: Honoraria; Celgene Corporation: Honoraria; Janssen: Honoraria. Sonneveld:Janssen-Cilag, Celgene, Onyx, Karyopharm: Honoraria, Research Funding; novartis: Honoraria.

Targeted Suppression of EVI1 Oncogene Expression by Sequence-Specific Pyrrole-Imidazole Polyamide

Human ectopic viral integration site 1 (EVI1) is an oncogenic transcription factor known to play a critical role in many aggressive forms of cancer. Its selective modulation is thought to alter the cancer-specific gene regulatory networks. Pyrrole-imidazole polyamides (PIPs) are a class of small DNA binders that can be designed to target any destined DNA sequence. Herein, we report a sequence-specific pyrrole-imidazole polyamide, PIP1, which can target specific base pairs of the REL/ELK1 binding site in the EVI1 minimal promoter. The designed PIP1 significantly inhibited EVI1 in MDA-MB-231 cells. Whole-transcriptome analysis confirmed that PIP1 affected a fraction of EVI1-mediated gene regulation. In vitro assays suggested that this polyamide can also effectively inhibit breast cancer cell migration. Taken together, these results suggest that EVI1-targeted PIP1 is an effective transcriptional regulator in cancer cells.

De novo acute myeloid leukemia subtype-M4 with initial trisomy 8 and later acquired t(3;12)(q26;p12) leading to ETV6/MDS1/EVI1 fusion transcript expression: A case report

The t(3;12)(q26;p13) translocation is a recurrent chromosomal aberration observed in myeloid malignancies. The translocation results in the generation of the ETV6/myelodysplastic syndrome 1 (MDS1)/ectopic viral integration site 1 (EVI1) fusion gene. However, the present case report is the first to present this rearrangement in acute myelogeneous leukemia (AML)-M4. Notably, this case is the first report of AML-M4 with an initial trisomy 8 and secondary acquired t(3;12)(q26;p13). Cells harboring the t(3;12) translocation were found to exhibit a higher proliferative capacity than cells with pure trisomy 8, which is consistent with the role of the ETV6/MDS1/EVI1 fusion transcript in the development and progression of malignancy.

Tissue-Specific Upregulation of MDS/EVI Gene Transcripts in the Intestine by Thyroid Hormone during Xenopus Metamorphosis

BackgroundIntestinal remodeling during amphibian metamorphosis resembles the maturation of the adult intestine during mammalian postembryonic development when the adult epithelial self-renewing system is established under the influence of high concentrations of plasma thyroid hormone (T3). This process involves de novo formation and subsequent proliferation and differentiation of the adult stem cells.Methodology/Principal FindingsThe T3-dependence of the formation of adult intestinal stem cell during Xenopus laevis metamorphosis offers a unique opportunity to identify genes likely important for adult organ-specific stem cell development. We have cloned and characterized the ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI generated via transcription from the upstream MDS promoter and alternative splicing. EVI and MDS/EVI have been implicated in a number of cancers including breast, leukemia, ovarian, and intestinal cancers. We show that EVI and MDS/EVI transcripts are upregulated by T3 in the epithelium but not the rest of the intestine in Xenopus laevis when adult stem cells are forming in the epithelium.Conclusions/SignificanceOur results suggest that EVI and MDS/EVI are likely involved in the development and/or proliferation of newly forming adult intestinal epithelial cells.

Overexpression of EVI1 interferes with cytokinesis and leads to accumulation of cells with supernumerary centrosomes in G0/1 phase

Ectopic viral integration site 1 (EVI1), a transcription factor frequently overexpressed in myeloid neoplasias, has been implicated in the generation of malignancy-associated centrosomal aberrations and chromosomal instability. Here, we sought to investigate the underlying cause of centrosome amplification in EVI1-overexpressing cells. We found that overexpression of EVI1-HA in U2OS cells induced supernumerary centrosomes, which were consistently associated with enlarged nuclei or binuclear cells. Live cell imaging experiments identified cytokinesis failure as the underlying cause of this phenotype. In accordance with previous reports, EVI1 overexpression induced a partial cell cycle arrest in G0/1 phase, accompanied by elevated cyclin D1 and p21 levels, reduced Cdk2 activity and activation of the p53 pathway. Supernumerary centrosomes predominantly occurred in resting cells, as identified by low levels of the proliferation marker Ki-67, leading to the conclusion that they result from tetraploidization after cytokinesis failure and are confined to G0/1-arrested tetraploid cells. Depletion of p53 using siRNA revealed that further polyploidization of these cells was inhibited by the p53-dependent tetraploidy checkpoint.

Unbiased proteomic analysis of proteins interacting with the HIV-1 5′LTR sequence: role of the transcription factor Meis

To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5′long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5′LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5′LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches.

Determination of Nucleotide Sequences of Mitochondrial Plasmid pGT704 of Paramecium caudatum

Many kinds of linear plasmids, or plasmid-like DNAs, in mitochondria have been reported in higher plants and fungi. Nevertheless, such plasmids were not previously known from the animal kingdom. Paramecium is the first example in which a mitochondrial plasmid-like DNA so far discovered among ciliates. In natural stocks, P. caudatum has one of three types of DNA : type I (6.4 kb), type II, a set of four DNAs (8.2 kb; 4.1 kb; 2.8kb; and 1.4 kb) and type III (8.6 kb). Paramecium caudatum stock GT704 only has type-II DNA. The 8.2 and 2.8 kb DNAs are dimers of the 4.1 and 1.4 kb DNA, respectively. The aim of this study was to determine the sequence of mitochondrial plasmid-like DNA in P. caudatum stock GT704 in order to identify potential gene products and other nucleotide sequences that might have relationship with integration of DNA and also the maintanance of DNA itself in mitochondria. The methods used in this study were isolation of the DNA, cloning and sequencing then followed by analysis of sequences obtained using available public domain. The dimers were found to be easily dissociated to monomers when treated with heat underconditions of low ionic strength. These results suggest that each monomer has a sticky end of single- stranded DNA that has complementary nucleotide sequences, and so attaches to each other at the end by base-pairing to form a dimer, leaving a nick that can be filled by ligation using ligase. Nucleotide sequences of some part of the DNA have been obtained randomly. Sequence analysis suggests that these dimers DNAs have 2 ORFs that code for putative RNA and DNA polymerases. It is assumed that the origin of this mitochondrial plasmid-like DNA is transposable element or some viruses since the motif EVI-1 (ectopic viral integration site-1) was found in the sequence. It is also assumed that this DNA can integrate into mtDNA since it also has some sequences homolog to the sequences of mt- DNA of some ciliates.

Targeted Degradation of the AML1/MDS1/EVI1 Oncoprotein by Arsenic Trioxide

Arsenic trioxide (ATO) has been found to be an effective treatment for acute promyelocytic leukemia patients and is being tested for treating other hematologic malignancies. We have previously shown that AML1/MDS1/EVI1 (AME), a fusion gene generated by a t(3;21)(q26;q22) translocation found in patients with chronic myelogenous leukemia during blast phase, myelodysplastic syndrome, or acute myelogenous leukemia (AML), impairs hematopoiesis and eventually induces an AML in mice. Both fusion partners of AME, AML1 and MDS1/EVI1, encode transcription factors and are also targets of a variety of genetic abnormalities in human hematologic malignancies. In addition, aberrant expression of ectopic viral integration site 1 (EVI1) has also been found in solid tumors, such as ovarian and colon cancers. In this study, we examined whether ATO could target AME and related oncoproteins. We found that ATO used at therapeutic levels degrades AME. The ATO treatment induces differentiation and apoptosis in AME leukemic cells in vitro as well as reduces tumor load and increases the survival of mice transplanted with these cells. We further found that ATO targets AME via both myelodysplastic syndrome 1 (MDS1) and EVI1 moieties and degrades EVI1 via the ubiquitin-proteasome pathway and MDS1 in a proteasome-independent manner. Our results suggest that ATO could be used as a part of targeted therapy for AME-, AML1/MDS1-, MDS1/EVI1-, and EVI1-positive human cancers.

Four human chromosome 3q and four human chromosome 21 loci map onto sheep chromosome 1q.

Eight new loci have been assigned to sheep Chromosome (Chr) 1q by use of a chromosomally characterized minipanel of sheep x hamster cell hybrids. Four loci, which have been mapped to the distal region of human Chr 3q, are ceruloplasmin (CP), sucrase isomaltase (SI), glucose transporter 2 (GLUT2), and ectopic viral integration site 1 (EVI1). The other four loci, on human Chr 21, include interferon alpha receptor (IFNAR); interferon inducible protein p78, murine (MX1); collagen type VI, alpha 1 (COL6A1); and S100 protein, beta polypeptide (S100B). All of these loci, except GLUT2 and MX1, have been mapped onto bovine Chr 1 or are syntenic with loci on this chromosome. The in situ localization of transferrin (TF) to sheep Chr 1q42-q45 confirms our previous assignment of this locus and independently anchors the eight new syntenic loci to sheep Chr 1q.