Repression of RUNX1 activity by EVI1: a new role of EVI1 in leukemogenesis

Recurring chromosomal translocations observed in human leukemia often result in the expression of fusion proteins that are DNA-binding transcription factors. These altered proteins acquire new dimerization properties that result in the assembly of inappropriate multimeric transcription complexes that deregulate hematopoietic programs and induce leukemogenesis. Recently, we reported that the fusion protein AML1/MDS1/EVI1 (AME), a product of a t(3;21)(q26;q22) associated with chronic myelogenous leukemia and acute myelogenous leukemia, displays a complex pattern of self-interaction. Here, we show that the 8th zinc finger motif of MDS1/EVI1 is an oligomerization domain involved not only in interaction of AME with itself but also in interactions with the parental proteins, RUNX1 and MDS1/EVI1, from which AME is generated. Because the 8th zinc finger motif is also present in the oncoprotein EVI1, we have evaluated the effects of the interaction between RUNX1 and EVI1 in vitro and in vivo. We found that in vitro, this interaction alters the ability of RUNX1 to bind to DNA and to regulate a reporter gene, whereas in vivo, the expression of the isolated 8th zinc finger motif of EVI1 is sufficient to block the granulocyte colony-stimulating factor-induced differentiation of 32Dcl3 cells, leading to cell death. As EVI1 is not detected in normal bone marrow cells, these data suggest that its inappropriate expression could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators, leading to their functional impairment

Ectopic Expression of Leishmanial DNA Polymerase β in Escherichia coli Confers Survival Advantage against Ultraviolet Radiation

Leishmania donovani encounters oxidative environment in the host macrophage and expected to have robust DNA repair mechanisms. Base Excision Repair (BER), a predominant repair pathway in L. donovani remains unexplored. Presence of mitochondria in eukaryotes has been projected as a symbiotic relationship since long and the role of DNA polymerase β in repair of mitochondrial DNA has gained importance in recent past. We ectopically expressed Leishmania DNA polymerase β (LdPolβ) under inducible promoter in E. coli and found it is biologically active in vitro by using pUC19 as substrate. Further we checked its effect on sensitivity of E. coli to UV rays. We find that heterologous LdPolβ slows down the growth of E. coli and surprisingly, could protect it from lethal effects of UV to a large extent. Co-expression of leishmania DNA Ligase IIIα (LdLigIIIα) has a synergistic effect on survival advantage offered by LdPolβ. Survival advantage given LdPolβ in E. coli is reconfirmed by FACS analysis. Our observations indicate that LdPolβ is crucial for handling ROS induced toxicity inside the mitochondria of the parasite and for its survival inside host macrophage. This studied may lead to explore for finding of the importance of LdPolβ in survival against DNA damaging agents in L. donovani and its role in pathogenesis of leishmaniasis, it would help to discover new target and development of newer drug against Leishmaniasis

RUNX1-RUNX1 homodimerization modulates RUNX1 activity and function

RUNX1 (AML1, CBFalpha2, PEBP2alphaB) is a transcription factor essential for the establishment of the hematopoietic stem cell. It is generally thought that RUNX1 exists as a monomer that regulates hematopoietic differentiation by interacting with tissue-specific factors and its DNA consensus through its N terminus. RUNX1 is frequently altered in human leukemia by gene fusions or point mutations. In general, these alterations do not affect the N terminus of the protein, and it is unclear how they consistently lead to hematopoietic transformation and leukemia. Here we report that RUNX1 homodimerizes through a mechanism involving C terminus-C terminus interaction. This RUNX1-RUNX1 interaction regulates the activity of the protein in reporter gene assays and modulates its ability to induce hematopoietic differentiation of hematopoietic cell lines. The promoters of genes regulated by RUNX1 often contain multiple RUNX1 binding sites. This arrangement suggests that RUNX1 could homodimerize to bring and hold together distant chromatin sites and factors and that if the dimerization region is removed by gene fusions or is altered by point mutations, as observed in leukemia, the ability of RUNX1 to regulate differentiation could be impaired

Oxidative Stress-Mediated Overexpression of Uracil DNA Glycosylase in Leishmania donovani

Leishmania donovani is an intracellular protozoan parasite that causes endemic tropical disease visceral leishmaniasis (VL). Present drugs used against this fatal disease are facing resistance and toxicity issues. Survival of leishmania inside the host cells depends on the parasite’s capacity to cope up with highly oxidative environment. Base excision repair (BER) pathway in L. donovani remains unexplored. We studied uracil DNA glycosylase (UNG), the key enzyme involved in BER pathway, and found that the glycosylase activity of recombinant LdUNG (Leishmania donovani UNG) expressed in E. coli is in sync with the activity of the parasite lysate under different reaction conditions. Overexpression of UNG in the parasite enhances its tolerance towards various agents which produce reactive oxygen species (ROS) and shows a higher infectivity in macrophages. Surprisingly, exposure of parasite to amphotericin B and sodium antimony gluconate upregulates the expression of UNG. Further, we found that the drug resistant parasites isolated from VL patients show higher expression of UNG. Mechanisms of action of some currently used drugs include accumulation of ROS. Our findings strongly suggest that targeting LdUNG would be an attractive therapeutic strategy as well as potential measure to tackle the problem of drug resistance in the treatment of leishmaniasis

Sequence analysis of Histone H4 and HAT2 of <i>L</i>. <i>donovani</i>.

<p>(1A) Comparison of N-terminus region of histone H4 of <i>L</i>. <i>donovani</i> (LdH4) with that of <i>L</i>. <i>infantum</i> (LiH4), <i>L</i>. <i>major</i> (LmH4), <i>L</i>. <i>mexicana</i> (LmxH4), <i>Trypanosoma brucei</i> (TbH4), <i>T</i>. <i>cruzi</i> (TcH4) and human (HuH4). Boxes represent conserved lysine residues. The arrow represents K16 of human histone H4 which is replaced by arginine (R14) in <i>L</i>. <i>donovani</i>. (1B) Zinc finger motif present in the sequence of HAT2 formed by two cysteine and two histidine residues (C₂H₂ type).</p

HAT2 mediates histone H4K4 acetylation and affects micrococcal nuclease sensitivity of chromatin in <i>Leishmania donovani</i>

<div><p>Histone post-translational modifications (PTMs) such as acetylation and methylation are known to affect chromatin higher order structures. Primary targets of these modifications include basic residues present at N-terminus tail region of core histones. Four histone acetyltransferase (HAT) genes have been identified in trypanosomatids. HAT1, HAT3 and HAT4 of <i>Leishmania donovani</i> have been partially characterized. However, there is no report about HAT2 of <i>Leishmania donovani</i>. Lysine residues present on the N-terminal tail of <i>Leishmania donovani</i> histone H4 are conserved in other trypanosomatids and humans. PTMs of lysines modulate various functions at chromatin level. The four histone acetyltransferases encoded in Leishmania genome were over-expressed to analyse their functional activity. All four HATs were found actively acetylating core histones H3/H4. Similar to <i>L</i>. <i>donovani</i> HAT3 and HAT4, HAT2 was found to be a member of MYST family protein and have SAS2 type domain. Over-expression of HAT2 significantly increases acetylation of H4K4. To analyse the effect of HAT2 over-expression on chromatin accessibility, micrococcal nuclease digestion assay was performed. MNase digestion resulted in a higher proportion of the mononucleosomes and dinucleosomes in HAT2 over-expressing cells as compared to WT <i>L</i>. <i>donovani</i> cells. Acetylation of lysine-4 neutralizes the amino terminal region of histone H4. This weakens its interaction with neighbouring nucleosomes and the linker DNA. HAT2 over-expression in <i>L</i>. <i>donovani</i> resulted in highly accessible chromatin suggesting chromatin decondensation. HAT2 may have an important role to play in global regulation of transcription in <i>L</i>. <i>donovani</i>. Better understanding of these epigenetic determinants of parasite would help in designing novel therapeutic strategies.</p></div

Occurrence of Antibiotic Resistance Gene Cassettes aac(6′)-Ib, dfrA5, dfrA12, and ereA2 in Class I Integrons in Non-O1, Non-O139 Vibrio cholerae Strains in India

Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated. Out of the 94 strains examined, 22 strains were found to have class I integrons. The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6′)-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance. To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6′)-Ib, and ereA2 cassettes in class I integrons of V. cholerae. Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids. Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both. Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance

HAT2 over-expression and activity analysis.

<p>(2A) RE double digestion of HAT2 cloned in pLPneo2. Clone was digested with HindIII and XhoI. Two fragments (~1800 base pairs for HAT2 and ~5500 base pairs for vector pLPneo2) were resolved in 1% agarose gel. (2B) WT and HAT2 over-expressing <i>L</i>. <i>donovani</i> cells were counted every eight hours for seven days and plotted. P <0.0001 was observed in two-way analysis of variance showing statistical significance. (2C) Relative histone acetylation activity assay for <i>L</i>. <i>donovani</i> cells over-expressing HAT1, HAT2, HAT3 and HAT4. The activities were compared to WT cells and vector (pLPneo2) alone transfected cells. Nuclear extract supplied with kit was taken as experimental control. Total histone acetylation was measured at 2 hours 30 minutes and 3 hours after start of reaction and relative activities were calculated per minute and per gram of total cell extract. The data shown represent average of three independent experiments. The p values < 0.0001 compared with control values is shown as ***.</p

Acetylation of Histones H3 and H4 in over-expressing <i>Leishmania donovani</i>.

<p>(3A) Western blot analysis of HAT2 over-expressing <i>L</i>. <i>donovani</i> (Lane 3) using Anti-acetyl-Histone H4 (Millipore, Cat. 06–866) antibody. WT (Lane 1) and vector (pLPneo2) transfected <i>L</i>. <i>donovani</i> (Lane 2) were used as controls. M indicates molecular weight marker. For nuclear loading control, blot was reprobed with anti H3 antibody. (3B) H4K4 acetylation assay using histones isolated from HATs over-expressing cells by colorimetric method. Histones of WT cells and pLPneo2 transfected cells were used as control. Relative levels of acetylation of H4K4 are shown in percentage assuming that of WT cells as 100%. The p values < 0.0001 compared with WT cells are shown as ***. (3C) Total histone H3 acetylation assay by fluorimetric method for HATs over-expressing cells. Histones isolated from WT cells and vector (pLPneo2) only transfected cell were taken as controls. H3 acetylation was presented in percentage considering 100% for WT cells. *** represents p values < 0.0001 compared to WT cells.</p

L-Asparaginase of Leishmania donovani: Metabolic target and its role in Amphotericin B resistance

Emergence of Amphotericin B (AmB) resistant Leishmania donovani has posed major therapeutic challenge against the parasite. Consequently, combination therapy aimed at multiple molecular targets, based on proteome wise network analysis has been recommended. In this regard we had earlier identified and proposed L-asparaginase of Leishmania donovani (LdAI) as a crucial metabolic target. Here we report that both LdAI overexpressing axenic amastigote and promastigote forms of L. donovani survives better when challenged with AmB as compared to wild type strain. Conversely, qRT-PCR analysis showed an upregulation of LdAI in both forms upon AmB treatment. Our data demonstrates the importance of LdAI in imparting immediate protective response to the parasite upon AmB treatment. In the absence of structural and functional information, we modeled LdAI and validated its solution structure through small angle X-ray scattering (SAXS) analysis. We identified its specific inhibitors through ligand and structure-based approach and characterized their effects on enzymatic properties (Km, Vmax, Kcat) of LdAI. We show that in presence of two of the inhibitors L1 and L2, the survival of L. donovani is compromised whereas overexpression of LdAI in these cells restores viability. Taken together, our results conclusively prove that LdAI is a crucial metabolic enzyme conferring early counter measure against AmB treatment by Leishmania. Keywords: Leishmania donovani, L-asparaginase, Amphotericin B resistance, Metabolic targe