ALL-1/MLL1, a homologue of Drosophila TRITHORAX, modifies chromatin and is directly involved in infant acute leukaemia.

Rearrangements of the ALL-1/MLL1 gene underlie the majority of infant acute leukaemias, as well as of therapy-related leukaemias developing in cancer patients treated with inhibitors of topoisomerase II, such as VP16 and doxorubicin. The rearrangements fuse ALL-1 to any of >50 partner genes or to itself. Here, we describe the unique features of ALL-1-associated leukaemias, and recent progress in understanding molecular mechanisms involved in the activity of the ALL-1 protein and of its Drosophila homologue TRITHORAX.

Expression profiles of acute lymphoblastic and myeloblastic leukemias with ALL-1 rearrangements.

The ALL-1 gene is directly involved in 5-10% of acute lymphoblastic leukemias (ALLs) and acute myeloid leukemias (AMLs) by fusion to other genes or through internal rearrangements. DNA microarrays were used to determine expression profiles of ALLs and AMLs with ALL-1 rearrangements. These profiles distinguish those tumors from other ALLs and AMLs. The expression patterns of ALL-1-associated tumors, in particular ALLs, involve oncogenes, tumor suppressors, antiapoptotic genes, drug-resistance genes, etc., and correlate with the aggressive nature of the tumors. The genes whose expression differentiates between ALLs with and without ALL-1 rearrangement were further divided into several groups, enabling separation of ALL-1-associated ALLs into two subclasses. One of the groups included 43 genes that exhibited expression profiles closely linked to ALLs with ALL-1 rearrangements. Further, there were evident differences between the expression profiles of AMLs in which ALL-1 had undergone fusion to other genes and AMLs with partial duplication of ALL-1. The extensive analysis described here pinpointed genes that might have a direct role in pathogenesis.

Upregulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4 : 11) abnormality.

Rearrangements of the human ALL-1 gene are frequently encountered in acute lymphocytic leukemias (ALL) and acute myeloid leukemias (AML). These rearrangements are mostly due to chromosome translocations and result in production of chimeric proteins composed of the N-terminal fragment of ALL-1 and the C-terminal segments of the partner proteins. The most common chromosome translocation involving ALL-1 is the t(4 : 11) associated with ALL. ALL-1 is the human homologue of Drosophila trithorax and directly activates transcription of multiple Hox genes. A preliminary DNA microarray screen indicated that the Meis1, HoxA9 and AC133 genes were overexpressed in ALLs with t(4 : 11), compared to ALLs with very similar phenotype but without the chromosomal abnormality. These genes, as well as additional five Hox genes, were subjected to comprehensive semi-quantitative or quantitative RT-PCR analysis in 57 primary ALL and AML tumors. Meis1 and HoxA9 were found expressed in 13/14 of ALLs with the t(4 : 11) and in 8/8 of AMLs with ALL-1 rearrangements. The two genes were not consistently transcribed in other types of ALL. AC133 was transcribed in 13/14 of ALLs with t(4 : 11), but in only 4/8 of AMLs with ALL-1 rearrangements. HoxA10 was expressed in most leukemias with ALL-1 alterations, but was also transcribed in PrePreB CD10(-) ALLs lacking the t(4 : 11). Expression of HoxA5, HoxA7, HoxC8 and HoxC10 did not correlate with ALL-1 rearrangements. Coexpression of Meis1 and HoxA9, overexpression of HoxA10, and overexpression or fusion of HoxA9 were previously implicated in certain acute myeloid leukemias in mice and humans. The present work suggests that upregulation of Meis1, HoxA9, and possibly HoxA10 might also play a role in pathogenesis of acute lymphocytic and acute myeloid leukemias associated with ALL-1 fusions.

huASH1 protein, a putative transcription factor encoded by a human homologue of the Drosophila ash1 gene, localizes to both nuclei and cell-cell tight junctions.

During animal development, regions of the embryo become committed to position-specific identities, which are determined by spatially restricted expression of Hox/homeotic genes. This expression pattern is initially established by the activity of the segmentation genes and is subsequently maintained during the proliferative stage through the action of transcription factors encoded by the trithorax (trx) and Polycomb (Pc) groups of genes. trithorax (trx)and ash1 (absent, small, or homeotic 1) are members of the Drosophila trx group. Their products are associated with chromosomes and are believed to activate transcription of target genes through chromatin remodeling. Recently, we reported molecular studies indicating that TRX and ASH1 proteins act in concert to bind simultaneously to response elements located at close proximity within the same set of target genes. Extension of these and other studies to mammalian systems required identification and cloning of the mammalian homologue of ash1 (the mammalian homologue of trx, ALL-1, was previously cloned). We have identified a human expressed sequence tag (EST) clone with similarity to the SET domain of Drosophila ASH1, and used it to clone the human gene. huASH1 resides at chromosomal band 1q21. The gene is expressed in multiple tissues as an approximately 10.5-kb transcript and encodes a protein of 2962 residues. The protein contains a SET domain, a PHD finger, four AT hooks, and a region with homology to the bromodomain. The last region is not present in Drosophila ASH1, and as such might confer to the human protein a unique additional function. Using several anti-huASH1 Ab for immunostaining of cultured cells, we found that the protein is distributed in intranuclear speckles, and unexpectedly also in intercellular junctions. Double-immunofluorescence labeling of huASH1 and several junctional proteins localized the huASH1 protein into tight junctions. The significance of huASH1 dual location is discussed. In particular, we consider the possibility that translocation of the protein between the junctional membrane and the nucleus may be involved in adhesion-mediated signaling.

Self-association of the SET domains of human ALL-1 and of Drosophila TRITHORAX and ASH1 proteins.

The human ALL-1 gene is involved in acute leukemia through gene fusions, partial tandem duplications or a specific deletion. Several sequence motifs within the ALL-1 protein, such as the SET domain, PHD fingers and the region with homology to DNA methyl transferase are shared with other proteins involved in transcription regulation through chromatin alterations. However, the function of these motifs is still not clear. Studying ALL-1 presents an additional challenge because the gene is the human homologue of Drosophila trithorax. The latter is a member of the trithorax-Polycomb gene family which acts to determine the body pattern of Drosophila by maintaining expression or repression of the Antennapedia-bithorax homeotic gene complex. Here we apply yeast two hybrid methodology, in vivo immunoprecipitation and in vitro 'pull down' techniques to show self association of the SET motifs of ALL-1, TRITHORAX and ASH1 proteins (Drosophila ASH1 is encoded by a trithorax-group gene). Point mutations in evolutionary conserved residues of TRITHORAX SET, abolish the interaction. SET-SET interactions might act in integrating the activity of ALL-1 (TRX and ASH1) protein molecules, simultaneously positioned at different maintenance elements and directing expression of the same or different target genes.

Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter.

Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development. TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers. Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins. We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes. Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements. Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s). This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other. Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts. Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription.

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex.

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

[Substrate properties of dioxolane analogs of 3'-deoxythymidine-5'-triphosphate in DNA synthesis reactions, catalyzed by various DNA polymerases]. / Substratnye svoistva dioksolanovykh analogov 3'-dezoksitimidin-5'-trifosfata v reaktsiiakh sinteza DNK, kataliziruemykh razlichnymi DNK-polimerazami.

The substrate properties of 3'-deoxythymidine 5'-triphosphate analogs prepared on the basis of 2,4-disubstituted 1,3-dioxolanes were investigated in reactions of the DNA synthesis catalyzed by various DNA polymerases. The 4'-triphosphates of (+/-)-cis-4-hydroxymethyl-2-(1-thyminylmethyl)-1,3-dioxolane and the corresponding (+/-)-trans-isomer were shown to be terminating substrates of terminal deoxynucleotidyl transferase. 4'-Triphosphate of (+/-)-cis-4-hydroxymethyl-2-(1-thyminylmethyl)- 1,3-dioxolane terminates the DNA synthesis catalyzed by HIV reverse transcriptase, whereas 2'-triphosphate of (+/-)-cis-2-hydromethyl-4-(1-thyminylmethyl)-1,3-dioxolane is a terminator in the DNA synthesis catalyzed by HIV reverse transcriptase and the Klenow fragment of DNA polymerase I.

[2'-Deoxynucleoside-5'-triphosphate, modified via the carbohydrate and triphosphate residues, in the DNA synthesis reaction]. / 2'-Dezoksinukleozid-5'-trifosfaty, modifitsirovannye po uglevodnomu i trifosfatnomu ostatkam, v reaktsii sinteza DNK.

We have investigated the substrate properties of deoxyribonucleoside 5'-triphosphate analogues, modified in the carbohydrate and triphosphate moieties, in DNA synthesis catalyzed by different DNA polymerases and reverse transcriptases. It was shown that (3'-azido-2',3'-dideoxythymidine-5'-O-methylenephosphonate) diphosphate, (3'-azido-2',3'-dideoxythymidine 5'-phosphate) dibromomethylenediphosphonate, (3'-azido-2',3'-dideoxythymidine 5'-phosphate) phosphonoacetate terminate DNA synthesis catalyzed by reverse transcriptases. (2'-Deoxythymidine 5'-phosphate) phosphonoacetate displays substrate properties for DNA polymerase beta, different reverse transcriptases, terminal deoxynucleotidyl transferase, but not for DNA polymerase alpha, Klenow's fragment DNA polymerase I. The Km value for this substance in DNA synthesis reactions catalyzed by reverse transcriptases was two of orders magnitude higher than that for native 2'-deoxythymidine 5'-triphosphate.

[Reverse transcriptase of the human immunodeficiency virus: isolation and substrate specificity]. / Obratnaia transkriptaza virusa immunodefitsita cheloveka: vydelenie i substratnaia spetsifichenost'.

Human immunodeficiency virus (HIV-I) reverse transcriptase was expressed in E. coli and purified to homogeneity (E. coli strain RRI (pRC-RT, pRK 248cIts)). We have investigated the substrate properties toward to DNA synthesis, catalyzed by this enzyme, of some nucleoside-5'-triphosphate analogues, previously studied in the same reactions, catalyzed by AMV and M-MLV reverse transcriptases. We have investigated substrate properties of new analogues of 2',3'-dideoxy-2',3'-didehydro- and 2',3'-dideoxytubercidin-5'-triphosphates. We have compared the relative efficiency of incorporation of different analogues tested in the DNA chain. It has been shown that expressed and purified HIV reverse transcriptase had the same specificity to analogues of 2'-deoxyribonucleoside-5'-triphosphates as was described for reverse transcriptases and natural HIV reverse transcriptase as well. These properties allow to apply the expressed HIV reverse transcriptase in different model systems.

[Acyclic analogs of 2',3'-dideoxy-2',3'-didehydronucleoside-5'-triphosphates--terminators of DNA synthesis, catalyzed by a broad set of DNA polymerases]. / Atsiklicheskie analogi 2',3'-didezoksi-2',3'-didegidronukleozid-5'-trifosfatov--terminatory sinteza DNK, kataliziruemogo shirokim naborom DNK-polimeraz.

O-4'-nor-2', 3'-dideoxy-2', 3'-didehydronucleoside 5'-triphosphates are shown to be effective termination substrates of DNA biosynthesis catalyzed by human placental DNA polymerases alpha and epsilon, rat liver DNA polymerase beta, reverse transcriptases of human immunodeficiency virus and avian myeloblastosis virus, and calf thymus terminal deoxynucleotidyl transferase. These compounds do not interact only with the Escherichia coli DNA polymerase I (Klenow fragment). The probable reasons of interaction of acyclo-d4NTP with the DNA synthesizing complexes are discussed.

[Fluorescent analogs of nucleoside-5'-phosphates for the study of nucleic acids by nonradioactive methods]. / Fluorestsentnye analogi nukleozid-5'-trifosfatov dlia izucheniia nukleinovykh kislot neradioaktivnymi metodami.

The synthesis of 2'-deoxyuridine 5'-triphosphate analogues with fluorescent residues of fluorescein and rhodamine nature at C5 of the uracil base was performed. Reverse transcriptase of avian myeloblastosis virus, DNA polymerase beta of rat liver, terminal deoxynucleotidyl transferase of calf thymus and E. coli DNA polymerase I, Klenow fragment, were shown to be capable to incorporate a nucleotide residue with fluorescent label into 3'-terminus of oligonucleotide. These fluorescent labeled oligonucleotides were used as primers for synthesis of (-)-chain of M13mp10 phage. Fluorescently labeling template-primer complexes were used for DNA sequencing.

[Conformation limited nucleoside-5'-phosphates as termination substrates for DNA-polymerases]. / Konformatsionno ogranichennye nukleozid-5'-trifosfaty kak terminatornye substraty DNK-polimeraz.

We have investigated the ability of some nucleoside 5'-triphosphate analogues to terminate the DNA synthesis catalyzed by calf thymus DNA polymerase alpha and terminal deoxynucleotidyl transferase, rat liver DNA polymerase beta, E. coli DNA polymerase I (Klenow's fragment) and AMV reverse transcriptase. It has been shown that lyxoanhydronucleoside 5'-triphosphates terminate DNA synthesis catalyzed by reverse transcriptase and terminal deoxynucleotydil transferase. 2',3'-O-Isopropylidenecytidine 5'-triphosphate inhibits the DNA synthesis catalyzed by reverse transcriptase and DNA polymerase beta and its moiety was incorporated in the place of dTMP residue. Riboanhydroadenosine 5'-triphosphate reveals the properties of an effective termination substrate for all the DNA polymerases studied. This is the first attempt to investigate nucleotide analogues with the restricted conformation of the carbohydrate moiety as termination substrates for several prokaryotic and eukaryotic DNA polymerases.

[Analogs of pyrophosphate in a pyrophosphorolysis reaction catalyzed by DNA polymerases]. / Analogi pirofosfata v reaktsii pirofosforoliza, kataliziruemoi DNK-polimerazami.

The reaction of pyrophosphorolysis catalyzed by Escherichia coli DNA polymerase I Klenov fragment, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and AMV reverse transcriptase was studied. Some pyrophosphate (PPi) analogs were taken as low molecular weight substrates. It was shown that only imidodiphosphonic acid acted as the PPi substrate analog for the reactions catalyzed by DNA polymerases I and alpha, both imidodiphosphonic acid and methylenediphosphonic acid were active in the case of DNA polymerase beta and reverse transcriptase. Other analogs tested were neither nucleotide residue acceptors, nor inhibitors of the pyrophosphorolysis reaction with PPi. The abilities of some PPi analogs to inhibit the DNA elongation catalyzed by reverse transcriptase were investigated. The principles of specificity of low molecular substrates recognition by DNA polymerases and some problems concerning the mechanisms of DNA synthesis inhibition by PPi analogues are discussed.

[DNA polymerase I, Klenow fragment of Escherichia coli: hydrolysis and pyrophosphorolysis of DNA containing phosphothioate groups]. / DNK-polimeraza I Escherichia coli, fragment Klenova: gidroliz i pirofosforoliz DNK, soderzhashchei fosfotioatnye gruppy.

Reaction of DNA synthesis catalyzed by DNA polymerase I KF in the presence of 2'-deoxynucleoside 5'-alpha-thiotriphosphates (dNTP alpha S) was investigated. DNA with thiophosphate groups (DNA[P=S]) obtained by such a way was studied in reactions of hydrolysis and pyrophosphorolysis catalyzed by DNA polymerase I KF. It is shown that the rate of DNA elongation is decreased both on the step of incorporation of dNMP alpha S residues and on the step of incorporation of the next dNMP residue. The rate of pyrophosphorolysis of 3'-terminal dNMP alpha S was demonstrated to be one order of magnitude less in comparison with the corresponding reaction with the natural dNMP residue. Contrary, the rate of 3'----5'-exonuclease hydrolysis of both DNA[P=S] and DNA of the same structure revealed no distinguishable differences.