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Despite treatment with intensive chemotherapy, acute myelogenous leukemia (AML) remains an aggressive malignancy with a dismal outcome in most patients. We found that AML cells exhibit an unusually rapid accumulation of the repressive histone mark H3K27me3 on nascent DNA. In cell lines, primary cells and xenograft mouse models, inhibition of the H3K27 histone methyltransferase EZH2 to decondense the H3K27me3-marked chromatin of AML cells enhanced chromatin accessibility and chemotherapy-induced DNA damage, apoptosis, and leukemia suppression. These effects were further promoted when chromatin decondensation of AML cells was induced upon S-phase entry after release from a transient G 1 arrest mediated by CDK4/6 inhibition. In the p53 -null KG-1 and THP-1 AML cell lines, EZH2 inhibitor and doxorubicin cotreatment induced transcriptional reprogramming that was, in part, dependent on derepression of H3K27me3-marked gene promoters and led to increased expression of cell death-promoting and growth-inhibitory genes.In conclusion, decondensing H3K27me3-marked chromatin by EZH2 inhibition represents a promising approach to improve the efficacy of DNA-damaging cytotoxic agents in patients with AML. This strategy might allow for a lowering of chemotherapy doses, with a consequent reduction of treatment-related side effects in elderly patients with AML or those with significant comorbidities. SIGNIFICANCE: Pharmacological inhibition of EZH2 renders DNA of AML cells more accessible to cytotoxic agents, facilitating leukemia suppression with reduced doses of chemotherapy. See related commentary by Adema and Colla, p. 359 ., (©2021 American Association for Cancer Research.)

Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila, polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. Here, we show that, in mouse embryonic stem cells (ESCs), H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones and reduced PRC2 occupancy. Further, we show H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. Our data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, our findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an "active" chromatin state., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. We devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Rearrangements of the MLL (ALL1) gene are very common in acute infant and therapy-associated leukemias. The rearrangements underlie the generation of MLL fusion proteins acting as potent oncogenes. Several most consistently up-regulated targets of MLL fusions, MEIS1, HOXA7, HOXA9, and HOXA10 are functionally related and have been implicated in other types of leukemias. Each of the four genes was knocked down separately in the human precursor B-cell leukemic line RS4;11 expressing MLL-AF4. The mutant and control cells were compared for engraftment in NOD/SCID mice. Engraftment of all mutants into the bone marrow (BM) was impaired. Although homing was similar, colonization by the knockdown cells was slowed. Initially, both types of cells were confined to the trabecular area; this was followed by a rapid spread of the WT cells to the compact bone area, contrasted with a significantly slower process for the mutants. In vitro and in vivo BrdU incorporation experiments indicated reduced proliferation of the mutant cells. In addition, the CXCR4/SDF-1 axis was hampered, as evidenced by reduced migration toward an SDF-1 gradient and loss of SDF-1-augmented proliferation in culture. The very similar phenotype shared by all mutant lines implies that all four genes are involved and required for expansion of MLL-AF4 associated leukemic cells in mice, and down-regulation of any of them is not compensated by the others.

Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.

Histone lysine methylation regulates genomic functions, including gene transcription. Previous reports found various degrees of methylation at H3K4, H3K9, and H4K20 within the transcribed region of active mammalian genes. To identify the enzymes responsible for placing these modifications, we examined ASH1L, the mammalian homolog of the Drosophila melanogaster Trithorax group (TrxG) protein Ash1. Drosophila Ash1 has been reported to methylate H3K4, H3K9, and H4K20 at its target sites. Here we demonstrate that mammalian ASH1L associates with the transcribed region of all active genes examined, including Hox genes. The distribution of ASH1L in transcribed chromatin strongly resembles that of methylated H3K4 but not that of H3K9 or H4K20. Accordingly, the SET domain of ASH1L methylates H3K4 in vitro, and knockdown of ASH1L expression reduced H3K4 trimethylation at HoxA10 in vivo. Notably, prior methylation at H3K9 reduced ASH1L-mediated methylation at H3K4, suggesting cross-regulation among these marks. Drosophila ash1 and trithorax interact genetically, and the mammalian TrxG protein MLL1 and ASH1L display highly similar distributions and substrate specificities. However, by using MLL null cell lines we found that their recruitments occur independently of each other. Collectively, our data suggest that ASH1L occupies most, if not all, active genes and methylates histone H3 in a nonredundant fashion at a subset of genes.

The recent discovery of a large number of histone demethylases suggests a central role for these enzymes in regulating histone methylation dynamics. Histone H3K27 trimethylation (H3K27me3) has been linked to polycomb-group-protein-mediated suppression of Hox genes and animal body patterning, X-chromosome inactivation and possibly maintenance of embryonic stem cell (ESC) identity. An imbalance of H3K27 methylation owing to overexpression of the methylase EZH2 has been implicated in metastatic prostate and aggressive breast cancers. Here we show that the JmjC-domain-containing related proteins UTX and JMJD3 catalyse demethylation of H3K27me3/2. UTX is enriched around the transcription start sites of many HOX genes in primary human fibroblasts, in which HOX genes are differentially expressed, but is selectively excluded from the HOX loci in ESCs, in which HOX genes are largely silent. Consistently, RNA interference inhibition of UTX led to increased H3K27me3 levels at some HOX gene promoters. Importantly, morpholino oligonucleotide inhibition of a zebrafish UTX homologue resulted in mis-regulation of hox genes and a striking posterior developmental defect, which was partially rescued by wild-type, but not by catalytically inactive, human UTX. Taken together, these findings identify a small family of H3K27 demethylases with important, evolutionarily conserved roles in H3K27 methylation regulation and in animal anterior-posterior development.

The trithorax and the polycomb group proteins are chromatin modifiers, which play a key role in the epigenetic regulation of development, differentiation and maintenance of cell fates. The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalysing the di- and tri-methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. Here we show that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, we demonstrate that ectopic expression of JMJD3 leads to a strong decrease of H3K27me3 levels and causes delocalization of polycomb proteins in vivo. Consistent with the strong decrease in H3K27me3 levels associated with HOX genes during differentiation, we show that UTX directly binds to the HOXB1 locus and is required for its activation. Finally mutation of F18E9.5, a Caenorhabditis elegans JMJD3 orthologue, or inhibition of its expression, results in abnormal gonad development. Taken together, these results suggest that H3K27me3 demethylation regulated by UTX/JMJD3 proteins is essential for proper development. Moreover, the recent demonstration that UTX associates with the H3K4me3 histone methyltransferase MLL2 (ref. 8) supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation.

Erythropoietin-producing hepatoma-amplified sequence (Eph) receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, function as a unique signaling system triggered by cell-to-cell interaction and have been shown to mediate neurodevelopmental processes. In addition, recent studies showed deregulation of some of Eph/ephrin genes in human malignancies, suggesting the involvement of this signaling pathway in tumorigenesis. The ALL1 (also termed MLL) gene on human chromosome 11q23 was isolated by virtue of its involvement in recurrent chromosome translocations associated with acute leukemias with poor prognosis. The translocations fuse ALL1 to any of >50 partner genes and result in production of chimeric proteins composed of the ALL1 N terminus and the C terminus of the partner protein. The most common translocations in ALL1-associated leukemias are t(4;11) and t(9;11), which generate ALL1/AF4 and ALL1/AF9 fusion protein, respectively. In the present study, we sought to determine whether ALL1 fusion proteins are involved in regulation of Eph/ephrin genes. Screening of K562 cells producing recombinant ALL1/AF4 or ALL1/AF9 fusion protein revealed transcriptional up-regulation of the EphA7. Consistent with this finding, siRNA-mediated suppression of ALL1/AF4 in SEMK2 cells carrying the t(4;11) chromosome translocation resulted in down-regulation of EphA7. ChIP analysis demonstrated the occupancy of tagged ALL1 fusion proteins on the EphA7 promoter, pointing to EphA7 as a direct target of the formers. Further studies demonstrate that EphA7 up-regulation is accompanied by ERK phosphorylation. Finally, we show apoptotic cell death, specific for leukemic cells carrying the t(4;11) chromosome translocation, after treatment of the cells with an ERK phosphorylation blocker.

MicroRNAs (miRNAs) are noncoding small RNA of approximately 22 bases, which suppress expression of target genes through translational block or degradation of a target's transcript. Recent studies uncovered specific miRNA expression profiles in human malignancies. Nevertheless, the mechanisms underlying cancer-specific miRNA expression are largely unknown. miRNA biogenesis consists of a series of steps beginning with generation of a primary transcript, termed pri-miRNA, and continuing into excision of a stem-loop hairpin structure within pri-miRNA by the nuclear RNaseIII enzyme Drosha, transportation to the cytoplasm, and further processing by a second RNaseIII enzyme Dicer, into a 22-base mature duplex RNA. In principle, alteration in any step during this maturation process could affect miRNA production. The ALL-1 (also termed MLL) gene was originally isolated by virtue of its involvement in recurrent chromosome translocations associated with acute leukemias, particularly in infant and therapy-related leukemias. These translocations result in the fusion of ALL-1 with partner genes and the consequent production of chimeric leukemogenic proteins. Here, we identify specific miRNAs up-regulated in leukemias triggered by All1 fusions. Further, we demonstrate coimmunoprecipitation of the All1/Af4 and All1/Af9 fusions with Drosha, disrupted by treatment with DNase I. Finally, we present evidence from ChIP experiments for All1 fusion protein-mediated recruitment of Drosha to target genes encoding miRNAs. Such recruitment may underlie the enhanced expression of the relevant miRNAs.

ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

The mixed-lineage leukemia (MLL1/ALL-1/HRX) histone methyltransferase is involved in the epigenetic maintenance of transcriptional memory and the pathogenesis of human leukemias. To understand its role in cell type specification, we determined the human genomic binding sites of MLL1. We found that MLL1 functions as a human equivalent of yeast Set1. Like Set1, MLL1 localizes with RNA polymerase II (Pol II) to the 5' end of actively transcribed genes, where histone H3 lysine 4 trimethylation occurs. Consistent with this global role in transcription, MLL1 also localizes to microRNA (miRNA) loci that are involved in leukemia and hematopoiesis. In contrast to the 5' proximal binding behavior at most protein-coding genes, MLL1 occupies an extensive domain within a transcriptionally active region of the HoxA cluster. The ability of MLL1 to serve as a start site-specific global transcriptional regulator and to participate in larger chromatin domains at the Hox genes reveals dual roles for MLL1 in maintenance of cellular identity.

The evolutionary conserved SET domain is present in many eukaryotic chromatin-associated proteins, including some members of the trithorax (TrxG) group and the polycomb (PcG) group of epigenetic transcriptional regulators and modifiers of position effect variegation. All SET domains examined exhibited histone lysine methyltransferase activity, implicating these proteins in the generation of epigenetic marks. However, the mode of the initial recruitment of SET proteins to target genes and the way that their association with the genes is maintained after replication are not known. We found that SET-containing proteins of the SET1 and SET2 families contain motifs in the pre-SET region or at the pre-SET-SET and SET-post-SET boundaries which very tightly bind single-stranded DNA (ssDNA) and RNA. These motifs also bind stretches of ssDNA generated by superhelical tension or during the in vitro transcription of duplex DNA. Importantly, such binding withstands nucleosome assembly, interfering with the formation of regular nucleosomal arrays. Two representatives of the SUV39 SET family, SU(VAR)3-9 and G9a, did not bind ssDNA. The trxZ11 homeotic point mutation, which is located within TRX SET and disrupts embryonic development, impairs the ssDNA binding capacity of the protein. We suggest that the motifs described here may be directly involved in the biological function(s) of SET-containing proteins. The binding of single-stranded nucleic acids might play a role in the initial recruitment of the proteins to target genes, in the maintenance of their association after DNA replication, or in sustaining DNA stretches in a single-stranded configuration to allow for continuous transcription.

The Mixed Lineage Leukemia (MLL) gene is involved in lymphoblastic and myeloid leukemia through chromosome translocations leading to fusion of MLL to partner genes, or through internal MLL rearrangements. MLL is the mammalian counterpart of the Drosophila trithorax (trx) gene, involved in maintaining active gene expression states. We have used transgenic Drosophila to assess the molecular targets and cellular processes affected by MLL and two of its leukemic fusion proteins. We find that whereas expression of normal human MLL in flies does not result in phenotypic alterations, overexpressing the human MLL-AF9 and MLL-AF4 proteins causes larval to pupal lethality, which interestingly resembles the phenotypes displayed by certain Drosophila trx mutant alleles. MLL-AF9 and MLL-AF4 transgenic flies exhibit antagonistic alterations in cell cycle progression. Additionally, flies expressing MLL-AF9 display impairment in higher order chromatin integrity, evidenced in decondensation of mitotic figures. The effects of MLL fusion proteins in Drosophila suggest that alteration of chromatin structure by MLL fusion proteins may contribute to the lethal phenotype. Our results indicate that the mode(s) of action of MLL-AF9 in Drosophila varies from that of MLL-AF4. Taken together, the expression of MLL fusion proteins in Drosophila provides a new and powerful system to reveal and characterize biological activities associated with MLL fusion proteins.

Rapid induction of the Drosophila melanogaster heat shock gene hsp70 is achieved through the binding of heat shock factor (HSF) to heat shock elements (HSEs) located upstream of the transcription start site (reviewed in ref. 3). The subsequent recruitment of several other factors, including Spt5, Spt6 and FACT, is believed to facilitate Pol II elongation through nucleosomes downstream of the start site. Here, we report a novel mechanism of heat shock gene regulation that involves modifications of nucleosomes by the TAC1 histone modification complex. After heat stress, TAC1 is recruited to several heat shock gene loci, where its components are required for high levels of gene expression. Recruitment of TAC1 to the 5'-coding region of hsp70 seems to involve the elongating Pol II complex. TAC1 has both histone H3 Lys 4-specific (H3-K4) methyltransferase (HMTase) activity and histone acetyltransferase activity through Trithorax (Trx) and CREB-binding protein (CBP), respectively. Consistently, TAC1 is required for methylation and acetylation of nucleosomal histones in the 5'-coding region of hsp70 after induction, suggesting an unexpected role for TAC1 during transcriptional elongation.

Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers moulting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homologue Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. Here we report that TRR is a histone methyltransferases capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. We propose that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters.

ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of > or =29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complex's H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter.

Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators, which is required to maintain active states of Hox gene expression during development. We have purified from Drosophila embryos a trithorax acetylation complex (TAC1) that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression.

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.

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 AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

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.

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of approximately 100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5-10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell-cell contacts and found that AF-6 accumulated at the cell-cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

The ALL1 gene (also called MLL, HRX, or Htrx1) at the cytogenetic band 11q23 is consistently altered by chromosome rearrangements in acute leukemias (ALs) of early infancy, in ALs developed after exposure to topoisomerase (topo) II-inhibitory drugs, and in a small subset of de novo ALs in children and adults. Because exposure to natural or medicinal substances blocking topo II during pregnancy have been proposed as etiological agents for infant leukemia, we have compared the distribution of ALL1 gene breakpoints in infant leukemias with an altered ALL1 gene configuration to those in secondary leukemia associated with prior exposure to topo II targeting drugs and in reference to the major topo consensus binding site in exon 9. ALL1 gene breakpoint distribution was determined by Southern blot hybridization and/or reverse transcription-PCR of the ALL1/AF4 fusion cDNA in 70 patients. Using restriction enzyme analysis, the 8.3-kb ALL1 breakpoint cluster region was divided in a centromeric portion of 3.5 kb (region A) and telomeric portion of a 4.8 kb (region B). ALL1 breakpoint were located in region A in 8 of 28 (28.5%) cases of infant ALs, 16 of 24 (66%) cases of de novo ALs, and 0 of 5 cases of therapy-related (TR) ALs. Conversely, ALL1 breakpoints in region B were detected in 20 of 28 (71.5%) cases of infant AL, 8 of 24 (33%) cases of de novo AL, and 5 of 5 (100%) cases of TR AL (P = 0.002). These results were confirmed by direct sequencing of the ALL1/AF4 fusion transcript in 30 cases (19 infants and 11 child and adult de novo cases). The analysis of ALL1/AF4 junction types showed that children and adults with de novo leukemia had ALL1 breakpoints in intron 6 (9 cases) or intron 7 (2 cases), whereas breakpoints in infant cases were mainly located in intron 8 (14 cases) and less frequently in intron 6 (4 cases) and intron 7 (1 case). The difference in ALL1 breakpoint location between infant and noninfant AL patients with ALL1/AF4 fusion was statistically significant (P = 0.00005). These data demonstrated that infant and TR ALs share a similar biased clustering of ALL1 gene breakpoints, which supports the possibility that topo II inhibitors may also operate in utero and play a crucial role in the etiology of infant leukemia.

The ALL-1 gene positioned at 11q23 is directly involved in human acute leukemia either through a variety of chromosome translocations or by partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which plays a critical role in maintaining proper spatial and temporal expression of the Antennapedia-bithorax homeotic genes determining the fruit fly's body pattern. Utilizing specific antibodies, we found that the ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of the chromosome translocations and expressed in transfected cells showed similar speckles. Dissection of the ALL-1 protein identified within its approximately 1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter spanned two short sequences conserved with TRITHORAX. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, resulted in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. We suggest that the speckles represent protein complexes which contain multiple copies of the ALL-1 protein and are positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes.

In this study we investigated the presence of structural lesions in the ALL-1, p53 and p16 (cyclin-dependent kinase 4 inhibitor) genes in leukaemic cells obtained from 22 patients with infant acute leukaemia (aged < 18 months). Of these, 18 cases were classified as acute lymphoblastic leukaemia (ALL) and four as acute myeloid leukaemia (AML). Tumour DNAs were analysed by a combination of Southern blot. polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequence analyses. The results showed ALL-1 gene rearrangements in 15/22 (68%) cases, p53 gene mutations in 5/22 (26%), and a homozygous deletion of p16 in a single T-ALL case. p53 and p16 alterations were all found in the group of patients with ALL-1 gene rearrangements. p53 mutations were more often associated with a myeloid phenotype (3/5). In summary, multiple molecular alterations were found in 6/15 (40%) infant acute leukaemias with ALL-1 rearrangements. As to the clinical course, patients with additional lesions had similar clinical outcome with respect to patients with ALL-1 gene rearrangement as the sole genetic aberration. This may support the hypothesis that ALL-1 alterations are genetic events per se sufficient to confer a fully malignant phenotype to the leukaemic clone.

The ALL-1 gene is involved in human acute leukemia through chromosome translocations and fusion to partner genes, or through partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which transregulates the homeotic genes of the Antennapedia and bithorax complexes controlling body segment identity. ALL-1 encodes a very large protein of 3968 amino acids which presumably interacts with many proteins. Here we applied yeast two hybrid screening to identify proteins interacting with the N-terminal segment of ALL-1. One protein obtained in this way was the product of the unr gene. This protein consists of multiple repeats homologous to the cold shock domain (CSD), a motif common to some bacterial and eukaryotic nucleic acids-binding proteins. The minimal region on unr required for the interaction with ALL-1 included two CSD and two intervening polypeptides. The interaction was confirmed by in vitro binding studies, and by coimmunoprecipitation from COS cells overexpressing the relevant segments of the two proteins. These results suggest that unr is involved in an interaction of ALL-1 with DNA or RNA.

In this study we used reverse transcriptase-polymerase chain reaction (RT-PCR) for the longitudinal monitoring of minimal residual disease in 12 patients with All-1/AF-4 positive ALL. Of these, seven also showed at presentation a typical t(4;11) cytogenetic translocation. Seven patients were infants <18 months of age and five were adults. Eleven patients were treated with high-dose intensive induction and consolidation chemotherapy without bone marrow transplantation and one received conservative treatment due to poor performance status. Three had resistant disease, four relapsed within 12 months after achieving complete remission, and five are in continuous complete remission (CCR) at 32, 39, 52, 53 and 61 months from diagnosis, respectively. The sequential analysis of the ALL-1/AF-4 hybrid transcript showed a persistently negative RT-PCR in the five CCR long-term survivors. The PCR analysis resulted persistently positive in the remaining seven cases, including the four cases who relapsed after the achievement of clinical CR. These data emphasize the clinical relevance of PCR monitoring analysis in t(4;11) ALL patients and should be considered in order to better determine variable post-remission treatment according to risk prediction.

Prevention of high frequency spontaneous T cell lymphoma development in AKR mice by mAb 18-5 treatment was shown to involve inhibition of the recombinant Class I MCF virus formation and elimination of the early occurring potential lymphoma cells (PLCs). A low B cell lymphoma incidence (16% at a mean latency of 540 days) and a low level of PLCs (yielding 12% B cell lymphoma development following lymphoid cell transfer) was observed in mAb 18-5 treated mice (in contrast to a high PLC level in thymectomized AKR mice that could be experimentally triggered to progress to overt CD5+ B cell lymphomas). Administration of anti CD8 mAb or IL-4 to 12-month-old mAb 18-5 pre-treated mice only slightly increased B cell lymphoma incidence (up to 30-40%). Exposure to split-dose irradiation resulted in 26% B cell lymphomas at a 250 day mean latency. The phenotypes of the B lymphomas developing in mAb 18-5 treated mice were: B220+ (14.8+, 6B2+), 6C3+, Mac2+, CD5-. Most lymphomas expressed l-a and surface IgM, pointing to their mature B cell characteristics. Moreover, in some of the lymphomas, high levels of IgM production and secretion were determined. A comparison of the morphological characteristics (based on light and ultrastructure microscopy) of CD5+ and CD5- B cell lymphomas developing in AKR mice indicated marked differences. Analysis of the IgH locus of representative CD5- B lymphomas showed an identical pattern of IgH rearrangement in some tumors (similar to previous findings among CD5+ lymphomas). The virological analysis of the CD5- B cell lymphomas (similar to those observed in the CD5+ B cell lymphomas of AKR origin) showed that their development did not require formation of the pathogenic MCF recombinant viruses. The differences observed between the CD5+ and CD5- B cell lymphomas developing in AKR mice (following prevention of spontaneous T cell lymphomagenesis) may be due to their origin of different B cell precursors or from B cells at different levels of differentiation.

We have analyzed by Southern blotting the ALL-1 (MLL, HRX, Hrtx 1) gene configuration in a series of 126 patients with acute myeloid leukemia (AML) representative of all ages and French-American-British Classification groups and correlated this genetic feature with clinical and biological features at diagnosis. ALL-1 gene rearrangements were detected in 17 of the 74 cases with M4-M5 (myelomonocytic and monocytic) AML and in 2 of the 52 cases with other leukemic subtypes (P < 0.01). Within the series of 74 M4-M5 patients, ALL-1 rearrangements were significantly associated with French-American-British Classification M5 (P = 0.009), high WBC (P = 0.002), and young age. In particular, all 5 infant (< 1.5 years) AML cases, 6 of the 19 (31%) patients between 1.5 and 18 years of age, and 6 of the 50 (12%) patients > 18 years old showed an altered ALL-1 genomic configuration (P < 0.001). Immunophenotypic characterization revealed coexpression of lymphoid and myeloid markers in 6 of 17 ALL-1 rearranged M4-M5 cases. The IgH gene configuration was studied in 77 of 126 AMLs. Five patients (6%) showed IgH clonal rearrangements and all were in the ALL-1 rearranged group (P < 0.0001). Our findings indicate that ALL-1 rearrangement is the commonest genetic alteration presently detectable in M4-M5 AML, particularly in childhood where it is found in up to one-third of all cases. The association of IgH rearrangements with ALL-1 alterations in AML, coupled to the frequent detection in this subset of lymphoid associated markers, further supports the origin of these tumors from a common multipotent precursor with bipotential lymphoid and monocytic differentiation capability.

Rearrangements of the ALL-1 gene by reciprocal translocations involving chromosome band 11q23 are frequently associated with human acute leukemia. We have previously reported the detection of ALL-1 gene rearrangements in adult patients with acute myeloid leukemia lacking cytogenetic evidence of 11q23 translocations. These included 2 of 19 patients with normal karyotypes as well as 3 of 4 patients with trisomy 11 as a sole cytogenetic abnormality. Rearrangement of the ALL-1 genes in two of the patients with trisomy 11 was shown to result from a direct tandem duplication of a portion of the gene spanning exons 2-6. Here we report the characterization of the ALL-1 gene rearrangement in one of the previously reported acute myeloid leukemia patients with a normal karyotype. ALL-1 rearrangement in this patient results from a direct tandem duplication of a portion of the gene spanning exons 2-8. RNA polymerase chain reaction and DNA sequence analysis show that the partially duplicated ALL-1 gene is transcribed into mRNA capable of encoding a partially duplicated protein. Sequence analysis of the genomic fusion region provides evidence for Alu-mediated homologous recombination as a mechanism for partial duplication of the ALL-1 gene.

DNA rearrangements caused by chromosome translocations between band 11q23 and various chromosomes can be detected by a single probe, B859, an 859-base pair complementary DNA fragment derived from the human ALL-1 gene. To try to understand why band 11q23 becomes a frequent target of the translocations, we have sequenced the entire breakpoint cluster region, a 8342-base pair BamHI genomic fragment delineated by B859. We found eight Alu repeats located within this region in the same orientation as the ALL-1 gene. We have also analyzed the sequences of the breakpoints in 10 patients with 6 different types of 11q23 aberration. In five patients the breaks coincided with Alu sequences on chromosome 11, but not on the partner chromosomes. Also, seven of the breaks occurred in the region delineated by exons 6 and 7, which is composed mainly of Alu sequences. In three patients topoisomerase II recognition site-like sequences, at different stringency levels, were identified at the breakpoints on chromosome 11. We conclude that while there is no specific sequence element present at all the breakpoints, the high density of Alu sequences in the breakpoint cluster region possibly makes the latter more prone to recombination events.

Adriamycin (Adr) is one of the most powerful antitumor drugs. Its therapeutic effect may be due to its cyclic reduction-oxidation and, thus, generation of oxygen radicals. Using the spin-trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and EPR we have demonstrated that in an enzymatic system consisting of NADPH, NADPH-cytochrome P-450 reductase, and Fe(EDTA)2 Adr stimulates formation of .OH radicals in the presence of DNA or RNA with equal efficiency. Incubation of nucleic acids in the Adr-dependent reaction generating .OH radicals resulted in extensive degradation of double- and single-stranded DNA, but did not effect RNA. In contrast, both DNA and RNA were effectively destroyed in a footprinting system, ascorbate-Fe(EDTA)2-H2O2, which generates .OH radicals in massive quantities. Fluorescence assays indicated that Adr forms stable complexes with ds- and ss-DNA but reacts only slightly with RNA. We conclude that the formation of Adr-nucleic acid complex is necessary for .OH radical-mediated cleavage of the latter, and thus, Adr may be regarded as a chemical nuclease acting in situ.

We have examined a t(9;11)(p22;q23) chromosome translocation in an acute myeloid leukemia of an infant. The breakpoints on the two chromosomes occurred within introns of the involved genes: AF-9 on chromosome 9, and ALL-1 on chromosome 11. Sequence analysis identified heptamers flanking the breakpoints on both chromosomes 9 and 11, suggesting that the V-D-J recombinase was involved in the translocation. The presence of an N-region between the two chromosomes supports the hypothesis that a mistake in V-D-J joining was involved in the genesis of the translocation and indicates that terminal deoxynucleotidyl transferase was expressed in the cells from which this acute myeloid leukemia originated. In addition, potential topoisomerase II DNA-binding sites were found near the breakpoints of both chromosomes, suggesting the involvement of altered topoisomerase II activity in this translocation. Altered topoisomerase II activity in the presence of an active V-D-J recombinase may be a pathogenetic mechanism of acute myeloid leukemia with rearrangements at 11q23.

The chromosome 11q23 band is a genetic region frequently involved in nonrandom karyotypic abnormalities of acute leukemia. A genomic locus named ALL-1 or MLL, where 11q23 breakpoints are clustered, has been recently cloned and characterized. We have made use of an ALL-1-specific probe in Southern blot experiments to analyze the configuration of this gene in a large series of acute leukemia patients, representative of all different myeloid and lymphoid subtypes. Nine of 145 cases (6.2%) showed abnormal ALL-1 restriction fragments in leukemic DNAs. Of these nine cases, five patients in whom karyotypic data were available displayed chromosome 11q23 aberrations, including t(4;11) (three cases) and t(9;11) (two cases). Immunophenotypic and morphocytochemical characterization of ALL-1-rearranged acute leukemia revealed prevalence of poorly differentiated B lymphoid and/or monoblastic features. Considering the whole series, ALL-1 rearrangements were significantly associated with female sex, higher white blood cell counts at presentation, and very poor clinical outcome. The presence of residual disease was molecularly documented in one case at the time of clinical remission after induction treatment and was followed by early relapse. We conclude that ALL-1 rearrangements are new molecular markers of human leukemia with considerable diagnostic and prognostic relevance.

AKR mice are highly susceptible to development of spontaneous T-cell lymphoma. Thymus removal at the age of 1-3 months greatly reduces T-cell lymphoma. Lymphomas that have the characteristics of T- and/or B-cells occur sporadically in peripheral lymphoid tissues of old thymectomized AKR/J mice. These thymectomized mice were shown to carry dormant potential lymphoma cells. Transplantation of lymphoid cells from 8-12-month-old AKR/J mice, thymectomized at the age of 6 to 8 weeks, into intact or thymectomized young recipients yielded 80-100% Ly-1+ pre-B or B-cell lymphomas. In the AKR-Fv-1b congenic strain the Fv-1n allele of AKR/J mice was substituted with the Fv-1b allele, thereby limiting viral replication and spread of the endogenous N-tropic murine leukemia virus. As a result of this restriction in virus spread, AKR-Fv-1b mice develop a low spontaneous incidence (7%) of T-cell lymphomas and about 28% of Ly-1+ B-cell lymphomas at old age. In spleens of 15-18-month-old thymectomized AKR/J mice and intact AKR-Fv-1b mice, 30-60% of the B-cells were of the Ly-1+ B type. Analysis of the IgH locus in these normal old spleens and Ly-1+ B lymphomas indicated mono- or oligoclonality. One particular IgH rearrangement was identified in many individual old spleens and tumors. A second specific IgH rearrangement was found in some tumors. Possible mechanisms involved in the expansion of Ly-1+ clones and their progression into tumors are discussed.