Identification of new translocations involving ETV6 in hematologic malignancies by fluorescence in situ hybridization and spectral karyotyping.

TEL/ETV6 is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners; 35 different chromosome bands have been involved in ETV6 translocations, of which 13 have been cloned. To identify additional ETV6 partner genes and to characterize the chromosomal abnormalities more fully, we studied bone marrow samples from patients known to have rearrangements of 12p, using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). FISH analysis was done with 14 probes located on 12p12.1 to 12p13.3. Nine ETV6 rearrangements were identified using FISH. The aberrations include t(1;12)(p36;p13), t(4;12)(q12;p13) (two patients), t(4;12)(q22;p13), t(6;12)(p21;p13), der(6)t(6;21)(q15;q?)t(12;21)(p13;q22), t(6;12)(q25;p13), inv(12)(p13q24), and t(2;2;5;12;17)(p25;q23;q31;p13;q12). Six new ETV6 partner bands were identified: 1p36, 4q22, 6p21, 6q25, 12q24, and 17q12. Our present data as well previous data from us and from other researchers suggest that ETV6 is involved in 41 translocations. The breakpoints in ETV6 were upstream from the exons coding for the HLH (helix-loop-helix) domain in six cases. Although cytogenetic analysis identified 12p abnormalities in all cases, FISH and SKY detected new and unexpected chromosomal rearrangements in many of them. Thus, complete characterization of the samples was achieved by using all three techniques in combination.

Cytogenetic and molecular analysis of the acute monocytic leukemia cell line THP-1 with an MLL-AF9 translocation.

Cell lines derived from patients with leukemia are used in many molecular biology studies. Here we report the cytogenetic analysis of the THP-1 cell line using G-banding, fluorescence in situ hybridization (FISH), and spectral karyotyping (SKY), and the molecular characterization of the MLL-AF9 rearrangement by RT-PCR. The THP-1 cell line was established from the peripheral blood of a 1-year-old boy with acute monocytic leukemia (AML-M5). THP-1 is near-diploid and consists of two related subclones with a number of aberrations, including the t(9;11), associated with AML M5. The use of FISH allowed us to identify and characterize otherwise hidden cytogenetic rearrangements, which include duplication of the 3' portion of MLL in the derivative 9 chromosome and a deletion of the 5' portion of the AF9 gene involved in the translocation. In addition to confirming the FISH results, SKY allowed for a more precise characterization of the karyotype of THP-1 and allowed us to identify other abnormalities in this cell line, including der(1)t(1;12), der(20)t(1;20), deletions 6p, 12p, and 17p, trisomy 8, and monosomy 10. Sequencing of the RT-PCR product showed a direct in-frame fusion product on the derivative chromosome 11 between exon 6 (exon 9) of MLL and exon 5 of AF9, which is most commonly involved in MLL-AF9 translocations. This study demonstrates that combining different techniques to achieve a more precise characterization of the THP-1 cell line provides important information that will be valuable for understanding the critical events required for leukemogenesis.

Binding of trithorax and Polycomb proteins to the bithorax complex: dynamic changes during early Drosophila embryogenesis.

In Drosophila, the maintenance of developmentally important transcription patterns is controlled at the level of chromatin structure. The Polycomb group (PcG) and trithorax group (trxG) genes encode proteins involved in chromatin remodelling. PcG genes have been proposed to act by packaging transcriptional repressed chromosomal domains into condensed heterochromatin-like structures. Some of the trxG proteins characterized so far are members of chromatin opening complexes (e.g. SWI/SNF and GAGA/NURF) which facilitate binding of transcription factors and components of the basal transcriptional machinery. Genetic and biochemical data suggest that these two groups of regulatory factors may act through a common set of DNA elements. In the present study, we have investigated the binding of Trithorax (TRX) and Polycomb (PC) protein in the bithorax complex (BX-C) during embryogenesis. In addition, we have identified the minimal fragments from the Ultrabithorax (Ubx) regulatory region that are capable of recruiting TRX to chromosomal sites containing them. Comparative analysis of the binding of the two proteins shows that TRX and PC bind target sequences (PcG-regulated elements, PREs) by cellular blastoderm, when BX-C transcription begins. At the same stage, TRX but not PC is strongly associated with core promoters. Later, at germ band extension, the time of derepression in Polycomb mutants, PC binding is also detected outside core PREs and additionally binds to the fragments containing promoters.

The Drosophila trithorax proteins contain a novel variant of the nuclear receptor type DNA binding domain and an ancient conserved motif found in other chromosomal proteins.

The products of the trithorax gene are required to stably maintain homeotic gene expression patterns established during embryo-genesis by the action of the transiently expressed segmentation genes. We have determined the intron/exon structure of the trx gene and the large alternatively spliced trx RNAs, which are capable of encoding only two protein isoforms. These very large trx proteins differ only in a long Ser- and Gly-rich N-terminal extension, encoded by exon II, which is present only in the larger trx isoform. We have identified a novel variant of the highly conserved nuclear receptor type of DNA binding domain. We have found that the previously identified Cys-rich central region contains multiple novel zinc finger motifs which are also present in the Polycomb-like protein and RBP2, a retinoblastoma binding protein. The trx proteins terminate with another novel conserved domain which we have identified in proteins from three kingdoms, including plants and fungi, indicating that has an ancient origin. Many of these proteins are chromosomally associated, suggesting that this domain may be involved in interactions between trx and other highly conserved components of chromatin involved in transcription regulation. The sequence alterations of trx mutations identify the highly conserved regions of trx as critical for the function of these large proteins. We show that zygotically expressed trx RNAs encoding the larger protein isoform are initially expressed in a spatially restricted pattern which overlaps the expression domains of the BX-C genes Ubx, abd-A and Abd-B. This pattern is transient and evolves into a broader expression domain encompassing the entire germ band during the extended germ band stage.

Trithorax is required to maintain engrailed expression in a subset of engrailed-expressing cells.

We show that maintenance but not initiation of engrailed (en) gene expression in the Drosophila embryo requires trithorax (trx), which is also required to maintain stable long-term expression of the homeotic genes throughout the development. Like the homeotic genes, en expression is dependent on trx in only a subset of embryonic cells normally expressing en, including specific cells in the nervous system and the dorsal fat body cells surrounding the gonad. Loss of en expression in the dorsal fat body is correlated with the sterility of en females which also carry trx mutations. In addition, trx is required for normal en expression in the posterior compartment of the developing wing, reflected in enhancement of en phenotypes in en adults which also carry trx mutations. trx appears to be dispensable for maintenance of en expression in other embryonic cells. The trx protein binds to the region of the polytene chromosomes which contains the en gene, suggesting that trx regulates en expression directly by binding to the en regulatory region.

The Drosophila trithorax protein binds to specific chromosomal sites and is co-localized with Polycomb at many sites.

trithorax is required to stably maintain homeotic gene expression patterns established during embryogenesis by the action of the transiently expressed products of the segmentation genes. The large trithorax proteins contain a number of highly conserved novel motifs, some of which have been hypothesized to interact directly with specific DNA sequences in their target genes. Using antibodies directed against trithorax proteins, we show that they are bound to 63 specific sites on the polytene chromosomes of the larval salivary gland. trithorax binding is detected at the sites of its known targets, the Bithorax and Antennapedia complexes, despite the transcriptionally repressed state of these loci in the salivary gland. A temperature-sensitive trithorax mutation greatly reduces the number of binding sites. Simultaneous localization of trithorax and Polycomb indicates that many of their chromosomal binding sites coincide. We localized one trithorax binding site within a portion of the large 5' regulatory region of the Ubx gene, to an interval which also contains binding sites for Polycomb group proteins. These results suggest that trithorax exerts its effects by binding directly or indirectly to specific DNA sequences in its target genes. Co-localization with Polycomb also suggests that interactions between these activators and repressors of the homeotic genes may be a significant feature of their mode of action.