Trithorax Gene Research Articles

Androgen-induced Epigenetic Profiles of Polycomb and Trithorax Genes in Prostate Cancer Cells.

Androgens are essential for the growth of most prostate cancers (PCa). As a result, androgen ablation is the mainstay of the treatment of PCa. Proteins of the polycomb and trithorax family are master epigenetic regulators of cell type specific gene expression including androgen receptor. We interrogated epigenetic changes of a 24-gene panel corresponding to polycomb and trithorax genes by PCR array and differential gene expression by quantitative real time-PCR on prostate cancer cell line (LNCaP) treated with the synthetic ligand R1881. We observed the highest methylation for CBX2, PCGF6, PHC2, EZH2 and TRIM27 genes and the lowest methylation for CBX8 and PCGF2 (p<0.05), and a modest decrease in the expression of EZH2. Differential methylation profiles of polycomb and trithorax genes may contribute to the dynamics of prostate carcinogenesis.

THE HOX NETWORK LEADS THE NEOPLASTIC TRANSFORMATION IN THE HUMAN SOLID TUMORS

Class I homeobox genes (HOX), are 39 transcription factors involved in the regulation of different cell processes: i) cell identity; ii) cell growth and proliferation; iii) cell communication; iv) determining anteroposterior axis during embryonic development; these genes are characterized by 183 nucleotide sequence (homeobox) encoding for a 61 amino acid domain (hoemodomain). HOX network controls the cell memory program during cell differentiation and is related to neoplastic transformation. Three gene families regulate the cell memory program: the Polycomb genes, able to block DNA-chromatin interaction leading to HOX genes silencing; the Trithorax genes, able to induce an open configuration of DNA-chromatin interaction and leading to HOX genes activation. Finally the HOX genes involved in the orchestration of the cell phenotype through the fine regulation of specific gene program. The proposal of this review, considering the recent literature and my personal research, is clarify the involvement Class I homeobox genes in the control of neoplastic transformation.

Drug Induced MLL Fusion Degradation Via Hsp90 Inhibition

The survival rate for infants is less than 50%. The fast majority of infant acute leukemias are characterized cytogenetically by balanced chromosomal translocations involving the mixed lineage leukemia (MLL) gene. Leukemic therapies that degrade the driver oncogene are associated with loss of cancer cell self-renewal and excellent cure rates. Therefore, therapy that degrades the MLL fusion gene would offer new hope to these patients.Recently it was shown that the Drosophila Trithorax gene, an analogue of the human MLL, is degraded by Radicicol. Radicicol is a natural compound and a well-known Hsp90 inhibitor.Here, we show that Radicicol is able to induce a dose dependent degradation of the MLL-fusion protein in a panel of human MLL rearranged cell lines and in human cord blood-derived MLL-AF9 immortalised myeloid cells. This drug induced degradation of the MLL-fusion gene results in down-regulation of the expression of MLL target genes, including HOXA9, MEIS1 and c-MYB. Functionally, this results in a loss of self-renewal of the leukemic stem cells, as shown by methylcellulose colony forming assays.Radicicol proved ineffective and too toxic for in vivo use. One of the best tolerated Hsp90 inhibitors is Ganetespib. It is currently in phase II/III clinical trials.We extended our Radicicol data to Ganetespib. Like Radicicol, Ganetespib induces MLL-fusion protein degradation and downregulation of MLL target genes. Treatment of MLL rearranged leukemia with Ganetespib results in a loss of leukemic stem cell activity.In conclusion, we show how the inactivation of the MLL-fusion and down-regulation of MLL target genes results in a block of leukemic stem cell self- renewal. We will validate these findings in a pre-clinical in vivo model in the near future. DisclosuresNo relevant conflicts of interest to declare.

Trithorax regulates systemic signaling during Drosophila imaginal disc regeneration.

Although tissue regeneration has been studied in a variety of organisms, from Hydra to humans, many of the genes that regulate the ability of each animal to regenerate remain unknown. The larval imaginal discs of the genetically tractable model organism Drosophila melanogaster have complex patterning, well-characterized development and a high regenerative capacity, and are thus an excellent model system for studying mechanisms that regulate regeneration. To identify genes that are important for wound healing and tissue repair, we have carried out a genetic screen for mutations that impair regeneration in the wing imaginal disc. Through this screen we identified the chromatin-modification gene trithorax as a key regeneration gene. Here we show that animals heterozygous for trithorax are unable to maintain activation of a developmental checkpoint that allows regeneration to occur. This defect is likely to be caused by abnormally high expression of puckered, a negative regulator of Jun N-terminal kinase (JNK) signaling, at the wound site. Insufficient JNK signaling leads to insufficient expression of an insulin-like peptide, dILP8, which is required for the developmental checkpoint. Thus, trithorax regulates regeneration signaling and capacity.

Polycomb Group Member Rybp Is a Functional Tumor Suppressor Repressed By Mir-9 in MLL-Rearranged AML

MicroRNAs (miRNAs), are small non-coding RNA molecules known to be important regulators of cancer biology. Notably, we and others have shown that miRNAs play important roles in Acute Myeloid Leukemia (AML), a heterogeneous malignancies with multiple chromosomal and molecular abnormalities. Patients with chromosomal rearrangements involving mixed lineage leukemia (MLL), the mammalian homology of trithorax gene, are associated with poor survival. Previously, we have found that MLL-rearranged AML drives aberrant expression of several miRNAs, most notably microRNA-9 (miR-9). Expression of miR-9 with MLL-AF9, a common MLL-translocation, was sufficient to promote transformation normal hematopoietic progenitor cells in vitro and leukemogenesis in vivo. We previously found that miR-9 reduces expression of several genes but we did not know which genes were critical tumor suppressors. We found that the polycomb group member RING1- and YY1-Bindin Protein (RYBP) was consistently inhibited upon miR-9 expression. To assess the regulation of RYBP we used publically available data from the Cancer Genome Atlas (TCGA) and looked at genome-wide Illumina 450K methylation data. We did not find a strong correlation with methylation and RYBP expression, suggesting that expression of RYBP is likely not regulated by the DNA methylation machinery in patients. Upon looking at copy number alterations we found that a small population of AML patients contained either homozygous or heterozygous loss of RYBP, suggesting a potential role of RYBP in leukemia pathogenesis. To assess the role of RYBP we did a series of in vitro experiments. We found that expression of RYBP was sufficient to attenuate colony-forming growth driven by MLL- AF9. Furthermore, RYBP expression was able to reduce proliferation, increase apoptosis, and significantly reduce immature cell population. To determine the role of RYBP expression in vivo, we transplanted lethally irradiated mice with progenitors retrovirally transduced with MLL-AF9 compared to MLL-AF9 and RYBP. We found that expression of RYBP was sufficient to reduce leukemia burden in vivo as well as induce differentiation as shown by flow cytometry and histological analysis. Thus, this demonstrates that RYBP is a functional tumor suppressor in MLL-rearranged AML. In conclusion, we have demonstrated that chromosomal rearrangements involving MLL, the mammalian homology of trithorax, downregulates a member of the polycomb complex through upregulation of miR-9. DisclosuresNo relevant conflicts of interest to declare.

The Trithorax group gene Ash1l regulates quiescence, self-renewal potential and niche occupancy in adult hematopoietic stem cells

Absent, small or homeotic discs 1 (Ash1) encodes a Trithorax group (TrxG) protein first identified in Drosophila. Mammalian Ash1l (Ash1-like) contains a conserved internal SET domain with in vitro H3K36 dimethyltransferase activity. Using a gene trap (GT) model, we discovered an essential role for Ash1l in adult hematopoietic stem cells (HSCs). Ash1lGT/GT mice have a 90% reduction in Ash1l transcripts. Despite preserved HSCs at birth, young adult Ash1l-deficient mice showed profound depletion of long-term HSCs (LT-HSCs) and markedly decreased quiescence (G0 cells). Ash1l-deficient HSCs had decreased expression of p27/p57, two critical regulators of HSC quiescence. In transplantation assays, Ash1l-deficient bone marrow was incapable of long-term reconstitution. Non-myeloablative transplantation of wild-type progenitors into non-irradiated Ash1l-deficient mice led to high engraftment of wild-type LT-HSCs, demonstrating abnormal availability of HSC niches. We assessed if wild-type progenitors engrafting in the non-irradiated Ash1lGT/GT mice retained HSC properties. Donor cells (GFP+) were injected into non-irradiated Ash1lGT/GT mice and allowed to reconstitute for 9 weeks before infusion of wild-type cells CD45.1+. Interestingly, most LT-HSCs in Ash1lGT/GT mice (≥93%) were derived from the initial GFP+ donor and had not been outcompeted by the later infusion of CD45.1+ cells. Thus, wild-type HSCs could stably engraft in the Ash1l-deficient HSC niche, consistent with normal niche function but decreased niche occupancy by Ash1l-deficient HSCs. We next assessed the mechanisms of Ash1l action and its functional interaction with the TrxG member Mixed Lineage Leukemia 1 (Mll1). Ash1l deficiency decreased expression of several posterior Hox genes in HSCs. Loss of both Ash1l and Mll1 led to overt hematopoietic failure, providing the first in vivo evidence that TrxG genes can synergize in mammals. We are currently exploring the molecular mechanisms underlying this cooperativity. Together, our results uncover a Trithorax gene network that controls quiescence, self-renewal and niche occupancy in adult HSCs. Absent, small or homeotic discs 1 (Ash1) encodes a Trithorax group (TrxG) protein first identified in Drosophila. Mammalian Ash1l (Ash1-like) contains a conserved internal SET domain with in vitro H3K36 dimethyltransferase activity. Using a gene trap (GT) model, we discovered an essential role for Ash1l in adult hematopoietic stem cells (HSCs). Ash1lGT/GT mice have a 90% reduction in Ash1l transcripts. Despite preserved HSCs at birth, young adult Ash1l-deficient mice showed profound depletion of long-term HSCs (LT-HSCs) and markedly decreased quiescence (G0 cells). Ash1l-deficient HSCs had decreased expression of p27/p57, two critical regulators of HSC quiescence. In transplantation assays, Ash1l-deficient bone marrow was incapable of long-term reconstitution. Non-myeloablative transplantation of wild-type progenitors into non-irradiated Ash1l-deficient mice led to high engraftment of wild-type LT-HSCs, demonstrating abnormal availability of HSC niches. We assessed if wild-type progenitors engrafting in the non-irradiated Ash1lGT/GT mice retained HSC properties. Donor cells (GFP+) were injected into non-irradiated Ash1lGT/GT mice and allowed to reconstitute for 9 weeks before infusion of wild-type cells CD45.1+. Interestingly, most LT-HSCs in Ash1lGT/GT mice (≥93%) were derived from the initial GFP+ donor and had not been outcompeted by the later infusion of CD45.1+ cells. Thus, wild-type HSCs could stably engraft in the Ash1l-deficient HSC niche, consistent with normal niche function but decreased niche occupancy by Ash1l-deficient HSCs. We next assessed the mechanisms of Ash1l action and its functional interaction with the TrxG member Mixed Lineage Leukemia 1 (Mll1). Ash1l deficiency decreased expression of several posterior Hox genes in HSCs. Loss of both Ash1l and Mll1 led to overt hematopoietic failure, providing the first in vivo evidence that TrxG genes can synergize in mammals. We are currently exploring the molecular mechanisms underlying this cooperativity. Together, our results uncover a Trithorax gene network that controls quiescence, self-renewal and niche occupancy in adult HSCs.

Polycomb and trithorax gene regulation—potential targets for β-cell regeneration

Polycomb and trithorax gene regulation—potential targets for β-cell regeneration

Combined modulation of polycomb and trithorax genes rejuvenates β cell replication

Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclin-dependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the age-dependent increase of Ink4a transcription. We generated an inducible pancreatic β cell-specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass.

The trithorax protein partner menin acts in tandem with EZH2 to suppress C/EBP and differentiation in MLL-AF9 leukemia

Trithorax and polycomb group proteins antagonistically regulate the transcription of many genes, and cancer can result from the disruption of this regulation. Deregulation of trithorax function occurs through chromosomal translocations involving the trithorax gene MLL, leading to the expression of MLL fusion proteins and acute leukemia. It is poorly understood how MLL fusion proteins block differentiation, a hallmark of leukemogenesis. We analyzed the effect of acute depletion of menin, a close partner of MLL that is critical for MLL and MLL-AF9 recruitment to target genes, on MLL-AF9 leukemia cell differentiation using an in vivo model. We performed cDNA microarray analysis of menin-regulated genes from primary leukemia cells to determine menin-regulated pathways involved in suppressing MLL-AF9 leukemia cell differentiation. We found that menin binds the promoter of the polycomb gene Ezh2, and promotes its expression. EZH2 interacts with the differentiation-promoting transcription factor C/EBPα and represses C/EBPα target genes. Menin depletion reduces MLL binding to the Ezh2 locus, EZH2 expression, and EZH2 binding and repressive H3K27 methylation at C/EBPα target genes, thereby inducing the expression of pro-differentiation C/EBPα targets. In conclusion, our results show that in contrast to its classical role antagonizing trithorax function, the polycomb group protein EZH2 collaborates with trithorax-associated menin to block MLL-AF9 leukemia cell differentiation, uncovering a novel mechanism for suppression of C/EBPα and leukemia cell differentiation, through menin-mediated upregulation of EZH2.

Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences

Orofacial malformations resulting from genetic and/or environmental causes are frequent human birth defects yet their etiology is often unclear because of insufficient information concerning the molecular, cellular and morphogenetic processes responsible for normal facial development. We have, therefore, derived a comprehensive expression dataset for mouse orofacial development, interrogating three distinct regions – the mandibular, maxillary and frontonasal prominences. To capture the dynamic changes in the transcriptome during face formation, we sampled five time points between E10.5–E12.5, spanning the developmental period from establishment of the prominences to their fusion to form the mature facial platform. Seven independent biological replicates were used for each sample ensuring robustness and quality of the dataset. Here, we provide a general overview of the dataset, characterizing aspects of gene expression changes at both the spatial and temporal level. Considerable coordinate regulation occurs across the three prominences during this period of facial growth and morphogenesis, with a switch from expression of genes involved in cell proliferation to those associated with differentiation. An accompanying shift in the expression of polycomb and trithorax genes presumably maintains appropriate patterns of gene expression in precursor or differentiated cells, respectively. Superimposed on the many coordinated changes are prominence-specific differences in the expression of genes encoding transcription factors, extracellular matrix components, and signaling molecules. Thus, the elaboration of each prominence will be driven by particular combinations of transcription factors coupled with specific cell:cell and cell:matrix interactions. The dataset also reveals several prominence-specific genes not previously associated with orofacial development, a subset of which we externally validate. Several of these latter genes are components of bidirectional transcription units that likely share cis-acting sequences with well-characterized genes. Overall, our studies provide a valuable resource for probing orofacial development and a robust dataset for bioinformatic analysis of spatial and temporal gene expression changes during embryogenesis.

Functional Characterization of the dRYBP Gene in Drosophila

The Drosophila dRYBP gene has been described to function as a Polycomb-dependent transcriptional repressor. To determine the in vivo function of the dRYBP gene, we have generated mutations and analyzed the associated phenotypes. Homozygous null mutants die progressively throughout development and present phenotypes variable both in their penetrance and in their expressivity, including disrupted oogenesis, a disorganized pattern of the syncytial nuclear divisions, defects in pattern formation, and decreased wing size. Although dRYBP mutations do not show the homeotic-like phenotypes typical of mutations in the PcG and trxG genes, they enhance the phenotypes of mutations of either the Sex comb extra gene (PcG) or the trithorax gene (trxG). Finally, the dRYBP protein interacts physically with the Sex comb extra and the Pleiohomeotic proteins, and the homeotic-like phenotypes produced by the high levels of the dRYBP protein are mediated through its C-terminal domain. Our results indicate that the dRYBP gene functions in the control of cell identity together with the PcG/trxG proteins. Furthermore, they also indicate that dRYBP participates in the control of cell proliferation and cell differentiation and we propose that its functional requirement may well depend on the robustness of the animal.

The Enhancer of Trithorax and Polycomb Corto Interacts with Cyclin G in Drosophila

Background Polycomb (PcG) and trithorax (trxG) genes encode proteins involved in the maintenance of gene expression patterns, notably Hox genes, throughout development. PcG proteins are required for long-term gene repression whereas TrxG proteins are positive regulators that counteract PcG action. PcG and TrxG proteins form large complexes that bind chromatin at overlapping sites called Polycomb and Trithorax Response Elements (PRE/TRE). A third class of proteins, so-called “Enhancers of Trithorax and Polycomb” (ETP), interacts with either complexes, behaving sometimes as repressors and sometimes as activators. The role of ETP proteins is largely unknown.Methodology/Principal FindingsIn a two-hybrid screen, we identified Cyclin G (CycG) as a partner of the Drosophila ETP Corto. Inactivation of CycG by RNA interference highlights its essential role during development. We show here that Corto and CycG directly interact and bind to each other in embryos and S2 cells. Moreover, CycG is targeted to polytene chromosomes where it co-localizes at multiple sites with Corto and with the PcG factor Polyhomeotic (PH). We observed that corto is involved in maintaining Abd-B repression outside its normal expression domain in embryos. This could be achieved by association between Corto and CycG since both proteins bind the regulatory element iab-7 PRE and the promoter of the Abd-B gene.Conclusions/SignificanceOur results suggest that CycG could regulate the activity of Corto at chromatin and thus be involved in changing Corto from an Enhancer of TrxG into an Enhancer of PcG.

Bladder exstrophy–epispadias complex: Investigation of suppressor of variegation, enhancer of zeste and Trithorax (SET) as a candidate gene in a large cohort of patients

The bladder exstrophy-epispadias complex (BEEC) describes a rare anterior midline defect with variable expression involving the infra-umbilical abdominal wall, including the pelvis, urinary tract and external genitalia. It is assumed that the underlying cause of BEEC is a multifactorial mode of inheritance; however, its aetiology remains unknown. Only a few BEEC patients with distinctive cytogenetic features such as numeric or structural chromosomal abnormalities have been reported. The observation of translocations concerning the region of chromosome 9q32-q34.1 in two patients implies that this region bears a candidate gene which, during early blastogenesis, governs the development of this primary field. In this context, we considered the suppressor of variegation, enhancer of zeste and Trithorax (SET) gene, located at chromosome 9q34, to be a good candidate, as the protein encoded is involved in the regulation of cell proliferation and differentiation. Moreover, SET expression was detected in embryonic kidney. A total of 33 patients affected with BEEC were analysed for mutations in the SET gene. SET analysis did not reveal either a mutation or the presence of four single-nucleotide polymorphisms (dbSNP124) already described in the database. The data obtained in this study most likely exclude the SET gene as a possible genetic cause of BEEC. Hence, other genes in this region may contribute to the development of this midline defect.

Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL

Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.

CALM-AF10 Positive T-ALLs Show a Pattern of Expression Similar to MLL-Translocated Acute Leukemias.

The t(10;11) translocation is recurrent in T-ALL and AML. The AF10 gene on chromosome 10 is rearranged either with MLL or CALM located on chromosome 11. CALM-AF10 fusion gene is found in T-ALLs in immature (IM) and TCRγδ-expressing (TCRGD+) T-ALLs. We compared 6 CALM-AF10+ T-ALLs cases (4 IM, 2 TCRGD+) to 17 CALM-AF10 negative T-ALLs cases (14 IM, 3 TCRGD+) using Affymetrix U133A microarrays.44 genes were significantly overexpressed in CALM-AF10+ T-ALLs, the most significant being HOXA9, a homeobox gene overexpressed in MLL-translocated acute leukemias (MLL-t AL), BMI1, a polycomb family member whose function in regulation of HOX genes expression is opposite to Trithorax genes (whose MLL belongs), SOX4, a frequent insertion site in retroviral-induced leukemogenesis, SFRS6 and COMMD3 (p≤0.001). Only two other HOX genes, HOXA5 and HOXA10, were significantly increased. 89 genes were significantly underexpressed in CALM-AF10+ T-ALLs, the most significant being GGH, ARL6IP4, NBS1, OGFR and TUBB (p≤0.001).An independent analysis of the expression of HOXA5, HOXA9, HOXA10 and BMI1 genes was done by quantitative RT-PCR in 10 CALM-AF10+ T-ALLs and 27 CALM-AF10 negative T-ALLs. These were compared to 19 MLL-translocated acute leukemias (2 MLL-AF10, 5 MLL-AF4, 3 MLL-AF6, 5 MLL-AF9, 3 MLL-ELL and 1 MLL-ENL), since HOXA9 overexpression had been previously associated with MLL-t AL.HOXA5, HOXA9 and HOXA10 expression were higher in CALM-AF10+ T-ALLs than in CALM-AF10 negative T-ALLs (p<0.001), confirming the microarray results. HOXA5 and HOXA9 expressions in CALM-AF10+ T-ALLs were similar to those detected in MLL-t AL and lower for HOXA10 in CALM-AF10+ T-ALLs as compared to the values of MLL-t AL (p=0.008). BMI1 expression in CALM-AF10+ T-ALLs was significantly higher than in CALM-AF10 negative T-ALLs and MLL-t AL (p<0.001).Additionally, MEIS1 expression was determined as this gene was associated in MLL-t AL with the overexpression of HOXA9. As for BMI1, MEIS1 expression was significantly higher in CALM-AF10+ T-ALLs compared to CALM-AF10 negative T-ALLs and MLL-t AL (p<0.001 and p=0.019, respectively).In summary, we demonstrated here the association between CALM-AF10 in T-ALLs and overexpression of HOXA5, HOXA9, HOXA10, BMI1 and MEIS1 genes. Overexpression of BMI1 is restricted to CALM-AF10+ T-ALLs. Although no obvious similarities are apparent between MLL and CALM proteins, the activation of HOXA and MEIS1 genes represent a highly recurrent pattern of expression in CALM-AF10+ T-ALLs and MLL-t AL. Consequently, the leukemias resulting in the activation of HOXA9 (MLL-t AL, CALM-AF10+ AL, NUP98-HOXA9 AML) should be seen as an independent group of acute leukemias and may benefit from common therapeutic protocols.

Expression of leukemic MLL fusion proteins in Drosophila affects cell cycle control and chromosome morphology.

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.

Mechanisms of Transformation by MLL

Rearrangements of the mixed-lineage leukemia gene MLL1 (MLL, HRX, ALL1), the human homologue of the Drosophila gene trithorax, are associated with aggressive acute leukemias in both children and adults. Transformation by rearranged forms of MLL1, including in-frame fusion proteins, partial tandem duplications, and amplification of MLL1 through upregulation of Hox gene and cofactor expression apparently results in a block in hematopoietic differentiation. MLL1 regulates Hox gene expression via direct promoter binding and histone H3 Lys 4 methylation mediated by the intrinsic methyltransferase activity of the SET domain. Mll1 knockout leads to loss of Hox gene expression, defects in hematopoiesis, and embryonic lethality. A close homologue, MLL2 is amplified in some solid tumors. MLL2 also has histone H3 Lys 4 methyltransferase activity that is dependent on menin, a protein mutated in multiple neoplasia type I (MEN1) and which is required for normal Hox expression. These findings underscore the importance of the MLL histone methyltransferases in development and disease.

COMPASS: a complex of proteins associated with a trithorax-related SET domain protein.

The trithorax genes encode an evolutionarily conserved family of proteins that function to maintain specific patterns of gene expression throughout cellular development. Members of this protein family contain a highly conserved 130- to 140-amino acid motif termed the SET domain. We report the purification and molecular identification of the subunits of a protein complex in the yeast Saccharomyces cerevisiae that includes the trithorax-related protein Set1. This protein complex, which we have named COMPASS (Complex Proteins Associated with Set1), consists of seven polypeptides ranging from 130 to 25 kDa. The same seven proteins were identified in COMPASS purified either by conventional biochemical chromatography or tandem-affinity tagging of the individual subunits of the complex. Null mutants missing any one of the six nonessential subunits of COMPASS grow more slowly than wild-type cells under normal conditions and demonstrate growth sensitivity to hydroxyurea. Furthermore, gene expression profiles of strains missing either of two nonessential subunits of COMPASS are altered in similar ways, suggesting these proteins have similar roles in gene expression in vivo. Molecular characterization of trithorax complexes will facilitate defining the role of this class of proteins in the regulation of gene expression and how their misregulation results in the development of human cancer.

Truncation of the Mll gene in exon 5 by gene targeting leads to early preimplantation lethality of homozygous embryos.

The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives.

Two Arabidopsis homologs of the animal trithorax genes: a new structural domain is a signature feature of the trithorax gene family

Two Arabidopsis genes have been characterized as first examples of plant genes homologous to the animal trithorax genes. The Arabidopsis genes are highly similar but display different tissue and development expression patterns. One of them was ubiquitously expressed, with highest levels registered in young seedlings. The other gene was less active in all tested tissues, was not expressed in mature leaves but was highly expressed in roots. A new structural motif common to all TRX-related proteins has been identified. This new architectural element was found only in genes of multicellular species and is present in all genes belonging to the trithorax family. Along with the SET domain and the PHD fingers, this new element is a signature feature for the trithorax gene family.