Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of B-ALL often associated with genetic variants that alter cytokine receptor signaling, including mutations in the interleukin-7 receptor (IL7R). To investigate whether IL7R variants are leukemia-initiating, we built mouse models expressing activated Il7r (aIL7R). B-cell intrinsic aIL7R mice developed spontaneous B-ALL, demonstrating sufficiency of Il7r activating mutations in leukemogenesis. Concomitant introduction of a knock-out allele in the associated adapter protein Lnk (encoded by Sh2b3) or a dominant-negative variant of the transcription factor Ikaros (Ikzf1) increased disease penetrance. The resulting murine leukemias displayed monoclonality and recurrent somatic Kras mutations and efficiently engrafted into immunocompetent mice. Phosphoproteomic analyses of aIL7R leukemic cells revealed constitutive Stat5 signaling and B cell receptor (BCR)-like signaling despite the absence of surface pre-BCR. Finally, in vitro treatment of aIL7R leukemic B-cells with Jak, mTOR, or Syk inhibitors blocked growth, confirming that each pathway is active in this mouse model of IL7R-driven B-ALL.

Generation and characterization of mice with mesenchyme-specific deletion of the entire ESET histone methyltransferase protein.

ESET protein (also known as SETDB1) catalyzes methylation of histone H3 at lysine 9 (H3-K9). In addition to the full-length transcript, mouse ESET gene also gives rise to alternative spicing variants encoding truncated proteins capable of retaining interaction with other epigenetic enzymes. To completely eliminate full-length ESET and its splicing variants, we have generated a conditional ESET allele with exon 4 flanked by two loxP sites for Cre-mediated DNA deletion and downstream frame-shift mutation of the entire coding region. Mating with Prx1-Cre mice and analysis of the resultant embryos revealed that mesenchyme-specific knockout of exon 4 completely eliminates full-length ESET and its truncated protein products, leading to profound defects in both the flat bones and long bones, ectopic hypertrophy of growth plate chondrocytes and downregulation of Indian hedgehog protein. In addition, exon 4 deletion results in reduced thickness of articular cartilage in E17.5 embryos, whereas deletion of exons 15-16 fails to do so. These findings offer us a useful tool to further study epigenetic regulation in a truly ESET-null background, and demonstrate that ESET plays a critical role in the control of chondrocyte hypertrophy and skeletal development.

ESET histone methyltransferase regulates osteoblastic differentiation of mesenchymal stem cells during postnatal bone development.

To investigate the effects of histone methyltransferase ESET (also known as SETDB1) on bone metabolism, we analyzed osteoblasts and osteoclasts in ESET knockout animals, and performed osteogenesis assays using ESET-null mesenchymal stem cells. We found that ESET deletion severely impairs osteoblast differentiation but has no effect on osteoclastogenesis, that co-transfection of ESET represses Runx2-mediated luciferase reporter while siRNA knockdown of ESET activates the luciferase reporter in mesenchymal cells, and that ESET is required for postnatal expression of Indian hedgehog protein in the growth plate. As the bone phenotype in ESET-null mice is 100% penetrant, these results support ESET as a critical regulator of osteoblast differentiation during bone development.

Mesenchyme-specific knockout of ESET histone methyltransferase causes ectopic hypertrophy and terminal differentiation of articular chondrocytes.

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.

ESET histone methyltransferase is essential to hypertrophic differentiation of growth plate chondrocytes and formation of epiphyseal plates.

The ESET (also called SETDB1) protein contains an N-terminal tudor domain that mediates protein-protein interactions and a C-terminal SET domain that catalyzes methylation of histone H3 at lysine 9. We report here that ESET protein is transiently upregulated in prehypertrophic chondrocytes in newborn mice. To investigate the in vivo effects of ESET on chondrocyte differentiation, we generated conditional knockout mice to specifically eliminate the catalytic SET domain of ESET protein only in mesenchymal cells. Such deletion of the ESET gene caused acceleration of chondrocyte hypertrophy in both embryos and young animals, depleting chondrocytes that are otherwise available to form epiphyseal plates for endochondral bone growth. ESET-deficient mice are thus characterized by defective long bone growth and trabecular bone formation. To understand the underlying mechanism for ESET regulation of chondrocytes, we carried out co-expression experiments and found that ESET associates with histone deacetylase 4 to bind and inhibit the activity of Runx2, a hypertrophy-promoting transcription factor. Repression of Runx2-mediated gene transactivation by ESET is dependent on its H3-K9 methyltransferase activity as well as its associated histone deacetylase activity. In addition, knockout of ESET is associated with repression of Indian hedgehog gene in pre- and early hypertrophic chondrocytes. Together, these results provide clear evidence that ESET controls hypertrophic differentiation of growth plate chondrocytes and endochondral ossification during embryogenesis and postnatal development.

FOXO1 is a direct target of EWS-Fli1 oncogenic fusion protein in Ewing's sarcoma cells.

Ewing's family tumors are characterized by a specific t(11;22) chromosomal translocation that results in the formation of EWS-Fli1 oncogenic fusion protein. To investigate the effects of EWS-Fli1 on gene expression, we carried out DNA microarray analysis after specific knockdown of EWS-Fli1 through transfection of synthetic siRNAs. EWS-Fli1 knockdown increased expression of genes such as DKK1 and p57 that are known to be repressed by EWS-Fli1 fusion protein. Among other potential EWS-Fli1 targets identified by our microarray analysis, we have focused on the FOXO1 gene since it encodes a potential tumor suppressor and has not been previously reported in Ewing's cells. To better understand how EWS-Fli1 affects FOXO1 expression, we have established a doxycycline-inducible siRNA system to achieve stable and reversible knockdown of EWS-Fli1 in Ewing's sarcoma cells. Here we show that FOXO1 expression in Ewing's cells has an inverse relationship with EWS-Fli1 protein level, and FOXO1 promoter activity is increased after doxycycline-induced EWS-Fli1 knockdown. In addition, we have found that direct binding of EWS-Fli1 to FOXO1 promoter is attenuated after doxycycline-induced siRNA knockdown of the fusion protein. Together, these results suggest that suppression of FOXO1 function by EWS-Fli1 fusion protein may contribute to cellular transformation in Ewing's family tumors.

Proper expression of helix-loop-helix protein Id2 is important to chondrogenic differentiation of ATDC5 cells.

The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. To identify novel factors that are involved in the control of chondrogenesis, we carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 (inhibitor of DNA-binding protein 2) gene. This retroviral insertion increased Id2 protein levels to twice those found in normal ATDC5 cells. To investigate whether an elevated level of Id2 protein was responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopic Id2 exhibited signs of de-differentiation, such as rapid growth, and insulin failed to induce expression of Sox9 (Sry-type high-mobility-group box 9) or matrix genes such as type II collagen (COL2) in these cells. When endogenous Id2 was knocked down by siRNA (small interfering RNA) in ATDC5 cells, expression of Sox9 and COL2 was increased and chondrogenic differentiation was accelerated. To examine how Id2 is expressed in chondrocytes in vivo, we carried out immunostaining of E16.5 mouse embryos and found that Id2 is expressed in articular chondrocytes and proliferating chondrocytes, but barely detectable in hypertrophic chondrocytes. Our results suggest that proper expression of Id2 is important to achieving a fine balance between growth and differentiation during chondrogenesis.

EWS/FLI1 suppresses retinoblastoma protein function and senescence in Ewing's sarcoma cells.

Ewing's Family Tumors (EFTs) most commonly harbor a specific t(11;22) translocation that generates the EWS/FLI1 fusion protein responsible for malignant transformation. Many potential downstream targets of EWS/FLI1 have been identified but a detailed mechanism by which the fusion protein brings about transformation remains unknown. In this report, we show that depletion of EWS/FLI1 in Ewing's cell lines results in a senescence phenotype, a marked increase in expression of the G1/S regulatory proteins p27(kip1) and p57(kip2), and a significant decrease in cyclin D1 and CDK2. We also demonstrate for the first time, to our knowledge, that knockdown of EWS/FLI1 leads to hypophosphorylation and functional activation of the retinoblastoma (pRb) family of proteins. Consistent with activation of the pRb proteins, E2F-responsive genes such as cyclin A are repressed in EWS/FLI1-depleted cells. Together, these results support the role of EWS/LI1 as an inhibitor of cellular senescence and implicate the retinoblastoma family of proteins as key mediators of this inhibition.

Rab23 regulates differentiation of ATDC5 chondroprogenitor cells.

Insulin treatment of mouse ATDC5 chondroprogenitors induces these cells to differentiate into mature chondrocytes. To identify novel factors that are involved in this process, we carried out mutagenesis of ATDC5 cells through retroviral insertion and isolated two mutant clones incapable of differentiation. Inverse PCR analysis of these clones revealed that the retroviral DNA was inserted into the promoter region of the Rab23 gene, resulting in increased Rab23 expression. To investigate whether an elevated level of Rab23 protein led to inhibition of chondrogenic differentiation, we characterized ATDC5 cells that either overexpress endogenous Rab23 or stably express ectopic Rab23. Our results revealed that up-regulation of Rab23 can indeed inhibit chondrogenic differentiation with a concomitant down-regulation of matrix genes such as type II collagen and aggrecan. In addition, stable small interfering RNA knockdown of Rab23 also resulted in inhibition of chondrogenic differentiation as well as down-regulation of Sox9, a master regulator of chondrogenesis. Interestingly, Sox9 expression has recently been linked to Gli1, and we found that Rab23 knockdown decreased Gli1 expression in chondrocytes. Because the phenotypes of Rab23 mutations in mice and humans include defects in cartilage and bone development, our study suggests that Rab23 is involved in the control of Sox9 expression via Gli1 protein.

TASR-1 regulates alternative splicing of collagen genes in chondrogenic cells.

During the differentiation of chondroprogenitors into mature chondrocytes, the alternative splicing of collagen genes switches from longer isoforms to shorter ones. To investigate the underlying mechanisms, we infected mouse ATDC5 chondroprogenitor cells with retrovirus for stable expression of two closely related SR splicing factors. RT-PCR analysis revealed that TASR-1, but not TASR-2, influenced alternative splicing of type II and type XI collagens in ATDC5 cells. The effect of TASR-1 on splicing could be reversed with the addition of insulin. Results from our microarray analysis of ATDC5 cells showed that TASR-1 and TASR-2 differentially affect genes involved in the differentiation of chondrocytes. Of special interest is the finding that TASR-1 could down-regulate expression of type X collagen, a hallmark of hypertrophic chondrocytes. Immunohistostaining demonstrated that TASR-1 protein is more abundantly expressed than TASR-2 in mouse articular chondrocytes, raising the possibility that TASR-1 might be involved in phenotype maintenance of articular chondrocytes.

The oncogenic TLS-ERG fusion protein exerts different effects in hematopoietic cells and fibroblasts.

The oncogenic TLS-ERG fusion protein is found in human myeloid leukemia and Ewing's sarcoma as a result of specific chromosomal translocation. To unveil the potential mechanism(s) underlying cellular transformation, we have investigated the effects of TLS-ERG on both gene transcription and RNA splicing. Here we show that the TLS protein forms complexes with RNA polymerase II (Pol II) and the serine-arginine family of splicing factors in vivo. Deletion analysis of TLS-ERG in both mouse L-G myeloid progenitor cells and NIH 3T3 fibroblasts revealed that the RNA Pol II-interacting domain of TLS-ERG resides within the first 173 amino acids. While TLS-ERG repressed expression of the luciferase reporter gene driven by glycoprotein IX promoter in L-G cells but not in NIH 3T3 cells, the fusion protein was able to affect splicing of the E1A reporter in NIH 3T3 cells but not in L-G cells. To identify potential target genes of TLS-ERG, the fusion protein and its mutants were stably expressed in both L-G and NIH 3T3 cells through retroviral transduction. Microarray analysis of RNA samples from these cells showed that TLS-ERG activates two different sets of genes sharing little similarity in the two cell lines. Taken together, these results suggest that the oncogenic TLS-ERG fusion protein transforms hematopoietic cells and fibroblasts via different pathways.

Targeting of EWS/FLI-1 by RNA interference attenuates the tumor phenotype of Ewing's sarcoma cells in vitro.

The defining cytogenetic abnormality of Ewing's sarcoma is the presence of a balanced t(11;22) translocation expressing the EWS/FLI-1 chimeric fusion protein. The effect of EWS/FLI-1 appears to be dominant negative since over-expression of EWS does not overcome the sarcoma phenotype. Previous studies have shown that EWS/FLI-1 as well as related sarcoma fusion proteins are necessary and sufficient to induce transformation both in vitro and in vivo. In this study we report that synthetic small interfering RNA (siRNA) specifically suppresses EWS/FLI-1 fusion gene expression in SK-ES Ewing's sarcoma cells. Knockdown of the EWS/FLI-1 fusion protein is correlated with decreased cell proliferation and increased apoptosis. We demonstrate that Ewing's sarcoma tumors as well as Ewing's sarcoma cell lines predominantly express the CXCR4 chemokine receptor. Using an in vitro invasion assay, the SDF-1 ligand of CXCR4 was shown to be a potent stimulus of invasion by SK-ES cells. Knockdown of EWS/FLI-1 by RNA interference abrogates the invasiveness of SK-ES cells. These experiments suggest that targeted silencing of the EWS/FLI-1 fusion gene by siRNA represents a promising strategy to study the loss of EWS/FLI-1 protein in Ewing's sarcoma cells of otherwise identical genetic background.

Genomic structure and expression of the mouse ESET gene encoding an ERG-associated histone methyltransferase with a SET domain.

ESET (ERG-associated protein with a SET domain, also called SETDB1) is a novel histone methyltransferase that catalyzes methylation of histone H3-lysine 9 (H3-K9). Here we describe the genomic structure and expression of the mouse ESET gene that gives rise to ESET protein and its alternative splicing product. ESET is a 36-kb single copy gene and full-length ESET transcript consisting of 22 exons. The splicing variant retains only the first 12 exons and thus lacks sequences encoding the methyl CpG-binding domain and the catalytic SET domain. The U2 type conserved GT/AG consensus sequence is present at all of the splicing junctions within the ESET gene. The transcription initiation site of the ESET gene was determined by 5'-RACE experiment and by primer extension. The 5'-flanking sequence of the ESET gene does not contain the consensus TATA box. Instead, this ESET promoter region has features such as SP1-binding sites that are typical of housekeeping genes. The ESET promoter was functionally active when tested in transfection and luciferase assay. Full-length ESET transcript appears to be ubiquitously expressed. While the SET domain-deficient splicing variant is present in immortalized cell lines, it is undetectable by RT-PCR in the majority of normal mouse tissues.

COL11A2 collagen gene transcription is differentially regulated by EWS/ERG sarcoma fusion protein and wild-type ERG.

A specific t(21;22) chromosomal translocation creates the chimeric EWS/ERG gene in some cases of Ewing's sarcoma. In the resultant EWS/ERG fusion protein, the N-terminal part of the ETS family protein ERG is replaced by the N terminus of the RNA-binding protein EWS. We found that both the EWS/ERG and COL11A2 genes are expressed in the Ewing's sarcoma cell line, CADO-ES1. To investigate a potential role for EWS/ERG in COL11A2 gene expression, we characterized the COL11A2 promoter and tested the ability of wild-type ERG and EWS/ERG sarcoma fusion protein to transactivate COL11A2 promoter using a luciferase assay. We found that expression of EWS/ERG, but not wild-type ERG, transactivated the COL11A2 promoter and that this transactivation required not only the N-terminal region of EWS but also an intact DNA-binding domain from ERG. Electrophoretic mobility shift assay using COL11A2 promoter sequence showed involvement of EWS/ERG in the formation of DNA-protein complexes, and chromatin immunoprecipitation assay revealed direct interaction between COL11A2 promoter and EWS/ERG fusion protein in vivo. EWS/ERG, but not wild-type ERG, bound to RNA polymerase II. Treatment of cells with the histone deacetylase inhibitor trichostatin A enabled ERG to transactivate the COL11A2 promoter, therefore abolishing the differential effects of EWS/ERG and ERG. Taken together, these findings indicate that the COL11A2 gene is regulated both by potential ERG association with a histone deacetylase complex and by direct EWS/ERG recruitment of RNA polymerase II.

An ERG (ets-related gene)-associated histone methyltransferase interacts with histone deacetylases 1/2 and transcription co-repressors mSin3A/B.

Covalent modifications of histone tails play important roles in gene transcription and silencing. We recently identified an ERG ( ets -related gene)-associated protein with a SET (suppressor of variegation, enhancer of zest and trithorax) domain (ESET) that was found to have the activity of a histone H3-specific methyltransferase. In the present study, we investigated the interaction of ESET with other chromatin remodelling factors. We show that ESET histone methyltransferase associates with histone deacetylase 1 (HDAC1) and HDAC2, and that ESET also interacts with the transcription co-repressors mSin3A and mSin3B. Deletion analysis of ESET reveals that an N-terminal region containing a tudor domain is responsible for interaction with mSin3A/B and association with HDAC1/2, and that truncation of ESET enhances its binding to mSin3. When bound to a promoter, ESET represses the transcription of a downstream luciferase reporter gene. This repression by ESET is independent of its histone methyltransferase activity, but correlates with its binding to the mSin3 co-repressors. In addition, the repression can be partially reversed by treatment with the HDAC inhibitor trichostatin A. Taken together, these data suggest that ESET histone methyltransferase can form a large, multi-protein complex(es) with mSin3A/B co-repressors and HDAC1/2 that participates in multiple pathways of transcriptional repression.

Characterization and expression of the human gene encoding two translocation liposarcoma protein-associated serine-arginine (TASR) proteins.

Translocation liposarcoma protein (TLS)-associated serine-arginine (TASR)-1 and -2 are two newly identified serine-arginine splicing factors. Our recent studies suggest that disruption of TASR-mediated pre-mRNA splicing is involved in the pathogenesis of human leukemia and sarcomas. The mRNA transcripts for TASR-1 and -2 share an identical sequence at the 5' untranslated region (5' UTR) and in part of the coding region; however the other regions of the transcripts diverge from each other and it was not clear whether the differences resulted from alternative splicing or transcription from two distinct genes. Here we describe the assignment of both TASR cDNAs to the same 16 kb DNA segment located on chromosome 1. Despite the presence of at least three retroposed products of TASR-1 mRNA in the human genome, only the 16 kb structural TASR gene on chromosome 1 is actively transcribed. In addition, multiple polyadenylation sites and a rare U12-type intron were found within the TASR gene. Transcription initiation site of the TASR gene was determined by primer extension; analysis of the TASR promoter revealed that it lacks the TATA box but contains a GC-rich sequence. When cloned into a luciferase reporter and transfected into human cells, the TASR promoter construct generated luciferase activity that was at least 2000 fold greater than the promoterless plasmid. Northern blot analysis showed that at least five different TASR-1 and -2 transcripts are expressed in a broad range of human tissues.