Mixed Lineage Leukemia Protein Research Articles

Molecular Basis of Menin-MLL Interaction: Implication for Targeted Therapies in MLL Leukemias.

Abstract 3775Poster Board III-711Chromosomal translocations that affect the proto-oncogene MLL (Mixed Lineage Leukemia) occur in aggressive human acute leukemias, both AML and ALL, affecting children and adults. The normal MLL protein plays a key role in regulation of HOX genes expression, which are required for proper hematopoiesis. This function is frequently impaired by a fusion of MLL with one of 60 alternative partner genes to form a chimeric oncogene encoding MLL fusion proteins. MLL fusions upregulate HOX genes expression resulting in a blockage of blood cell differentiation that ultimately leads to acute leukemia. Patients with MLL rearrangement poorly respond to available treatments, emphasizing the urgent need to develop novel therapies to treat these leukemias. The leukemogenic activity of MLL fusions is dependent on association with menin, a protein encoded by the MEN1 (Multiple Endocrine Neoplasia I) gene. The menin binding motif is localized at the N-terminus of MLL and therefore it is retained in all MLL fusion proteins. The removal of this motif from MLL oncoproteins abrogates the ability to develop leukemia in mice. Menin functions as an essential oncogenic cofactor in MLL related leukemias and selective targeting of the menin-MLL interaction might represent a novel valuable therapeutic approach for the treatment of the MLL-related leukemias. To understand the molecular basis of how MLL-fusion proteins interact with menin, we carried out detailed in vitro characterization of menin binding to N-terminus of MLL using a collection of biochemical, biophysical and structural biology approaches. We demonstrated that 46 long N-terminal amino acid fragment of MLL very strongly associates with menin with low-nanomolar binding affinity. Employing the NMR spectroscopy, we identified the presence of two separate menin binding motifs within this MLL fragment, MBM1 (menin binding motif 1) and MBM2 (menin binding motif 2), which are separated by a poly-glycine linker. Peptides corresponding to both motifs are capable to independently interact with menin indicating the presence of two separate MLL binding sites on menin. Furthermore, the MBM1 binds to menin with 20-fold higher affinity compared to MBM2. Interestingly, we demonstrated that binding of one of the MBM peptides to menin negatively regulates binding of the second peptide most likely through the mechanism of an allosteric regulation. To aid in rational design of small molecule inhibitors of the menin-MLL interaction we characterized the conformation of the high affinity motif (MBM1) of MLL in a menin bound conformation using NMR spectroscopy. Furthermore, by applying both mutational studies and binding affinity measurements we identified that the most critical amino acids of MBM1 involved in interaction with menin comprise the RFPARP fragment of MLL. Overall, for the first time, we are providing detailed characterization and molecular basis of the MLL interaction with menin, which will be invaluable for development of therapeutically useful inhibitors selectively targeting this interaction. Disclosures:No relevant conflicts of interest to declare.

In vitro hematopoietic differentiation of mouse embryonic stem cells requires the tumor suppressor menin and is mediated by Hoxa9

Inactivating mutations in the tumor suppressor gene MEN1 cause the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1). The ubiquitously expressed MEN1 encoded protein, menin, interacts with MLL (mixed-lineage leukemia protein), and together they are essential components of a multiprotein complex with histone methyl transferase activity. MLL is also essential for hematopoiesis, and plays a critical role in leukemogenesis via epigenetic regulation of Hoxa9 expression that also requires menin. Therefore we chose to explore the role of menin in hematopoiesis. We generated Men1 −/− embryonic stem (ES) cell lines, and induced them to differentiate in vitro. While these cells were able to form embryoid bodies (EBs) expressing the early markers Flk-1 and c-Kit, their ability to further differentiate into hematopoietic colonies was compromised. The Men1 −/− ES cells show reduced expression of Hoxa9 that can be recovered by reexpression of Menin. We demonstrate that the block in differentiation of Men1 −/− ES cell lines can be rescued not only by the expression of menin but also that of Hoxa9. These results suggest that, similar to MLL, menin is required for hematopoiesis, and this requirement may be mediated through regulation of Hoxa9 expression.

Transcriptional Activation of the Mixed Lineage Leukemia–p27Kip1 Pathway by a Somatostatin Analogue

Mixed lineage leukemia (MLL) is a histone methyltransferase that activates gene transcription and associates with menin. In multiple endocrine neoplasia type 1 (Men1), a mutation of menin caused decreased expression of the p27(Kip1) and p18(Ink4C) genes and deregulated cell growth. We hypothesized that the same pathway might be involved in sporadic pituitary adenomas. mRNA levels for MLL, Men1, p27(Kip1), and p18(Ink4C) were measured in specimens of several sporadic pituitary adenomas, and a search for clinical parameters revealed that octreotide treatment affected the level of expression of some genes tested. To study molecular mechanisms, we cloned and characterized the MLL promoter region and used small interfering RNA for MLL and specific inhibitors for signal transduction pathways. A strong correlation between MLL and p27(Kip1) mRNA levels was observed in prolactinomas and growth hormone-secreting adenomas, and these levels were attenuated except in growth hormone-secreting adenomas treated with a somatostatin analogue, octreotide. Conversely, the patients treated with octreotide showed high levels of MLL-p27(Kip1) mRNA. Experiments in vitro showed that octreotide increased MLL and p27(Kip1) protein and mRNA levels, and overexpression of MLL induced a marked increase in p27(Kip1)promoter activity. Furthermore, octreotide stimulated the promoter activity of the MLL gene through phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. In addition, incubation with an inhibitor for methyltransferase, MTA, and knockdown of MLL completely inhibited the octreotide-induced expression of p27(Kip1). The MLL-p27(Kip1) pathway was down-regulated in the pituitary adenomas, and octreotide increased the p27(Kip1) level, at least in part, by sequential transcriptional stimulation of the MLL and p27(Kip1) genes through phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways.

Menin regulates the function of hematopoietic stem cells and lymphoid progenitors

AbstractMen1 is a tumor suppressor gene mutated in endocrine neoplasms. Besides its endocrine role, the Men1 gene product menin interacts with the mixed lineage leukemia (MLL) protein, a histone H3 lysine 4 methyltransferase. Although menin and MLL fusion proteins cooperate to activate Homeobox (Hox) gene expression during transformation, little is known about the normal hematopoietic functions of menin. Here, we studied hematopoiesis after Men1 ablation. Menin loss modestly impaired blood neutrophil, lymphocyte, and platelet counts. Without hematopoietic stress, multilineage and myelo-erythroid bone marrow progenitor numbers were preserved, while B lymphoid progenitors were decreased. In contrast, competitive transplantation revealed a marked functional defect of long-term hematopoietic stem cells (HSC) in the absence of menin, despite normal initial homing of progenitors to the bone marrow. HoxA9 gene expression was only modestly decreased in menin-deficient HSCs. These observations reveal a novel and essential role for menin in HSC homeostasis that was most apparent during situations of hematopoietic recovery, suggesting that menin regulates molecular pathways that are essential during the adaptive HSC response to stress.

Menin regulates the function of hematopoietic stem cells and lymphoid progenitors

Men1 is a tumor suppressor gene mutated in endocrine neoplasms. Besides its endocrine role, the Men1 gene product menin interacts with the mixed lineage leukemia (MLL) protein, a histone H3 lysine 4 methyltransferase. Although menin and MLL fusion proteins cooperate to activate Homeobox (Hox) gene expression during transformation, little is known about the normal hematopoietic functions of menin. Here, we studied hematopoiesis after Men1 ablation. Menin loss modestly impaired blood neutrophil, lymphocyte, and platelet counts. Without hematopoietic stress, multilineage and myelo-erythroid bone marrow progenitor numbers were preserved, while B lymphoid progenitors were decreased. In contrast, competitive transplantation revealed a marked functional defect of long-term hematopoietic stem cells (HSC) in the absence of menin, despite normal initial homing of progenitors to the bone marrow. HoxA9 gene expression was only modestly decreased in menin-deficient HSCs. These observations reveal a novel and essential role for menin in HSC homeostasis that was most apparent during situations of hematopoietic recovery, suggesting that menin regulates molecular pathways that are essential during the adaptive HSC response to stress.

Structural Basis for the Requirement of Additional Factors for MLL1 SET Domain Activity and Recognition of Epigenetic Marks

The mixed-lineage leukemia protein MLL1 is a transcriptional regulator with an essential role in early development and hematopoiesis. The biological function of MLL1 is mediated by the histone H3K4 methyltransferase activity of the carboxyl-terminal SET domain. We have determined the crystal structure of the MLL1 SET domain in complex with cofactor product AdoHcy and a histone H3 peptide. This structure indicates that, in order to form a well-ordered active site, a highly variable but essential component of the SET domain must be repositioned. To test this idea, we compared the effect of the addition of MLL complex members on methyltransferase activity and show that both RbBP5 and Ash2L but not Wdr5 stimulate activity. Additionally, we have determined the effect of posttranslational modifications on histone H3 residues downstream and upstream from the target lysine and provide a structural explanation for why H3T3 phosphorylation and H3K9 acetylation regulate activity.

Menin, Histone H3 Methyltransferases, and Regulation of Cell Proliferation: Current Knowledge and Perspective

Menin is a tumor suppressor encoded by the MEN1 gene that is mutated in patients with an inherited syndrome, multiple endocrine neoplasia type 1 (MEN1). Loss of menin has potent impact on proliferation of endocrine and non-endocrine cells. However, until recently little has been known as to how menin regulates cell proliferation. Rapid research progress in the past several years suggests that menin represses proliferation of endocrine cells yet promotes proliferation in certain types of leukemia cells via interacting with various transcriptional regulators. Menin interacts with histone H3 methyltransferases such as MLL (mixed lineage leukemia) protein. Increasing evidence has linked the biological function of menin to epigenetic histone modifications, control of the pattern of gene expression, and regulation of cell proliferation in a cell type-specific manner. In light of these recent findings, an emerging model suggests that menin is a crucial regulator of histone modifiers by acting as a scaffold protein to coordinate gene transcription and cell proliferation in a cell context-dependent manner. This recent progress unravels the coordinating role of menin in epigenetics and regulation of cell cycle, providing novel insights into understanding regulation of beta cell functions and diabetes, as well as the development and therapy of endocrine tumors and leukemia.

Previously Unknown Interactions Between AML1 and MLL Provide Epigenetic Regulation of Gene Expression in Normal Hematopoiesis and in Leukemia

In all organisms, the fundamental process of transcriptional regulation requires transcription factors, which bind to DNA in response to extra-cellular signals and regulate transcription of target genes. In eukaryotes, this process also involves epigenetic regulation, which includes DNA and histone modifications. Hematopoiesis and leukemia are excellent model systems for studying the higher eukaryotic regulations of gene expression and for identifying important molecules involved in genetic and epigenetic transcriptional regulation. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcriptional factor AML1/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. We have previously shown that the regulation of PU.1 by AML1 is mediated by 3 AML1 binding sites in the PU.1 upstream regulatory element (URE), located −14 kb relative to the transcription start site in mice (Huang et al. Nat Genet. 2008). To understand whether AML1 plays a critical role in regulating the PU.1 locus at the chromatin level, we have utilized this PU.1 regulation system as a model to study the potential interplay between AML1/CBFβ and MLL. We found that MLL binds to the evolutionarily conserved Runt-domain of AML1, enhances the formation of the AML1/CBFβ heterodimer, and blocks the ubiquitin-proteasome mediated degradation of AML1. We also found that AML1/CBFβ is required for maintenance of the H3K4-me3 histone mark at the PU.1 upstream regulatory element (URE) and promoter region, and that MLL is required for AML1 to regulate PU.1 expression. In contrast, we found that several leukemia-associated MLL fusion proteins, including MLL-AF4, MLL-AF9, MLL-ENL, and MLL-AF10, no longer stabilize AML1 but rather induce AML1 degradation and downregulate PU.1 expression. Taken together, our data indicate that MLL and some of its leukemia-associated fusion proteins physically and functionally interact with AML1. Their effects on target genes, particularly PU.1, may be critical for the normal regulation of hematopoiesis and for the aberrant regulation that underlies acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL).

Role of Jade-1 in the Histone Acetyltransferase (HAT) HBO1 Complex

Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

Menin Critically Links MLL Proteins with LEDGF on Cancer-Associated Target Genes

Menin displays the unique ability to either promote oncogenic function in the hematopoietic lineage or suppress tumorigenesis in the endocrine lineage; however, its molecular mechanism of action has not been defined. We demonstrate here that these discordant functions are unified by menin's ability to serve as a molecular adaptor that physically links the MLL (mixed-lineage leukemia) histone methyltransferase with LEDGF (lens epithelium-derived growth factor), a chromatin-associated protein previously implicated in leukemia, autoimmunity, and HIV-1 pathogenesis. LEDGF is required for both MLL-dependent transcription and leukemic transformation. Conversely, a subset of menin mutations in multiple endocrine neoplasia type 1 patients abrogate interaction with LEDGF while preserving MLL interaction but nevertheless compromise MLL/menin-dependent functions. Thus, LEDGF critically associates with MLL and menin at the nexus of transcriptional pathways that are recurrently targeted in diverse diseases.

The MLL SET domain is a histone H3 lysine 4 mono‐methyltransferase that interacts with WDR5 in the pre‐SET ATA2 domain.

Di‐ and Trimethylation of histone H3 Lysine 4 (H3K4) are associated with transcriptional activation, and are deposited by a family of proteins sharing a conserved SET domain. One of the chief regulators of H3K4 methylation is the Mixed Lineage Leukemia protein (MLL), which is required for the regulation of Hox genes in hematopoiesis and development. MLL interacts with WDR5, RbBP5, and Ash2L to form a core complex of proteins that are required for the regulation of MLL’s H3K4 methylation activity. It has been suggested that WDR5 plays a central role in maintaining the structural integrity of the MLL core complex. However, it is not currently understood how WDR5 interacts with MLL and how it regulates MLL’S catalytic properties. Here we report on the hydrodynamic properties of WDR5 and an MLL SET domain construct consisting of residues 3745‐3969 (MLL3745). We show that MLL3745 forms a 1:1 hetero‐dimer with WDR5 with a dissociation constant (Kd) of 0.26 ± 0.02 ÂμM. Furthermore, this interaction requires conserved residues in the pre‐SET ATA2 region of MLL3745. We also show that the recombinant MLL SET domain is a mono‐methyltransferase, and that interaction with WDR5 does not change MLL’S product specificity, but does enhance MLL’S thermostability. These results reveal a new function for the pre‐Set ATA2 domain of MLL in that it is important for the recruitment of proteins that regulate the catalytic activity of the SET domain.

Mechanisms of transcriptional regulation by MLL and its disruption in acute leukemia

Fusion of the mixed lineage leukemia protein (MLL) to one of over 50 different translocation partners converts it into a potent leukemogenic oncoprotein. The resulting fusion proteins transform primarily through upregulation of A-cluster Hox genes, including Hoxa9 and the Hox cofactor Meis1. Considerable progress has been made in delineating the differences between normal Hox gene regulation by MLL and deregulated transcription in MLL-induced leukemias. Some MLL translocation partners dimerize the truncated MLL molecule. Other translocation partners appear to recruit nuclear coactivator complexes that have diverse enzymatic activities that impinge on transcriptional elongation pathways. These enzymatic activities, including RNA polymerase II phosphorylation as well as histone H3 lysine 79 methylation present attractive targets for the development of future MLL-directed therapy.

Bimodal Degradation of MLL Protein by the Cell Cycle SCFSkp2 and APCCdc20 E3 Lilgases Assures Cell Cycle Execution: A Critical Regulatory Circuit Lost in Leukemogenic MLL-Fusions.

The MLL (mixed lineage leukemia) gene encodes a highly conserved 3,969 aa histone H3 K4 methyl transferase, of which chromosome translocations result in poor prognostic infant and therapy-related leukemias. Mysteriously, the pathognomonic MLL leukemia fusions consist of a common amino(N)-terminal ∼1400 aa of MLL fused in frame with more than 60 different partners with no shared characteristics. The most well known genetic function of MLL is to antagonize polycomb group of proteins for proper Hox gene expression. Consequently deregulated Hox genes caused by MLL translocations contribute to the MLL leukemogenesis. Besides Hox genes, it remains largely undetermined whether MLL participates in other biological processes. The 500kD MLL precursor undergoes evolutionarily conserved proteolytic maturation mediated by Taspase1 (Hsieh et al. 2003, MCB, 23, 186–194; Hsieh et al. 2003, Cell, 115, 293–303). Our recent studies on Taspase1 knockout cells established an MLL-E2F axis in orchestrating core cell cycle gene expression including Cyclins and possibly Cdk inhibitors (Takeda et al. 2006, Genes & Development , 20, 2397–2409). As MLL actively participates in the cell cycle regulation, we investigated the regulation of MLL through cell cycle transition. We uncovered a unique biphasic expression of MLL conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases. Specifically, SCFSkp2 and APCCdc20 mark MLL for degradation at S phase and late M phase, respectively. Abolished peak expression of MLL incurs corresponding defects in G1/S transition and M phase progression. Conversely, over-expression of MLL blocks S phase progression. Remarkably, MLL degradation initiates at its N-terminal ∼1400 aa that is retained in all MLL leukemia fusions. We examined prevalent MLL-fusions, including MLL-AF4, MLL-AF9, MLL-ELL and MLL-ELL, and observed their increased resistance to degradation. Furthermore, the same resistance was observed with the leukemogenic MLL-lacZ but not the non-leukemogenic MLL-Myc tag fusion. Thus, non-oscillating expression of MLL-fusions through the cell cycle, resulted from impaired degradation, likely constitutes the universal mechanism underlying all MLL leukemias. Our data conclude an essential post-translational regulation of MLL by the cell cycle ubiquitin/proteasome system (UPS) to assure the temporal necessity of MLL in coordinating cell cycle progression. Future studies aim at providing a comprehensive analysis on the cell cycle consequences associated with MLL-fusions using genetically modified cells derived from mice carrying various MLL-fusion knockin alleles, including MLL-AF4, MLL-AF9, and MLL-CBP.

Species Selectivity of Mixed-Lineage Leukemia/Trithorax and HCF Proteolytic Maturation Pathways

Site-specific proteolytic processing plays important roles in the regulation of cellular activities. The histone modification activity of the human trithorax group mixed-lineage leukemia (MLL) protein and the cell cycle regulatory activity of the cell proliferation factor herpes simplex virus host cell factor 1 (HCF-1) are stimulated by cleavage of precursors that generates stable heterodimeric complexes. MLL is processed by a protease called taspase 1, whereas the precise mechanisms of HCF-1 maturation are unclear, although they are known to depend on a series of sequence repeats called HCF-1(PRO) repeats. We demonstrate here that the Drosophila homologs of MLL and HCF-1, called Trithorax and dHCF, are both cleaved by Drosophila taspase 1. Although highly related, the human and Drosophila taspase 1 proteins display cognate species specificity. Thus, human taspase 1 preferentially cleaves MLL and Drosophila taspase 1 preferentially cleaves Trithorax, consistent with coevolution of taspase 1 and MLL/Trithorax proteins. HCF proteins display even greater species-specific divergence in processing: whereas dHCF is cleaved by the Drosophila taspase 1, human and mouse HCF-1 maturation is taspase 1 independent. Instead, human and Xenopus HCF-1PRO repeats are cleaved in vitro by a human proteolytic activity with novel properties. Thus, from insects to humans, HCF proteins have conserved proteolytic maturation but evolved different mechanisms.

Molecular Characterization of HTLV-1 Tax Interaction with the KIX Domain of CBP/p300

The viral oncoprotein Tax mediates transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1). Both Tax and the cellular transcription factor CREB bind to viral cyclic AMP response elements (vCREs) located in the viral promoter. Tax and serine 133 phosphorylated CREB (pCREB) bound to the HTLV-1 promoter facilitate viral transcription via the recruitment of the large cellular coactivators CBP/p300. While the interaction between the phosphorylated kinase inducible domain (pKID) of pCREB and the KIX domain of CBP/p300 has been well characterized, the molecular interactions between KIX, full-length Tax, and pCREB have not been examined. Here we biochemically characterized the interaction between Tax and KIX in a physiologically relevant complex containing pCREB and vCRE DNA. Our data show that Tax and pCREB simultaneously and independently bind two distinct surfaces on the KIX domain: Tax binds KIX at the previously characterized mixed-lineage leukemia (MLL) protein interaction surface while pCREB binds KIX at the pKID–KIX interface. These results provide evidence for a model in which Tax and pCREB bind distinct surfaces of KIX for effective CBP/p300 recruitment to the HTLV-1 promoter. We also show that MLL competes with Tax for KIX binding, suggesting a novel mechanism of Tax oncogenesis in which normal MLL function is disrupted by Tax.

Uncleaved TFIIA Is a Substrate for Taspase 1 and Active in Transcription

In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIAalphabeta gene and posttranslationally cleaved into alpha and beta subunits. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1-/- MEF cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset of genes during differentiation and development.

Organizing Motility

See related article, pages 378–385 Vascular smooth muscle cells (SMCs) acquire a highly specialized cytoskeleton during development that is organized for efficient transmission of contractile force. This SMC cytoskeleton must be extensively reorganized to support directed cell migrations that are required for repair of arterial injury. Particularly important in this reorganization process are adaptor proteins that mediate the assembly of multiprotein complexes involved in cell adhesion, lamellipodial extensions, signal transduction, and transcriptional activation. One such adaptor protein is called lipoma-preferred partner (LPP), a member of the LIM domain–containing protein family, most closely related to zxyin, ajuba, LIM domain–containing protein-1 (LIMD1), and thyroid receptor-interacting protein-6 (TRIP6).1 Evidence to suggest that LPP is a smooth muscle–restricted LIM protein that plays an important role in SMC migration after arterial injury is reported by Gorenne et al in this issue of Circulation Research .2 LPP was discovered as a component of t(3:12) chromosome translocations found in benign tumors of human adipose tissue (lipomas).3 The chromosome 3 breakpoint occurred in a 400-kb genomic locus that encoded a protein with proline-rich sequences and leucine zipper motifs at its N terminus and three LIM domains at its C terminus. The t(3:12) translocation produced a fusion protein containing the N-terminal DNA binding domain of HMGIC fused to the C-terminal LIM domains of a protein encoded by the chromosome 3 locus. The latter was given the name LIM domain–containing lipoma-preferred partner (LPP).3 The lipoma-associated fusion protein is localized to the nucleus4 and can function as a transcription activator.5 Translocations involving the LIM domains of LPP are the most common chromosomal translocations found in human tumors. In addition to lipomas, they have been found in various benign and malignant tumors of mesenchymal origin, including leukemias (fused with the mixed lineage leukemia protein (MLL), …

The tumor suppressor menin regulates hematopoiesis and myeloid transformation by influencing Hox gene expression

Menin is the product of the tumor suppressor gene Men1 that is mutated in the inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1). Menin has been shown to interact with SET-1 domain-containing histone 3 lysine 4 (H3K4) methyltransferases including mixed lineage leukemia proteins to regulate homeobox (Hox) gene expression in vitro. Using conditional Men1 knockout mice, we have investigated the requirement for menin in hematopoiesis and myeloid transformation. Men1 excision causes reduction of Hoxa9 expression, colony formation by hematopoietic progenitors, and the peripheral white blood cell count. Menin directly activates Hoxa9 expression, at least in part, by binding to the Hoxa9 locus, facilitating methylation of H3K4, and recruiting the methylated H3K4 binding protein chd1 to the locus. Consistent with signaling downstream of menin, ectopic expression of both Hoxa9 and Meis1 rescues colony formation defects in Men1-excised bone marrow. Moreover, Men1 excision also suppresses proliferation of leukemogenic mixed lineage leukemia-AF9 fusion-protein-transformed myeloid cells and Hoxa9 expression. These studies uncover an important role for menin in both normal hematopoiesis and myeloid transformation and provide a mechanistic understanding of menin's function in these processes that may be used for therapy.

MLL Translocation Partners AF4, AF5q31, and ENL Associate in a Higher Order Protein Complex with CDK9 and Cyclin T1.

The Mixed Lineage Leukemia (MLL) gene is frequently involved in chromosomal translocations that cause acute leukemia. More than 40 different genes have been identified as MLL translocation partners, with the expression of corresponding MLL fusion proteins. The MLL protein has histone methyltransferase activity and is required for embryonic development and hematopoiesis. Several proteins have been demonstrated to associate with MLL in a macromolecular complex, which is believed to have chromatin remodeling function. However, the C-terminal SET domain of MLL, which carries the histone methyltransferase activity, is lost in all MLL fusion proteins, thus making the biochemical functions of the fusion proteins unclear. Moreover, the promiscuity of MLL translocation partners, most of them with no known functions, further complicates an understanding of MLL leukemogenic mechanisms. In this study, we purified a protein complex containing AF4, the most common MLL translocation partner, using a combination of conventional column chromatography and immunoaffinity techniques. The AF4 protein complex contains AF5q31 and ENL, two other MLL translocation partners, as well as CDK9 and Cyclin T1, a heterodimer that regulates transcriptional elongation. Gel filtration confirmed that these five proteins co-fractionate with an estimated overall size of 0.8 MDa. All protein-protein interactions were further confirmed by immunoprecipitation-western blotting from K562 cell nuclear extract. To investigate whether these protein-protein interactions are retained in corresponding MLL fusion proteins, immunoprecipitation-western blotting assays were carried out in human leukemia cell lines harboring MLL chromosomal translocations. We found that MLL-AF4, MLL-AF5q31, MLL-ENL and MLL-AF9 each associate with wild type AF4 complex components, including CDK9 and Cyclin T1. In contrast, MLL-AF6 does not associate with any of the AF4 complex components. We propose that the four nuclear MLL translocation partner proteins (AF4, AF5q31, ENL/AF9), whose translocations are found in over 75% of MLL leukemias, associate in a higher order protein complex with CDK9 and Cyclin T1 and thus function in part to regulate transcriptional elongation. The association of CDK9 and Cyclin T1 with the four MLL fusion proteins suggests a common leukemogenic mechanism that may involve transcriptional elongation, which we are currently investigating. Conversely, MLL-cytosolic fusions, e.g. MLL-AF6, appear to function independently of association with the AF4 protein complex, possibly through a homo-dimerization pathway.

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.