Biomedical subjects

Michael L Cleary

Publications and source records attributed to Michael L Cleary.

32 records · Page 2Linked to original sources

PubMed2003

Pbx1 is essential for adrenal development and urogenital differentiation.

Pbx1 encodes a TALE (three amino acid loop extension) class homeodomain protein that participates in multimeric transcriptional complexes to regulate developmental gene expression. Previous studies demonstrate a critical role for Pbx1 as a developmental regulator whose absence results in embryonic lethality and multiple tissue and organ system abnormalities. Here we report a requirement for Pbx1 in the differentiation of urogenital organs, where Pbx1 is widely expressed in mesenchymal tissues. The complete lack of adrenal glands and formation of gonads displaying rudimentary sexual differentiation correlated with decreased cellular proliferation in Pbx1(-/-) genital ridges. Furthermore, expression of steroidogenic factor-1 (SF-1), a nuclear receptor essential for adrenal organogenesis, was reduced to minimal levels in Pbx1 mutants, indicating an upstream function for Pbx1 in adrenocortical development. Finally, loss of Pbx1 markedly reduces urogenital ridge outgrowth and results in impaired differentiation of the mesonephros and kidneys and the absence of Müllerian ducts. These findings establish a Pbx1-dependent pathway that regulates the expansion of SF-1 positive cells essential for adrenal formation and gonadal differentiation and demonstrate an early requirement for Pbx1 in urogenital development.

Adrenal Glands↗

PubMed2003

Bmi-1 regulation of INK4A-ARF is a downstream requirement for transformation of hematopoietic progenitors by E2a-Pbx1.

Loss-of-function alterations of INK4A are commonly observed in lymphoid malignancies, but are consistently absent in pre-B cell leukemias induced by the chimeric oncoprotein E2a-Pbx1 created by t(1;19) chromosomal translocations. We report here that experimental induction of E2a-Pbx1 enhances expression of BMI-1, a lymphoid oncogene whose product functions as a transcriptional repressor of the INK4A-ARF tumor suppressor locus. Bmi-1-deficient hematopoietic progenitors are resistant to transformation by E2a-Pbx1; however, the requirement for Bmi-1 is alleviated in cells deficient for both Bmi-1 and INK4A-ARF. Furthermore, the adverse effects of E2a-Pbx1 on pre-B cell survival and differentiation are partially bypassed by forced expression of p16(Ink4a). These results link E2a-Pbx1 with Bmi-1 on an oncogenic pathway that is likely to play a role in the pathogenesis of human lymphoid leukemias through downregulation of the INK4A-ARF gene.

Apoptosis↗

PubMed2003

MLL-GAS7 transforms multipotent hematopoietic progenitors and induces mixed lineage leukemias in mice.

A specific association with mixed lineage leukemias suggests that MLL oncoproteins may selectively target early multipotent hematopoietic progenitors or stem cells. We demonstrate here that a representative MLL fusion protein, MLL-GAS7, impairs the differentiation and enhances the in vitro growth of murine hematopoietic cells with multipotent features. The multilineage differentiation potential of these cells was suggested by their immuno-phenotypes and transcriptional programs and confirmed by their ability to induce three pathologically distinct leukemias in mice, including an acute biphenotypic leukemia (ABL) that recapitulates the distinctive hallmark features of many MLL-associated leukemias in humans. This experimental modeling of ABL in mice highlights its origin from multipotential progenitors that arrest at a bipotential stage specifically targeted or induced by MLL oncogenes.

Animals↗

PubMed2003

MOZ-TIF2-induced acute myeloid leukemia requires the MOZ nucleosome binding motif and TIF2-mediated recruitment of CBP.

The MOZ-TIF2 fusion is associated with acute myeloid leukemia (AML) with inv(8)(p11q13). MOZ is a MYST family histone acetyltransferase (HAT), whereas TIF2 is a nuclear receptor coactivator that associates with CREB binding protein (CBP). Here we demonstrate that MOZ-TIF2 has transforming properties in vitro and causes AML in a murine bone marrow transplant assay. The C2HC nucleosome recognition motif of MOZ is essential for transformation, whereas MOZ HAT activity is dispensable. However, MOZ-TIF2 interaction with CBP through the TIF2 CBP interaction domain (CID) is essential for transformation. These results indicate that nucleosomal targeting by MOZ and recruitment of CBP by TIF2 are critical requirements for MOZ-TIF2 transformation and indicate that MOZ gain of function contributes to leukemogenesis.

Acetyltransferases↗

PubMed2003

Dimerization contributes to oncogenic activation of MLL chimeras in acute leukemias.

MLL is a histone methyltransferase that can be converted into an oncoprotein by acquisition of transcriptional effector domains following heterologous protein fusions with a variety of nuclear transcription factors, cofactors, or chromatin remodeling proteins in acute leukemias. Here we demonstrate an alternative mechanism for activation of MLL following fusions with proteins (AF1p/Eps15 and GAS7) that normally reside in the cytoplasm. The coiled-coil oligomerization domains of these proteins are necessary and sufficient for leukemogenic transformation induced by the respective MLL fusion proteins. Furthermore, homodimerization of MLL by synthetic dimerization modules mimics bona fide MLL fusion proteins resulting in Hox gene activation and enhanced self-renewal of hematopoietic progenitors. Our studies support an oligomerization-dependent mechanism for oncogenic conversion of MLL, presumably in part by recruitment of accessory factors through the dimerized MLL moiety of the chimeric protein.

Acute Disease↗

PubMed2003

Novel SWI/SNF chromatin-remodeling complexes contain a mixed-lineage leukemia chromosomal translocation partner.

The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin. Several complexes of this family have been reported for humans. In this study, two additional complexes are described that belong to the same SWI/SNF family. These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity. But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes. This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3. Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia. The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL. Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.

Amino Acid Sequence↗

PubMed2002

Common mechanism for oncogenic activation of MLL by forkhead family proteins.

The mixed lineage leukemia (MLL) gene undergoes fusions with a diverse set of genes as a consequence of chromosomal translocations in acute leukemias. Two of these partner genes code for members of the forkhead subfamily of transcription factors designated FKHRL1 and AFX. We demonstrate here that MLL-FKHRL1 enhances the self-renewal of murine myeloid progenitors in vitro and induces acute myeloid leukemias in syngeneic mice. The long latency (mean = 157 days), reduced penetrance, and hematologic features of the leukemias were very similar to those observed for the forkhead fusion protein MLL-AFX and contrasted with the more aggressive features of leukemias induced by MLL-AF10. Transformation mediated by MLL-forkhead fusion proteins required 2 conserved transcriptional effector domains (CR2 and CR3), each of which alone was not sufficient to activate MLL. A synthetic fusion of MLL with FKHR, a third mammalian forkhead family member that contains both effector domains, was also capable of transforming hematopoietic progenitors in vitro. A comparable requirement for 2 distinct transcriptional effector domains was also displayed by VP16, which required its proximal minimal transactivation domain (MTD/H1) and distal H2 domain to activate the oncogenic potential of MLL. The functional importance of CR2 was further demonstrated by its ability to substitute for H2 of VP16 in domain-swapping experiments to confer oncogenic activity on MLL. Our results, based on bona fide transcription factors as partners for MLL, unequivocally establish a transcriptional effector mechanism to activate its oncogenic potential and further support a role for fusion partners in determining pathologic features of the leukemia phenotype.

Animals↗

PubMed2002

Leukemia proto-oncoprotein MLL is proteolytically processed into 2 fragments with opposite transcriptional properties.

MLL (mixed lineage leukemia; also ALL-1 or HRX) is a proto-oncogene that is mutated in a variety of acute leukemias. Its product is normally required for the maintenance of Hox gene expression during embryogenesis and hematopoiesis through molecular mechanisms that remain poorly defined. Here we demonstrate that MLL (mixed lineage leukemia) is proteolytically processed into 2 fragments (MLL(N) and MLL(C)) that display opposite transcriptional properties and form an intramolecular MLL complex in vivo. Proteolytic cleavage occurs at 2 amino acids (D2666 and D2718) within a consensus processing sequence (QXD/GZDD, where X is a hydrophobic amino acid and Z is an alanine or a valine) that is conserved in TRX, the Drosophila homolog of MLL, and in the MLL-related protein MLL2, suggesting that processing is important for MLL function. Processed MLL(N) and MLL(C) associate with each other via N-terminal (1253-2254 amino acids) and C-terminal (3602-3742 amino acids) intramolecular interaction domains. MLL processing occurs rapidly within a few hours after translation and is followed by the phosphorylation of MLL(C). MLL(N) displays transcriptional repression activity, whereas MLL(C) has strong transcriptional activation properties. Leukemia-associated MLL fusion proteins lack the MLL processing sites, do not undergo cleavage, and are unable to interact with MLL(C). These observations suggest that posttranslational modifications of MLL may participate in regulating its activity as a transcription factor and that this aspect of its function is perturbed by leukemogenic fusions.

Cloning, Molecular↗

PubMed2002

The AF10 leucine zipper is required for leukemic transformation of myeloid progenitors by MLL-AF10.

The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of the MLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fusion partners of unknown function. In this report, we demonstrate that retroviral-mediated transduction of an MLL-AF10 complementary DNA into primary murine myeloid progenitors enhanced their clonogenic potential in serial replating assays and led to their efficient immortalization at a primitive stage of myeloid differentiation. Furthermore, MLL-AF10-transduced cells rapidly induced acute myeloid leukemia in syngeneic or severe combined immunodeficiency recipient mice. Structure/function analysis showed that a highly conserved 82-amino acid portion of AF10, comprising 2 adjacent alpha-helical domains, was sufficient for immortalizing activity when fused to MLL. Neither helical domain alone mediated immortalization, and deletion of the 29-amino acid leucine zipper within this region completely abrogated transforming activity. Similarly, the minimal oncogenic domain of AF10 exhibited transcriptional activation properties when fused to the MLL or GAL4 DNA-binding domains, while neither helical domain alone did. However, transcriptional activation per se was not sufficient because a second activation domain of AF10 was neither required nor competent for transformation. The requirement for alpha-helical transcriptional effector domains is similar to the oncogenic contributions of unrelated MLL partners ENL and ELL, suggesting a general mechanism of myeloid leukemogenesis by a subset of MLL fusion proteins, possibly through specific recruitment of the transcriptional machinery.

Amino Acid Motifs↗

PubMed2002

Pbx1 inactivation disrupts pancreas development and in Ipf1-deficient mice promotes diabetes mellitus.

Pbx1 is a member of the TALE (three-amino acid loop extension) class of homeodomain transcription factors, which are components of hetero-oligomeric protein complexes thought to regulate developmental gene expression and to maintain differentiated cell states. In vitro studies have shown that Pbx1 regulates the activity of Ipf1 (also known as Pdx1), a ParaHox homeodomain transcription factor required for the development and function of the pancreas in mice and humans. To investigate in vivo roles of Pbx1 in pancreatic development and function, we examined pancreatic Pbx1 expression, and morphogenesis, cell differentiation and function in mice deficient for Pbx1. Pbx1-/- embryos had pancreatic hypoplasia and marked defects in exocrine and endocrine cell differentiation prior to death at embryonic day (E) 15 or E16. In these embryos, expression of Isl1 and Atoh5, essential regulators of pancreatic morphogenesis and differentiation, was severely reduced. Pbx1+/- adults had pancreatic islet malformations, impaired glucose tolerance and hypoinsulinemia. Thus, Pbx1 is essential for normal pancreatic development and function. Analysis of trans-heterozygous Pbx1+/- Ipf1+/- mice revealed in vivo genetic interactions between Pbx1 and Ipf1 that are essential for postnatal pancreatic function; these mice developed age-dependent overt diabetes mellitus, unlike Pbx1+/- or Ipf1+/- mice. Mutations affecting the Ipf1 protein may promote diabetes mellitus in mice and humans. This study suggests that perturbation of Pbx1 activity may also promote susceptibility to diabetes mellitus.

Animals↗

PubMed2002

MLL-AFX requires the transcriptional effector domains of AFX to transform myeloid progenitors and transdominantly interfere with forkhead protein function.

MLL-AFX is a fusion gene created by t(X;11) chromosomal translocations in a subset of acute leukemias of either myeloid or lymphoid derivation. It codes for a chimeric protein consisting of MLL fused to AFX, a forkhead transcription factor that normally regulates genes involved in apoptosis and cell cycle progression. We demonstrate here that forced expression of MLL-AFX enhances the self-renewal of hematopoietic progenitors in vitro and induces acute myeloid leukemias after long latencies in syngeneic recipient mice. MLL-AFX interacts with the transcriptional coactivator CBP, which is also a fusion partner for MLL in human leukemias. A potent minimal transactivation domain (CR3) at the C terminus of AFX mediates interactions with the KIX domain of CBP and is necessary for transformation of myeloid progenitors by MLL-AFX. However, CR3 alone is not sufficient, suggesting that simple acquisition of a transactivation domain per se does not activate the oncogenic potential of MLL. Rather, two conserved transcriptional effector domains (CR2 and CR3) of AFX are required for full oncogenicity of MLL-AFX and also endow it with the potential to competitively interfere with transcription and apoptosis mediated by wild-type forkhead proteins. Furthermore, a dominant-negative mutant of AFX containing CR2 and CR3 enhances the growth of myeloid progenitors in vitro, although considerably less effectively than does MLL-AFX. Taken together, these data suggest that recruitment of transcriptional cofactors utilized by forkhead proteins is a critical requirement for oncogenic action of MLL-AFX, which may impact both MLL- and forkhead-dependent transcriptional pathways.

3T3 Cells↗

PubMed2002

Transformation of bone marrow B-cell progenitors by E2a-Hlf requires coexpression of Bcl-2.

The chimeric transcription factor E2a-Hlf is an oncoprotein associated with a subset of acute lymphoblastic leukemias of early B-lineage derivation. We employed a retroviral transduction-transplantation approach to evaluate the oncogenic effects of E2a-Hlf on murine B-cell progenitors harvested from adult bone marrow. Expression of E2a-Hlf induced short-lived clusters of primary hematopoietic cells but no long-term growth on preformed bone marrow stromal cell layers comprised of the AC6.21 cell line. Coexpression with Bcl-2, however, resulted in the sustained self-renewal of early preB-I cells that required stromal and interleukin-7 (IL-7) support for growth in vitro. Immortalized cells were unable to induce leukemias after transplantation into nonirradiated syngeneic hosts, unlike the leukemic properties and cytokine independence of preB-I cells transformed by p190(Bcr-Abl) under identical in vitro conditions. However, bone marrow cells expressing E2a-Hlf in combination with Bcl-2, but not E2a-Hlf alone, induced leukemias in irradiated recipients with long latencies, demonstrating both a requirement for suppression of apoptosis and the need for further secondary mutations in leukemia pathogenesis. Coexpression of IL-7 substituted for Bcl-2 to induce the in vitro growth of pre-B cells expressing E2a-Hlf, but leukemic conversion required additional abrogation of undefined stromal requirements and was associated with alterations in the Arf/Mdm2/p53 pathway. Thus, E2a-Hlf enhances the self-renewal of bone marrow B-cell progenitors without inciting a p53 tumor surveillance response or abrogating stromal and cytokine requirements for growth, which are nevertheless abrogated during progression to a leukemogenic phenotype.

Animals↗

PubMed2002

Male infertility, impaired spermatogenesis, and azoospermia in mice deficient for the pseudophosphatase Sbf1.

Pseudophosphatases display extensive sequence similarities to phosphatases but harbor amino acid alterations in their active-site consensus motifs that render them catalytically inactive. A potential role in substrate trapping or docking has been proposed, but the specific requirements for pseudophosphatases during development and differentiation are unknown. We demonstrate here that Sbf1, a pseudophosphatase of the myotubularin family, is expressed at high levels in seminiferous tubules of the testis, specifically in Sertoli's cells, spermatogonia, and pachytene spermatocytes, but not in postmeiotic round spermatids. Mice that are nullizygous for Sbf1 exhibit male infertility characterized by azoospermia. The onset of the spermatogenic defect occurs in the first wave of spermatogenesis at 17 days after birth during the synchronized progression of pachytene spermatocytes to haploid spermatids. Vacuolation of the Sertoli's cells is the earliest observed phenotype and is followed by reduced formation of spermatids and eventual depletion of the germ cell compartment in older mice. The nullizygous phenotype in conjunction with high-level expression of Sbf1 in premeiotic germ cells and Sertoli's cells is consistent with a crucial role for Sbf1 in transition from diploid to haploid spermatocytes. These studies demonstrate an essential role for a pseudophosphatase and implicate signaling pathways regulated by myotubularin family proteins in spermatogenesis and germ cell differentiation.

Animals↗

PubMed2001

A trithorax-group complex purified from Saccharomyces cerevisiae is required for methylation of histone H3.

Histone methylation has emerged as an important mechanism for regulating the transcriptional accessibility of chromatin. Several methyltransferases have been shown to target histone amino-terminal tails and mark nucleosomes associated with either euchromatic or heterochromatic states. However, the biochemical machinery responsible for regulating histone methylation and integrating it with other cellular events has not been well characterized. We report here the purification, molecular identification, and genetic and biochemical characterization of the Set1 protein complex that is necessary for methylation of histone H3 at lysine residue 4 in Saccharomyces cerevisiae. The seven-member 363-kDa complex contains homologs of Drosophila melanogaster proteins Ash2 and Trithorax and Caenorhabditis elegans protein DPY-30, which are implicated in the maintenance of Hox gene expression and regulation of X chromosome dosage compensation, respectively. Mutations of Set1 protein comparable to those that disrupt developmental function of its Drosophila homolog Trithorax abrogate histone methylation in yeast. These studies suggest that epigenetic regulation of developmental and sex-specific gene expression are species-specific readouts for a common chromatin remodeling machinery associated mechanistically with histone methylation.

Animals↗