ABSTRACT
BACKGROUND: To gain insight into the pathogenesis of collapsing glomerulopathy, a rare form of FSGS that often arises in the setting of viral infections, we performed a genome-wide association study (GWAS) among inbred mouse strains using a murine model of HIV-1 associated nephropathy (HIVAN). METHODS: We first generated F1 hybrids between HIV-1 transgenic mice on the FVB/NJ background and 20 inbred laboratory strains. Analysis of histology, BUN, and urinary NGAL demonstrated marked phenotypic variation among the transgenic F1 hybrids, providing strong evidence for host genetic factors in the predisposition to nephropathy. A GWAS in 365 transgenic F1 hybrids generated from these 20 inbred strains was performed. RESULTS: We identified a genome-wide significant locus on chromosome 13-C3 and multiple additional suggestive loci. Crossannotation of the Chr. 13 locus, including single-cell transcriptomic analysis of wildtype and HIV-1 transgenic mouse kidneys, nominated Ssbp2 as the most likely candidate gene. Ssbp2 is highly expressed in podocytes, encodes a transcriptional cofactor that interacts with LDB1 and LMX1B, which are both previously implicated in FSGS. Consistent with these data, older Ssbp2 null mice spontaneously develop glomerulosclerosis, tubular casts, interstitial fibrosis, and inflammation, similar to the HIVAN mouse model. CONCLUSIONS: These findings demonstrate the utility of GWAS in mice to uncover host genetic factors for rare kidney traits and suggest Ssbp2 as susceptibility gene for HIVAN, potentially acting via the LDB1-LMX1B transcriptional network.
Subject(s)
AIDS-Associated Nephropathy/genetics , DNA-Binding Proteins/genetics , Genetic Loci/genetics , Genetic Predisposition to Disease/genetics , Glomerulosclerosis, Focal Segmental/genetics , Animals , Disease Models, Animal , Female , Genome-Wide Association Study , Male , Mice , Mice, TransgenicABSTRACT
Squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2) and 3 (SSBP3), in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease. First, we found that the relative abundance of LMO4, LDB1, and the two SSBPs correlated very significantly in a panel of human HNSCC cell lines. Second, expression of these proteins in tumor primaries and lymph nodes involved by metastasis were concordant in 3 of 3 sets of tissue. Third, using a Matrigel invasion and organotypic reconstruct assay, CRISPR/Cas9-mediated deletion of LDB1 in the VU-SCC-1729 cell line, which is highly invasive of basement membrane and cellular monolayers, reduced tumor cell invasiveness and migration, as well as proliferation on tissue culture plastic. Finally, inactivation of the LDB1 gene in these cells decreased growth and vascularization of xenografted human tumor cells in vivo. These data show that LMO4, LDB1, and SSBP2 and/or SSBP3 regulate metastasis, proliferation, and angiogenesis in HNSCC and provide the first evidence that SSBPs control LMO4 and LDB1 protein abundance in a cancer context.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carcinoma, Squamous Cell/pathology , DNA-Binding Proteins/metabolism , LIM Domain Proteins/metabolism , Mouth Neoplasms/pathology , Transcription Factors/metabolism , Animals , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Mice , Mouth Neoplasms/metabolism , Neoplasm Metastasis , Neoplasm Transplantation , Tissue Culture TechniquesABSTRACT
CD5 is a pan-T-cell surface marker and is rarely expressed in diffuse large B-cell lymphoma (DLBCL). Large-scale studies of de novo CD5+ DLBCL are lacking in Western countries. In this study by the DLBCL Rituximab-CHOP Consortium, CD5 was expressed in 5.5% of 879 DLBCL patients from Western countries. CD5+ DLBCL was associated with higher frequencies of >1 ECOG performance status, bone marrow involvement, central nervous system relapse, activated B-cell-like subtype, Bcl-2 overexpression, and STAT3 and NF-κB activation, whereas rarely expressed single-stranded DNA-binding protein 2 (SSBP2), CD30 or had MYC mutations. With standard R-CHOP chemotherapy, CD5+ DLBCL patients had significantly worse overall survival (median, 25.3 months vs. not reached, P< .0001) and progression-free survival (median, 21.3 vs. 85.8 months, P< .0001) than CD5- DLBCL patients, which was independent of Bcl-2, STAT3, NF-κB and the International Prognostic Index. Interestingly, SSBP2 expression abolished the prognostic significance of CD5 expression, suggesting a tumor-suppressor role of SSBP2 for CD5 signaling. Gene-expression profiling demonstrated that B-cell receptor signaling dysfunction and microenvironment alterations are the important mechanisms underlying the clinical impact of CD5 expression. This study shows the distinctive clinical and biological features of CD5+ DLBCL patients in Western countries and underscores important pathways with therapeutic implications.
Subject(s)
CD5 Antigens/biosynthesis , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , CD5 Antigens/genetics , Female , Gene Expression Profiling , Humans , Immunohistochemistry , Lymphoma, Large B-Cell, Diffuse/genetics , Male , Middle Aged , Prognosis , Tissue Array Analysis , Treatment OutcomeABSTRACT
Mesoderm Inducer in Xenopus Like1 (MIXL1), a paired-type homeobox transcription factor induced by TGF-ß family of ligands is required for early embryonic specification of mesoderm and endoderm. Retrovirally transduced Mixl1 is reported to induce acute myelogenous leukemia (AML) with a high penetrance. But the mechanistic underpinnings of MIXL1 mediated leukemogenesis are unknown. Here, we establish the protooncogene c-REL to be a transcriptional target of MIXL1 by genome wide chromatin immune precipitation. Accordingly, expression of c-REL and its downstream targets BCL2L1 and BCL2A2 are elevated in MIXL1 expressing cells. Notably, MIXL1 regulates c-REL through a zinc finger binding motif, potentially by a MIXL1-Zinc finger protein transcriptional complex. Furthermore, MIXL1 expression is detected in the cancer genome atlas (TCGA) AML samples in a pattern mutually exclusive from that of HOXA9, CDX2 and HLX suggesting the existence of a core, yet distinct HOX transcriptional program. Finally, we demonstrate MIXL1 to be induced by BMP4 and not TGF-ß in primary human hematopoietic stem and progenitor cells. Consequently, MIXL1 expressing AML cells are preferentially sensitive to the BMPR1 kinase inhibitor LDN-193189. These findings support the existence of a novel MIXL1-c REL mediated survival axis in AML that can be targeted by BMPR1 inhibitors. (MIXL1- human gene, Mixl1- mouse ortholog, MIXL1- protein).
Subject(s)
Bone Morphogenetic Protein 4/genetics , Homeodomain Proteins/genetics , Leukemia, Myeloid, Acute/genetics , Animals , Bone Morphogenetic Protein Receptors, Type I/antagonists & inhibitors , Cell Differentiation/physiology , Cell Line, Tumor , Genes, Homeobox , Genes, rel , HEK293 Cells , HL-60 Cells , Homeodomain Proteins/biosynthesis , Humans , K562 Cells , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Mice , Molecular Targeted Therapy , U937 CellsABSTRACT
Transcriptional mechanisms governing hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation are not fully understood. Sequence-specific ssDNA-binding protein 2 (SSBP2) is a candidate acute myelogenous leukemia (AML) suppressor gene located at chromosome 5q14. SSBP2 binds the transcriptional adaptor protein Lim domain-binding protein 1 (LDB1) and enhances LDB1 stability to regulate gene expression. Notably, Ldb1 is essential for HSC specification during early development and maintenance in adults. We previously reported shortened lifespan and greater susceptibility to B cell lymphomas and carcinomas in Ssbp2(-/-) mice. However, whether Ssbp2 plays a regulatory role in normal HSC function and leukemogenesis is unknown. In this study, we provide several lines of evidence to demonstrate a requirement for Ssbp2 in the function and transcriptional program of hematopoietic stem and progenitor cells (HSPCs) in vivo. We found that hematopoietic tissues were hypoplastic in Ssbp2(-/-) mice, and the frequency of lymphoid-primed multipotent progenitor cells in bone marrow was reduced. Other significant features of these mice were delayed recovery from 5-fluorouracil treatment and diminished multilineage reconstitution in lethally irradiated bone marrow recipients. Dramatic reduction of Notch1 transcripts and increased expression of transcripts encoding the transcription factor E2a and its downstream target Cdkn1a also distinguished Ssbp2(-/-) HSPCs from wild-type HSPCs. Finally, a tendency toward coordinated expression of SSBP2 and the AML suppressor NOTCH1 in a subset of the Cancer Genome Atlas AML cases suggested a role for SSBP2 in AML pathogenesis. Collectively, our results uncovered a critical regulatory function for SSBP2 in HSPC gene expression and function.
Subject(s)
Cell Differentiation , DNA-Binding Proteins/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Stress, Physiological , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Bone Marrow/metabolism , Bone Marrow/pathology , Bone Marrow Transplantation , Cell Differentiation/genetics , DNA-Binding Proteins/genetics , Gene Expression , Hematopoiesis/genetics , Homeostasis/genetics , Immunophenotyping , Mice , Mice, Knockout , Phenotype , Receptor, Notch1/genetics , Receptor, Notch1/metabolismABSTRACT
The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.
Subject(s)
Gene Expression , Muscle, Skeletal/metabolism , Muscular Dystrophies/genetics , Myotonic Disorders/genetics , Myotonic Dystrophy/genetics , RNA Splicing , Adult , Aged , Aged, 80 and over , Child , Exons , Female , Humans , Male , Middle Aged , Muscular Dystrophies/metabolism , Myotonic Disorders/metabolism , Myotonic Dystrophy/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Young AdultABSTRACT
The developmental activity of LIM homeodomain transcription factors (LIM-HDs) is critically controlled by LIM domain-interacting cofactors of LIM-HDs (CLIM, also known as NLI or LDB). CLIM cofactors associate with single-stranded DNA binding proteins (SSDPs, also known as SSBPs) thereby recruiting SSDP1 and/or SSDP2 to LIM-HD/CLIM complexes. Although evidence has been presented that SSDPs are important for the activity of specific LIM-HD/CLIM complexes, the developmental roles of SSDPs are unclear. We show that SSDP1a and SSDP1b mRNAs are widely expressed early during zebrafish development with conspicuous expression of SSDP1b in sensory trigeminal and Rohon-Beard neurons. SSDP1 and CLIM immunoreactivity co-localize in these neuronal cell types and in other structures. Over-expression of the N-terminal portion of SSDP1 (N-SSDP1), which contains the CLIM-interaction domain, increases endogenous CLIM protein levels in vivo and impairs the formation of eyes and midbrain-hindbrain boundary. In addition, manipulation of SSDP1 via N-SSDP1 over-expression or SSDP1b knock down impairs trigeminal and Rohon-Beard sensory axon growth. We show that N-SSDP1 is able to partially rescue the inhibition of axon growth induced by a dominant-negative form of CLIM (DN-CLIM). These results reveal specific functions of SSDP in neural patterning and sensory axon growth, in part due to the stabilization of LIM-HD/CLIM complexes.
Subject(s)
Axons/physiology , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Neurogenesis/physiology , Sensory Receptor Cells/physiology , Transcription Factors/metabolism , Zebrafish/embryology , Animals , Blotting, Western , Cell Differentiation/physiology , DNA Primers/genetics , DNA-Binding Proteins/genetics , Immunohistochemistry , In Situ Hybridization , Reverse Transcriptase Polymerase Chain Reaction , Sensory Receptor Cells/metabolismABSTRACT
Transient expression of adenoviral oncoprotein E1B55K in normal cells induces aggresome formation and sequestration of critical host proteins in aggresomes. Our previous studies reported that Sequence Specific Binding Protein 2 (SSBP2), a candidate tumor suppressor is recruited to aggresomes in adenovirally transformed human embryonal kidney 293 (HEK293) cells. To understand the extent and significance of the E1B55K-SSBP2 interactions in these cells, we have examined SSBP2 localization under conditions of stress in HEK293 cells. SSBP2 localizes to PML- Nuclear Bodies (PML-NBs) in response to inhibition of nuclear export, treatment with etoposide, hydroxyurea or gamma irradiation only in HEK293 cells. Furthermore, the PML-NBs grow in size and number in response to radiation over a 24 hour period in HEK293 cells analogous to previous findings for other cell types. Nonetheless, we conclude that E1B55K subverts SSBP2 function in HEK293 cells. These findings demonstrate the limitations in using HEK293 cells to study DNA damage response and other cellular processes since SSBP2 and similar regulatory proteins are aberrantly localized due to constitutive E1B55K expression.
ABSTRACT
Several, acquired, non-random chromosomal deletions have been characterized in acute myelogenous leukemia (AML). While the deletion limits vary among patients, there are consistent regions of overlap among the deleted segments between patients. Furthermore, chromosomal deletions are achieved frequently by unbalanced translocations between two and more chromosomes resulting loss of candidate leukemia suppressor loci from the affected chromosomes. Most deletions occurring as sole anomalies are associated with good-intermediate clinical outcome, but complex cytogenetic anomalies signify an aggressive clinical course. Thanks to the exciting development in microarray, siRNA technologies, a number of candidate AML suppressor genes localizing to the critical regions of overlap within the deletions have been identified recently. Most of the candidate genes do not function by the classical "two hits," namely loss of an allele unmasking inactivating mutations in the remaining allele. Gene dosage, epigenetic silencing, and uniparental disomy appear to be common mechanisms of gene inactivation in AML. While several of the newly discovered candidate genes lead to new pathways, a few of them affect previously known leukemogenic targets. Thus the investments made over the years on leukemia suppressor gene discovery are beginning to yield reasonable results at the present time. Future beholds promise for targeted therapy of these poorly characterized AMLs, as we uncover the mutations driving their clonal evolution.
Subject(s)
Chromosome Deletion , Chromosomes, Human/ultrastructure , Leukemia, Myeloid, Acute/genetics , Cell Transformation, Neoplastic/genetics , Chromosome Aberrations , Chromosomes, Human/genetics , Genes, Neoplasm , Genes, Tumor Suppressor , Humans , Leukemia, Myeloid, Acute/pathologyABSTRACT
The ZNF198-fibroblast growth factor receptor-1 (FGFR1) fusion kinase is a constitutively activated tyrosine kinase associated with a specific atypical myeloproliferative disease. The chimeric protein localizes to the cytoplasm, unlike the wild type FGFR1 receptor kinase, and presumably inappropriately phosphorylates specific targets as part of the oncogenic signaling cascade. Other than known targets of the FGFR1 kinase itself, few specific targets of ZNF198-FGFR1 have been identified. Using a genetically engineered HEK 293 cell system, we have identified proteins that are specifically phosphorylated in the presence of the fusion kinase using anti-phosphotyrosine immunoprecipitation and MS. Compared with 293 cells expressing exongenous wild type FGFR1, ZNF198-FGFR1 is associated with phosphorylation of several proteins including SSBP2, ABL, FLJ14235, CALM and TRIM4 proteins. The specificity of the phosphorylation events in the SSBP2 and ABL proteins, which have previously been implicated in leukemogenesis, was further confirmed independently using immunoprecipitation with protein-specific antibodies and Western blotting. The MS analysis also identified the phosphorylation events in the ZNF198 moiety in the chimeric protein that might be related to its function. These studies identify the intersection of several different leukemia-related pathways in the development of this myeloproliferative disorder and provide new insights into the substrates of FGFR1 under defined conditions.
Subject(s)
DNA-Binding Proteins/metabolism , Mass Spectrometry/methods , Myeloproliferative Disorders/enzymology , Myeloproliferative Disorders/metabolism , Proto-Oncogene Proteins c-abl/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Transcription Factors/metabolism , Blotting, Western , Cell Line , DNA-Binding Proteins/genetics , Humans , Immunoprecipitation , Monomeric Clathrin Assembly Proteins/metabolism , Myeloproliferative Disorders/genetics , Phosphorylation , Receptor, Fibroblast Growth Factor, Type 1/genetics , Transcription Factors/geneticsABSTRACT
A family of single-stranded DNA-binding proteins (or SSBPs) has been shown to augment the function of LIM-homeodomain (LIM-HD) transcription factors in embryogenesis by interaction with LIM domain-binding protein-1 (LDB1). No DNA-binding complex has been described, however, containing a LIM-HD protein, LDB1, and SSBP, and the mechanism by which SSBPs affect LIM-HD function had not been elucidated. Through use of electrophoretic mobility shift, antibody supershift, and ChIP analyses, we show that an Lhx2-Ldb1-Ssbp3 complex binds a specific element in the Lhx2 target gene Cga (encoding the alpha subunit of glycoprotein hormones) in the alphaT3-1 pituitary cell line. Using overexpression and knockdown approaches, we demonstrate that SSBP3 inhibits Lhx2 and Ldb1 turnover, stimulates assembly of this DNA-binding complex, promotes its recruitment to the Cga promoter, and enhances Cga transcription. These studies provide novel insights into the regulation of pituitary gene expression and LIM-HD function more generally.
Subject(s)
DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Pituitary Gland/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Cell Line , Chromatin Immunoprecipitation , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Glycoprotein Hormones, alpha Subunit/genetics , Homeodomain Proteins/antagonists & inhibitors , LIM Domain Proteins , LIM-Homeodomain Proteins , Mice , Pituitary Gland/cytology , Promoter Regions, Genetic , Transcription Factors/antagonists & inhibitorsABSTRACT
The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.
Subject(s)
DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Animals , Beta-Globulins/genetics , CHO Cells , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Cricetinae , Cricetulus , DNA, Single-Stranded/metabolism , Erythroid Precursor Cells/chemistry , GATA1 Transcription Factor/metabolism , Mice , Multiprotein Complexes/metabolism , Promoter Regions, Genetic , Protein Structure, Tertiary , RNA Interference , Repressor Proteins/metabolismABSTRACT
The Mix1 homeobox-like (MIXL1) gene encodes a paired class homeobox transcription factor that is involved in embryogenesis. Previous studies have shown that the MIXL1 gene product is expressed in B- and T-cell progenitors of normal bone marrow and, in some cell lines derived from hematopoietic neoplasms. The status of MIXL1 expression and subcellular localization in human lymphomas is unknown. Using a highly specific antibody, we assessed for MIXL1 expression in lymphoma cell lines of B- and T-cell lineage by reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. We also assessed for MIXL1 expression using immunohistochemical methods in 193 lymphoid tumors, including 140 B-cell non-Hodgkin lymphomas (NHL), 36 T-cell NHL, and 17 Hodgkin lymphomas (HL). MIXL1 was detected predominantly in the nuclear fraction of all cell lines tested and was predominantly nuclear in primary tumor specimens. Based on the distribution of the staining results (histogram), a 50% cutoff was selected for high versus low MIXL1 expression. High MIXL1 expression was detected more frequently in Burkitt lymphoma and diffuse large B-cell lymphoma compared with other types of B-cell NHL (P < .0001, chi(2) test). Most cases of T-cell NHL and all cases of HL also highly expressed MIXL1. Most plasma cell myelomas were negative for MIXL1, but rare cases had low MIXL1 expression. MIXL1 expression significantly correlated with proliferation index (Ki-67) in B-cell NHL (P < .0001). The frequent and high expression of MIXL1 in aggressive B-cell NHL, T-cell NHL, and HL suggests that MIXL1 may be involved in lymphomagenesis.
Subject(s)
Gene Expression Regulation, Neoplastic , Hodgkin Disease/genetics , Homeodomain Proteins/genetics , Lymphoma, Non-Hodgkin/genetics , Cell Line, Tumor , Humans , Lymphoid Tissue/metabolismABSTRACT
Seven members of the Mix family of paired-type homeoproteins regulate mesoderm/endoderm differentiation in amphibians. In mammals, the MIXL1 (Mix. 1 homeobox [Xenopus laevis]-like gene 1) gene is the sole representative of this family. Unlike the amphibian Mix genes that encode an open reading frame of >300 amino acids, mammalian MIXL1 encodes a smaller protein (approximately 230aa). However, mammalian MIXL1 contains a unique proline-rich domain (PRD) with a potential to interact with signal transducing Src homolgy 3 (SH3) domains. Notably, human MIXL1 also contains a unique tyrosine residue Tyr20 that is amino-terminal to the PRD. Here we report that mammalian MIXL1 protein is phosphorylated at Tyr20 and the phosphorylation is dramatically reduced in the absence of PRD. Our findings are consistent with Tyr20 phosphorylation of MIXL1 being a potential regulatory mechanism that governs its activity.
ABSTRACT
Acquired complete and partial deletions of chromosome 7 are associated with several malignancies. In acute myelogenous leukemia (AML) and preleukemic myelodysplasia (MDS), loss of chromosome 7 portends a poor clinical outcome. The identity of a classical leukemia suppressor gene, however, has been elusive. Previously, we defined a candidate suppressor locus of approximately 6 Mb in the 7q31 interval. Here we report an island of retention of heterozygosity within this interval in a case of monosomy 7. Allelotyping of AML cell lines revealed that ML3 and HEL cells, karyotypically diploid for chromosome 7, are hemizygous for all the 7q31 loci, implicating loss of the wild type and duplication of the remaining chromosome 7. Based on the completed genomic sequence of chromosome 7, we have generated a transcript map of the critical region of loss (between the D7S525 and D7S2502 loci). Notably, a recently characterized tumor suppressor gene, DOCK4, and an evolutionarily conserved zinc finger gene, ZNF277, localize to this interval, head to head, within <0.5 kb of each other. Thus, the reagents generated in this study will be valuable in elucidating the role of loss of 7q31 loci in the pathogenesis of AML.
Subject(s)
Chromosomes, Human, Pair 7 , Monosomy , Amino Acid Sequence , Chromosome Deletion , Chromosome Mapping , Humans , Karyotyping , Leukemia, Myeloid, Acute , Loss of Heterozygosity , Microsatellite Repeats , Molecular Sequence Data , Myeloproliferative Disorders , Polymorphism, Genetic , Tumor Cells, CulturedABSTRACT
Acute myelogenous leukemia (AML) is the most common leukemia in adults with clonal proliferation of myeloid stem cells. Two or more cooperating mechanisms, namely block in differentiation, enhanced proliferation and resistance to programmed cell death, underlie this neoplastic transformation. Nonrandom, complete and partial deletions of chromosome 5 are common anomalies in AML. Using positional cloning strategies, we characterized an evolutionarily conserved candidate myeloid leukemia suppressor gene encoding sequence-specific single-stranded DNA binding protein 2 (SSBP2) from chromosome 5q13.3, a locus that is frequently deleted in AML. Recent studies in Drosophila and Xenopus demonstrate a pivotal role for SSBPs in embryonic differentiation. In mammals, SSBP2 is one of three highly related and ubiquitously expressed genes. Here, we identify frequent loss of SSBP2 protein expression in human AML cell lines using highly specific antibodies. Furthermore, inducible expression of SSBP2 in the AML cell line U937 leads to loss of clonogenicity, G1 arrest and partial differentiation. Remarkably, inducible expression of SSBP2 is accompanied by downregulation of C-MYC expression. Our findings are consistent with human SSBP2 being a novel regulator of hematopoietic growth and differentiation, whose loss confers a block in differentiation advantage to myeloid leukemic cells.
Subject(s)
Cell Differentiation , DNA-Binding Proteins/genetics , Leukemia, Myeloid, Acute/genetics , Animals , COS Cells , Cell Proliferation , Chlorocebus aethiops , DNA-Binding Proteins/metabolism , Down-Regulation , G1 Phase/drug effects , Gene Expression Regulation , Genes, Tumor Suppressor , Genes, myc/drug effects , Humans , Leukemia, Myeloid, Acute/metabolism , Transfection , U937 CellsABSTRACT
Complete and partial deletions of chromosome 5 are recurrent anomalies associated with refractory myelogenous leukemia. Recent evidence suggests that these deletions arise from unbalanced two- or three-way translocations, rather than from interstitial breaks or segregation errors; however, very little is known about the molecular mechanisms underlying this multistep genomic instability. We have analyzed a complex rearrangement of chromosome band 5q both in the primary leukemic cells of the patient from whom the acute myelogenous leukemia (AML) cell line HL-60 was derived and in the HL-60 cells in culture. This highly stable rearrangement is a product of multiple events in which a small single-copy fragment flanking the 3' end of the GMCSF gene is juxtaposed to novel L1Hs sequences. The resulting genomic fragment is found inserted into a telomeric locus (D5S89), with loss of 4.1 Mbp of in-between sequences, encoding one or more candidate myeloid leukemia suppressor genes. The findings are consistent with a dynamic role for L1Hs in mediating instability that results in a complex chromosomal rearrangement. Furthermore, we provide what may be the first example of multiple L1Hs-associated deletions involving both a growth factor gene and a tumor suppressor locus in a primary leukemic clone.
Subject(s)
Chromosomal Instability/genetics , Chromosome Deletion , Chromosomes, Human, Pair 5/genetics , Base Sequence , Chromosome Mapping , Cloning, Molecular , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , HL-60 Cells , Humans , Molecular Sequence Data , Restriction MappingABSTRACT
Complete and partial deletions of chromosome 5q are recurrent cytogenetic anomalies associated with aggressive myeloid malignancies. Earlier, we identified an approximately 1.5-Mb region of loss at 5q13.3 between the loci D5S672 and D5S620 in primary leukemic blasts. A leukemic cell line, ML3, is diploid for all of chromosome 5, except for an inversion-coupled translocation within the D5S672-D5S620 interval. Here, we report the development of a bacterial artificial chromosome (BAC) contig to define the breakpoint and the identification of a novel gene SSBP2, the target of disruption in ML3 cells. A preliminary evaluation of SSBP2 as a tumor suppressor gene in primary leukemic blasts and cell lines suggests that the remaining allele does not undergo intragenic mutations. SSBP2 is one of three members of a closely related, evolutionarily conserved, and ubiquitously expressed gene family. SSBP3 is the human ortholog of a chicken gene, CSDP, that encodes a sequence-specific single-stranded DNA-binding protein. SSBP3 localizes to chromosome 1p31.3, and the third member, SSBP4, maps to chromosome 19p13.1. Chromosomal localization and the putative single-stranded DNA-binding activity suggest that all three members of this family are capable of potential tumor suppressor activity by gene dosage or other epigenetic mechanisms.
Subject(s)
Chromosomes, Human, Pair 5 , DNA-Binding Proteins/genetics , Multigene Family , Amino Acid Sequence , Chromosomes, Artificial, Bacterial , Conserved Sequence , Evolution, Molecular , Gene Expression , Humans , Molecular Sequence Data , Physical Chromosome Mapping , Sequence Alignment , Sequence Deletion , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
Molecular events involved in specification of early hematopoietic system are not well known. In Xenopus, a paired-box homeodomain family (Mix.1-4) has been implicated in this process. Although Mix-like homeobox genes have been isolated from chicken (CMIX) and mice (Mml/MIXL1), isolation of a human Mix-like gene has remained elusive. We have recently isolated and characterized a novel human Mix-like homeobox gene with a predicted open reading frame of 232 amino acids designated the Mix.1 homeobox (Xenopus laevis)-like gene (MIXL). The overall identity of this novel protein to CMIX and Mml/MIXL1 is 41% and 69%, respectively. However, the identity in the homeodomain is 66% to that of Xenopus Mix.1, 79% to that of CMIX, and 94% to that of Mml/MIXL1. In normal hematopoiesis, MIXL expression appears to be restricted to immature B- and T-lymphoid cells. Several acute leukemic cell lines of B, T, and myeloid lineage express MIXL suggesting a survival/block in differentiation advantage. Furthermore, Xenopus animal cap assay revealed that MIXL could induce expression of the alpha-globin gene, suggesting a functional conservation of the homeodomain. Isolation of the MIXL gene is the first step toward understanding novel regulatory circuits in early hematopoietic differentiation and malignant transformation.