Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation.

Pbx1 and a subset of homeodomain proteins collaboratively bind DNA as higher-order molecular complexes with unknown consequences for mammalian development. Pbx1 contributions were investigated through characterization of Pbx1-deficient mice. Pbx1 mutants died at embryonic day 15/16 with severe hypoplasia or aplasia of multiple organs and widespread patterning defects of the axial and appendicular skeleton. An obligatory role for Pbx1 in limb axis patterning was apparent from malformations of proximal skeletal elements, but distal structures were unaffected. In addition to multiple rib and vertebral malformations, neural crest cell-derived skeletal structures of the second branchial arch were morphologically transformed into elements reminiscent of first arch-derived cartilages. Although the skeletal malformations did not phenocopy single or compound Hox gene defects, they were restricted to domains specified by Hox proteins bearing Pbx dimerization motifs and unaccompanied by alterations in Hox gene expression. In affected domains of limbs and ribs, chondrocyte proliferation was markedly diminished and there was a notable increase of hypertrophic chondrocytes, accompanied by premature ossification of bone. The pattern of expression of genes known to regulate chondrocyte differentiation was not perturbed in Pbx1-deficient cartilage at early days of embryonic skeletogenesis, however precocious expression of Col1a1, a marker of bone formation, was found. These studies demonstrate a role for Pbx1 in multiple developmental programs and reveal a novel function in co-ordinating the extent and/or timing of proliferation with terminal differentiation. This impacts on the rate of endochondral ossification and bone formation and suggests a mechanistic basis for most of the observed skeletal malformations.

The Hox cofactor and proto-oncogene Pbx1 is required for maintenance of definitive hematopoiesis in the fetal liver.

Pbx1 is the product of a proto-oncogene originally discovered at the site of chromosomal translocations in acute leukemias. It binds DNA as a complex with a broad subset of homeodomain proteins, but its contributions to hematopoiesis have not been established. This paper reports that Pbx1 is expressed in hematopoietic progenitors during murine embryonic development and that its absence results in severe anemia and embryonic lethality at embryonic day 15 (E15) or E16. Definitive myeloerythroid lineages are present in Pbx1(-/-) fetal livers, but the total numbers of colony-forming cells are substantially reduced. Fetal liver hypoplasia reflects quantitative as well as qualitative defects in the most primitive multilineage progenitors and their lineage-restricted progeny. Hematopoietic stem cells from Pbx1(-/-) embryos have reduced colony-forming activity and are unable to establish multilineage hematopoiesis in competitive reconstitution experiments. Common myeloid progenitors (CMPs), the earliest known myeloerythroid-restricted progenitors, are markedly depleted in Pbx1(-/-) embryos at E14 and display clonogenic defects in erythroid colony formation. Comparative cell-cycle indexes suggest that these defects result largely from insufficient proliferation. Megakaryocyte- and erythrocyte-committed progenitors are also reduced in number and show decreased erythroid colony-forming potential. Taken together, these data indicate that Pbx1 is essential for the function of hematopoietic progenitors with erythropoietic potential and that its loss creates a proliferative constriction at the level of the CMP. Thus, Pbx1 is required for the maintenance, but not the initiation, of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets through which mature erythrocytes are generated. (Blood. 2001;98:618-626)

Expression of Pbx1b during mammalian organogenesis.

Mammalian Pbx genes (Pbx1-3) encode a family of TALE homeodomain proteins that function as transcriptional regulators in numerous cell types (Curr. Opin. Genet. Dev. 8 (1998) 423). The present study highlights distinctive features of Pbx1b expression during mouse embryonic development as a framework to understand its biological functions. Immunohistochemical analyses demonstrate extensive expression of Pbx1b throughout post-implantation development, with highest levels observed during early to mid-gestation. Its initial distribution is predominantly associated with condensing mesoderm, however, Pbx1b displays dynamic expression patterns in derivatives of all principal germ layers. In particular, Pbx1b localizes to sites of mesenchymal-epithelial interactions during periods of active morphogenesis in tissues such as the lung, kidney, tooth buds and vibrissae follicles. Furthermore, BrdU labeling studies reveal that Pbx1b expression domains partially overlap with regions of cellular proliferation. Taken together, these data suggest that Pbx1b contributes to multiple cellular processes during embryogenesis, which may include roles in cell-autonomous regulation as well as in the mediation of tissue interactions.

Molecular cytogenetics localizes two new breakpoints on 11q23.3 and 21q11.2 in myelodysplastic syndrome with t(11;21) translocation.

Translocation t(11;21)(q24;q11.2) is a rare but recurrent chromosomal abnormality associated with myelodysplastic syndrome (MDS) that until now has not been characterized at the molecular level. We report here results of a molecular cytogenetic analysis of this translocation in a patient with refractory anemia. Using FISH with a panel of 11q and 21q cosmid/YAC probes, we localized the chromosome 11 breakpoint at q23.3 in a region flanked by CP-921G9 and CP-939H3 YACs, distal to the HRX/MLL locus frequently involved in acute leukemias. The chromosome 21 breakpoint was mapped in a 800-kb fragment inserted into the CP-145E3 YAC at 21q11.2, proximal to the AML1 gene. It is noteworthy that in all four cases with a t(11;21) reported until now, a second der(11)t(11;21) and loss of normal chromosome 11 could be observed either at diagnosis or during the course of the disease. Since in our case heteromorphism was detected by FISH on the centromeric region of the two der(11), the second der(11) chromosome could be the result of a mitotic recombination that had occurred on the long arm of chromosome 11, rather than of duplication of the original der(11). Constancy of secondary karyotypic changes resulting in an extra copy of the putative chimeric gene at der(11), loss of 11 qter sequences, and partial trisomy 21 suggest that neoplastic progression of MDS cases with a t(11;21) may be driven by the same mechanism(s).

Organization and regulatory aspects of the human intestinal mucin gene (MUC2) locus.

The human MUC2 gene maps to chromosome 11p15, where three additional mucin genes have been located, and encodes the most abundant gastrointestinal mucin normally expressed in the intestinal goblet cell lineage. However, in pathological conditions, including colorectal cancer, MUC2 can be abnormally expressed. Therefore, it is of considerable interest to understand the regulation of the MUC2 gene and how the mechanism is altered in colon cancer. Toward this goal, we have isolated a group of overlapping clones (contig) spanning 85 kilobases harboring the entire MUC2 locus, including sequences located upstream of the gene. Detection of two DNase I-hypersensitive sites in the 5' region of the MUC2 gene suggests the presence of DNA regulatory elements. To better characterize this region, we have sequenced 12 kilobases of the upstream region and analyzed it for functional activity by cloning portions of it into a luciferase reporter vector and assaying for promoter/enhancer activity using a transient transfection assay. A fragment from the AUG translational initiation codon +1 to -848 confers maximal transcriptional activity in several intestinal cell lines. Elements located further upstream exert a negative effect on the expression of the reporter gene when tested in conjunction with homologous or heterologous promoters. The same pattern of expression is observed when the MUC2/luciferase constructs are transfected into HeLa cells, which do not express the endogenous MUC2 gene. However, the level of activity in HeLa cells is at least an order of magnitude higher, suggesting that additional sequences singularly or in combination are responsible for the tissue- and cell lineage-specific expression of MUC2. Finally, we have identified an additional mucin-like gene (MUCX), located upstream of MUC2. We show that this MUCX gene, that is transcribed in opposite orientation to that of MUC2, is expressed with a pattern distinct from that of MUC2, yet similar to that of MUC5B and MUC6, two additional mucin genes located at chromosome 11p15. Recent information on the order of the mucin genes at chromosome 11p15 suggests that MUCX may be MUC6, one of the already identified mucin genes, or a novel one, yet to be fully characterized.

A novel L23-related gene 40 kb downstream of the imprinted H19 gene is biallelically expressed in mid-fetal and adult human tissues.

The closely linked IGF2 and H19 genes on human chromosome 11p15.5 are monoallelically expressed as a result of genomic imprinting and show altered expression in Wilms' tumors (WTs). To map regional imprinting we have sought to isolate additional human genes close to IGF2/H19 and to characterize their allelic expression patterns. Here we report a novel gene, provisionally named L23MRP [L23 (mitochondrial)-related protein], which is oriented 'tail-to-tail' with H19 and is transcribed to within 40 kb of the last H19 exon. L23MRP is expressed biallelically in many mid-fetal and adult human tissues. This gene is also expressed at normal levels in WTs which have lost expression of H19 either via loss of the maternal chromosome 11p15.5 or via an epigenetic pathway involving site-specific DNA hypermethylation. These data indicate that, at least in post-embryonic stages, L23MRP is functionally insulated from the IGF2/H19 imprinted domain.

Translocation (11;15)(q23;q14) in three patients with acute non-lymphoblastic leukemia (ANLL): clinical, cytogenetic and molecular studies.

We report on three patients with acute non-lymphoblastic leukemia (ANLL) displaying the same chromosomal translocation t(11;15)(q23;q14). The clinical course of the disease was aggressive, and survival was short. The FAB subtype was M-2 in two cases, and M-1 in the remaining patient. Immunologically two cases showed aberrant expression of a lymphoid antigen (CD19 and TdT, respectively). HTRX1/MLL gene was rearranged in one patient studied at the time of diagnosis. These results plus data scattered in the literature show that the t(11;15)(q23;q14) can be added to the list of recurrent rearrangements in ANLL involving 11q23.

High-resolution physical mapping of a 250-kb region of human chromosome 11q24 by genomic sequence sampling (GSS).

A physical map of the region of human chromosome 11q24 containing the FLI1 gene, disrupted by the t(11;22) translocation in Ewing sarcoma and primitive neuroectodermal tumors, was analyzed by genomic sequence sampling. Using a 4- to 5-fold coverage chromosome 11-specific library, 22 region-specific cosmid clones were identified by phenol emulsion reassociation hybridization, with a 245-kb yeast artificial chromosome clone containing the FLI1 gene, and by directed "walking" techniques. Cosmid contigs were constructed by individual clone fingerprinting using restriction enzyme digestion and assembly with the Genome Reconstruction and AsseMbly (GRAM) computer algorithm. The relative orientation and spacing of cosmid contigs with respect to the chromosome was determined by the structural analysis of cosmid clones and by direct visual in situ hybridization mapping. Each cosmid clone in the contig was subjected to "one-pass" end sequencing, and the resulting ordered sequence fragments represent approximately 5% of the complete DNA sequence, making the entire region accessible by PCR amplification. The sequence samples were analyzed for putative exons, repetitive DNAs, and simple sequence repeats using a variety of computer algorithms. Based upon the computer predictions, Southern and Northern blot experiments led to the independent identification and localization of the FLI1 gene as well as a previously unknown gene located in this region of chromosome 11q24. This approach to high-resolution physical analysis of human chromosomes allows the assembly of detailed sequence-based maps and provides a tool for further structural and functional analysis of the genome.

Spontaneous loss of Ph chromosome with maintenance of clonal hemopoiesis in an untreated patient with myeloproliferative disease and a long survival.

The unusual case of myeloproliferative disease described here is characterized by the following features: (1) a clinically completely silent course for 11 years without splenomegaly, marrow fibrosis, or cellular morphologic alterations; (2) the presence, at the onset, of a Philadelphia (Ph) chromosome without DNA breakpoints in the M-bcr region; (3) the spontaneous loss of detectable Ph-positive cells, 5 years after the first finding of leukocytosis, in the absence of any therapy; (4) the maintenance of the clonal nature of hematopoiesis, as revealed by the PGK X-linked inactivation pattern, in the absence of the Ph chromosome; and (5) a biphasic trend in the levels of leukocytes, red cells, and platelets during the years of observation.

Yeast artificial chromosome cloning of 3.2 megabases within chromosomal band 11q24 closely linking c-ets 1 and Fli-1 and encompassing the Ewing sarcoma breakpoint.

Human chromosome 11 harbors many genes of medical significance and cancer-related rearrangements. The availability of cloned DNA in cosmids and in yeast artificial chromosomes (YACs), combined with fluorescence in situ hybridization analysis, has led to the cloning of genes at sites of chromosomal breakpoints in acute leukemias in 11q23 and in Ewing tumors in 11q24. YAC cloning has facilitated the construction of contigs covering large portions of chromosomes for the detailed analysis of disease gene regions. Here we have cloned in YACs approximately 3.2 Mb of DNA within band 11q24, spanning the Ewing sarcoma breakpoint. Landmark cosmids 23.2 (D11S374) and 5.8 (D11S372), shown by FISH to flank the breakpoint within a 1.5- to 1.8-Mb segment, were used to seed two YAC "walks" both centromeric and telomeric to the breakpoint by YAC-end cloning and screening of two total genomic YAC libraries. The centromeric YAC contig, which consists of 23 overlapping YACs and orders 19 sequence-tagged sites (STSs), covers a minimum of 2.2 Mb and spans the Ewing sarcoma breakpoint. c-ets 1 and Fli-1, two members of the ets family, have been linked within 400 kb of intervening DNA within this contig, which also comprises a polymorphic microsatellite, D11S912 (CA)n, which we have localized within the Fli-1 gene. The telomeric YAC contig, which consists of 11 overlapping YACs, comprises 5 STSs and covers a minimum of 1 Mb distal to the breakpoint. Taken together, the two contigs, which consist of a total of 34 YACs and comprise 24 STSs, are separated by a maximum gap of 200-400 kb and cover as a whole 3.2 Mb of DNA. This represents about 70% of human chromosomal band 11q24, which extends over approximately 4.4 Mb of DNA.

Cloning of the entire FLI1 gene, disrupted by the Ewing's sarcoma translocation breakpoint on 11q24, in a yeast artificial chromosome.

FLI1 (Friend leukemia virus integration 1), a member of the Ets gene family, is disrupted on 11q24 by the Ewing's Sarcoma (ES) and Peripheral Neuroepithelioma (PNE) t(11;22)(q24;q12) translocation. ES and PNE are Primitive Neuroectodermal Tumors (PNETs) and the consistent translocation t(11;22)(q24;q12) can be used for differential diagnosis. In PNETs the 3' part of human FLI1 is translocated from 11q24 to 22q12, where it is juxtaposed to the 5' end of the Ewing's Sarcoma gene (EWS). A fusion transcript, resulting in a chimeric protein, is generated. Here, we present the isolation and detailed characterization of a 250-kb colinear YAC, B45C11, which encompasses the ES and PNE breakpoint on 11q24, as shown by FISSH on ES and PNE chromosomes and interphase nuclei. This YAC represents a new reagent for potential use in rapid differential diagnosis by FISSH on tumor biopsies and on paraffin embedded samples, particularly when DNA and/or RNA are not available for molecular analysis. YAC B45C11, which spans 250 kb of contiguous DNA around the ES and PNE breakpoint, contains the entire FLI1 gene. Three potential HpaII-tiny-fragment (HTF) islands are revealed within the YAC. One of these islands appears to be associated with the 5' end of FLI1, which extends over approximately 120 kb of DNA on 11q24. In addition, we demonstrate that YAC B45C11 contains other transcribed sequences in addition to FLI1, by "cross-species" Northern blot hybridizations, which suggests the presence of additional genes in the immediate vicinity of the ES breakpoint on 11q24.

A novel zinc finger gene on human chromosome 1qter that is alternatively spliced in human tissues and cell lines.

DNA-binding proteins that share the conserved C2-H2 zinc finger motif have been shown to have important roles as transcriptional regulators of gene expression and have been implicated in several hereditary human diseases. In order to define potential candidate genes for inherited disorders characterized by aberrant gene expression, we utilized Kruppel-related sequences to isolate zinc finger-containing cDNAs. We isolated and characterized two novel zinc finger-encoding cDNAs from a human hepatoblastoma cell line, which demonstrate DNA sequence homology to a recently described human Kruppel-related gene HZF-3 and appear to be derived from a single gene by alternate mRNA splicing. This gene, denoted "HZF-16," give rise to at least two gene products. One cDNA (i.e., HZF-16.2) has nine zinc finger domains, while alternative splicing of the message gives rise to a smaller product (i.e., HZF-16.1) that has four domains. Despite the internal splicing event, both the 5'- and 3'-untranslated sequences in both cDNAs are identical, as are the first three domains. In the HZF-16.1 cDNA, the fourth zinc finger domain is a fusion product of domains four and nine of HZF-16.2 and could potentially give rise to a new DNA-binding specificity. These alternatively spliced transcripts are differentially regulated in human tissues and transformed cell lines and show a different distribution of expression between human cell lines and normal human tissue. This novel gene was mapped to human chromosome 1q44 by chromosomal in situ suppression hybridization and thus represents a candidate gene for trisomy 1q syndrome and for several other disorders.

Localization of the human oncostatin M gene (OSM) to chromosome 22q12, distal to the Ewing's sarcoma breakpoint.

Using fluorescence in situ hybridization, a cosmid clone containing the gene for oncostatin M (OSM) was mapped to human chromosome 22q12, placing the OSM gene in the same chromosome band as the leukemia-inhibitory factor gene (LIF). The location of the OSM gene was determined relative to the t(11;22)(q24;q12) of Ewing's sarcoma and found to be distal to the translocation breakpoint on chromosome 22. Analysis of physical distances by pulsed-field gel electrophoresis demonstrated further that the two genes lie within 500 kb of each other.

Tandem linkage of genes coding for leukemia inhibitory factor (LIF) and oncostatin M (OSM) on human chromosome 22.

Leukemia-inhibitory factor (LIF) and oncostatin M (OSM) are members of a family of structurally similar growth factors presenting overlapping and specific functions. Although the genes coding for IL-6, CSF3 and CNTF are scattered in the human and mouse genome, human LIF and OSM genes have conserved synteny in the course of evolution. Through isolation of a YAC and a cosmid clone containing both LIF and OSM we demonstrate that the two genes are linked in tandem on human chromosome 22q12, separated by 16 kilobases of intervening genomic DNA and transcribed in the same head-to-tail orientation. The close physical linkage between LIF and OSM genes brings new evidence of their evolutionary relationship.

Isolation and expression of linked zinc finger gene clusters on human chromosome 11q.

Proteins that share conserved "zinc finger" motifs represent a class of DNA-binding proteins that have been shown to play a fundamental role in regulating gene expression and to be involved in a number of human hereditary and malignant disease states. We have isolated, characterized, and mapped zinc finger-encoding genes specific to human chromosome 11q to investigate their possible association in the molecular pathogenesis of several disease loci mapped to this chromosome. An arrayed chromosome 11q cosmid library was screened using a degenerate oligonucleotide corresponding to the H/C link consensus sequence of the Drosophila Kruppel zinc finger gene, resulting in the isolation of six putative zinc finger genes. Three of the genes (ZNF123, ZNF125, and ZNF126) were analyzed and shown to contain tandemly repeated zinc finger motifs of the C2-H2 class. All three novel genes were found to be expressed in normal adult human tissues, although the tissue-specific pattern of expression differs markedly. Isolated zinc finger genes were regionally mapped on chromosome 11 using fluorescence in situ suppression hybridization and demonstrated clustering of the genes at 11q13.3-11q13.4 and 11q23.1-11q23.2. Analysis of in situ hybridization to interphase nuclei demonstrated a maximum distance of 1 Mb separating distinct finger genes. This analysis defines two linked multigene families of zinc finger genes to chromosome bands associated with a high frequency of specific translocations associated with malignancies.

Interphase cytogenetics for the detection of the t(11;22)(q24;q12) in small round cell tumors.

Among the small round cell tumors differential diagnosis is particularly difficult for their undifferentiated or primitive character. In this mixed group of tumors, only the primitive neuroectodermal tumors, which include Ewing's sarcoma (ES), show the unique and consistent feature of the (11;22)(q24;q12) translocation, which can therefore be considered a hallmark of these neoplasias. We analyzed four primitive neuroectodermal tumor cell lines, one osteosarcoma cell line, and 11 patients by fluorescent in situ hybridization with cosmid clones 23.2 and 5.8, bracketing the t(11;22) at 11q24. Metaphase spreads from tumor cell lines, and from biopsy specimens of three patients with ES were analyzed. In the remaining eight patients comprising five ES, two small cell osteosarcomas and one chronic osteomyelitis, only nuclei preparations were available for analysis. We detected the t(11;22) in interphase nuclei of the four primitive neuroectodermal tumor cell lines, of three patients in which the karyotype demonstrated the translocation and in five cases of ES in which cytogenetic analysis had not been possible. Two cases of small cell osteosarcoma and one chronic osteomyelitis were also analyzed and were both normal with respect to the t(11;22). By analyzing cell lines and small round cell tumor samples by fluorescent in situ hybridization, we established that interphase cytogenetics is a rapid alternative to chromosomal analysis for the detection of the t(11;22) and represents an invaluable tool for the differential diagnosis of small round cell tumors.

A trithorax-like gene is interrupted by chromosome 11q23 translocations in acute leukaemias.

Some acute lymphocytic leukaemias, particularly those in young children, are associated with a t(4;11)(q21;q23) reciprocal translocation. We have cloned the translocation breakpoint on chromosome 11q23 and isolated corresponding RNA transcripts from this region. The translocation occurs within a cluster of Alu repetitive elements located within an intron of a gene that gives rise to 11.5 (kb) transcript spanning the translocation breakpoint. The 11.5 kb transcript encodes a protein that is highly homologous to the Drosophila trithorax gene, a developmental regulator. An analysis of a series of leukaemic patients carrying t(4;11) and t(9;11) translocations indicate that the majority of breakpoints in infant leukaemias lie within a 5 kb region.

Dek-can rearrangement in translocation (6;9)(p23;q34).

The translocation (6;9)(p23;q34) is mainly found in specific subtypes of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The diagnosis of this translocation is not easy since the cytogenetic change is quite subtle. The two genes involved in this translocation were recently isolated and diagnosis at the DNA-level became an additional option. Both the dek gene on chromosome 6 and the can gene on chromosome 9 contain one specific intron where breakpoints of t(6;9) patients were found to cluster. The translocation results in a consistent chimeric dek-can mRNA which is generated from the 6p- derivative. Five centers participated in a study to estimate the incidence of t(6;9) in leukemic patients using conventional Southern blot analysis. Patients (n = 320) with either acute undifferentiated leukemia (AUL), AML, MDS or acute lymphoblastic leukemia (ALL) were screened for rearrangement of the genes involved in this translocation. Four of these 320 patients showed rearrangement of the can gene on chromosome 9, of which one also had a rearranged dek gene on chromosome 6. A further 20 patients were studied with karyotypic aberrations in which either the short arm of chromosome 6 or the long arm of chromosome 9 were specifically involved. Both conventional Southern blot analysis and contour-clamped homogeneous electric field (CHEF) analysis failed to show dek-can rearrangement in any of these patients. The results of our study indicate that the incidence of the t(6;9) is a low as reported based on cytogenetic data and that rearrangement of the dek and can genes is mainly restricted to this specific translocation.

Human herpesvirus-6 in human lymphomas: identification of specific sequences in Hodgkin's lymphomas by polymerase chain reaction.

In search of a possible involvement of the human herpesvirus type 6 (HHV-6) in human Hodgkin's and non-Hodgkin's lymphomas, we studied the levels of anti-HHV-6 antibodies in the sera of 94 cases by an immunofluorescence assay, as well as the presence of HHV-6 sequences in the affected tissues of 66 cases by polymerase chain reaction, using one set of primer oligonucleotides. Our results showed higher anti-HHV-6 antibody titers in human lymphomas than in normal blood donors, but the difference is statistically significant only when normal donors are compared with Hodgkin's lymphoma cases. HHV-6 sequences were detected in 3 of 25 Hodgkin's lymphomas and 0 of the 41 cases of non-Hodgkin's lymphomas studied. The three cases positive for HHV-6 sequences belong to the nodular sclerosis-lymphocyte depletion histologic subtype and share remarkable similarities in their clinical features. Furthermore, Southern blot analysis of total genomic DNA obtained from the neoplastic tissues of two of the three patients showed the same restriction fragment length polymorphism. Our results suggest that: (1) the high level of anti-HHV-6 antibodies in Hodgkin's disease is due to an activation of the immune system not related to the presence of HHV-6 sequences in affected lymph nodes; (2) the presence of HHV-6 sequences in human lymphoid tissues is not a frequent event, rather it is in fact a very rare event in non-Hodgkin's lymphomas, while in Hodgkin's cases it is more frequent than previously reported on the basis of Southern blot analysis; and (3) the presence of HHV-6 sequences in Hodgkin's lymphomas may have a relation with the clinical presentation of the disease.