Leukemia with distinct phenotypes in transgenic mice expressing PML/RAR alpha, PLZF/RAR alpha or NPM/RAR alpha.

Recurrent chromosomal translocations involving the RAR alpha locus on chromosome 17 are the hallmark of acute promyelocytic leukemia (APL). The RAR alpha gene fuses to variable partners (PML, PLZF, NPM, NuMA and STAT5B: X genes) leading to the expression of APL-specific fusion proteins with identical RAR alpha moieties. To analyse whether the variable X moiety could affect the activity of the fusion protein in vivo, we generated and characterized, on a comparative basis, NPM/RAR alpha transgenic mice (TM) in which the fusion gene is expressed under the control of a human Cathepsin G (hCG) minigene. We compared the features of the leukemia observed in these TM with those in hCG-PML/RAR alpha and hCG-PLZF/RAR alpha TM. In all three transgenic models, leukemia developed after a variably long latency, with variable penetrance. However, the three leukemias displayed distinct cytomorphological features. hCG-NPM/RAR alpha leukemic cells resembled monoblasts. This phenotype contrasts with what was observed in the hCG-PML/RAR alpha TM model in which the leukemic phase was characterized by the proliferation of promyelocytic blasts. Similarly, hCG-PLZF/RAR alpha TM displayed a different phenotype where terminally differentiated myeloid cells predominated. Importantly, the NPM/RAR alpha oncoprotein was found to localize in the nucleolus, unlike PML/RAR alpha and PLZF/RAR alpha, thus possibly interfering with the normal function of NPM. Similarly to what was observed in human APL patients, we found that NPM/RAR alpha and PML/RAR alpha, but not PLZF/RAR alpha leukemia, was responsive to all-trans retinoic acid (ATRA) or As2O3 treatments. Taken together, our results underscore the critical relevance of the X moiety in dictating the biology of the disease and the activity of the APL fusion oncoprotein.

Two critical hits for promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations that always involve the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes). Due to the reciprocity of the translocation, X-RARalpha and RARalpha-X fusion proteins coexist in APL blasts. PLZF-RARalpha transgenic mice (TM) develop leukemia that lacks the differentiation block at the promyelocytic stage that characterizes APL. We generated TM expressing RARalpha-PLZF and PLZF-RARalpha in their promyelocytes. RARalpha-PLZF TM do not develop leukemia. However, PLZF-RARalpha/RARalpha-PLZF double TM develop leukemia with classic APL features. We demonstrate that RARalpha-PLZF can interfere with PLZF transcriptional repression and that this is critical for APL pathogenesis, since leukemias in PLZF(-/-)/PLZF-RARalpha mutants and in PLZF-RARalpha/RARalpha-PLZF TM are indistinguishable. Thus, both products of a cancer-associated translocation are crucial in determining the distinctive features of the disease.

The murine Bapx1 homeobox gene plays a critical role in embryonic development of the axial skeleton and spleen.

Our previous studies in both mouse and human identified the Bapx1 homeobox gene, a member of the NK gene family, as one of the earliest markers for prechondrogenic cells that will subsequently undergo mesenchymal condensation, cartilage production and, finally, endochondral bone formation. In addition, Bapx1 is an early developmental marker for splanchnic mesoderm, consistent with a role in visceral mesoderm specification, a function performed by its homologue bagpipe, in Drosophila. The human homologue of Bapx1 has been identified and mapped to 4p16.1, a region containing loci for several skeletal diseases. Bapx1 null mice are affected by a perinatal lethal skeletal dysplasia and asplenia, with severe malformation or absence of specific bones of the vertebral column and cranial bones of mesodermal origin, with the most severely affected skeletal elements corresponding to ventral structures associated with the notochord. We provide evidence that the failure of the formation of skeletal elements in Bapx1 null embryos is a consequence of a failure of cartilage development, as demonstrated by downregulation of several molecular markers required for normal chondroblast differentiation (&agr; 1(II) collagen, Fgfr3, Osf2, Indian hedgehog, Sox9), as well as a chondrocyte-specific alpha1 (II) collagen-lacZ transgene. The cartilage defects are correlated with failed differentiation of the sclerotome at the time when these cells are normally initiating chondrogenesis. Loss of Bapx1 is accompanied by an increase in apoptotic cell death in affected tissues, although cell cycling rates are unaltered.

Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL.

Acute promyelocytic leukaemia (APL), associated with chromosomal translocations involving the retinoic acid receptor alpha gene (RARA) and the PML gene, is sensitive to retinoic acid (RA) treatment, while APL patients harbouring translocations between RARA and the PLZF gene do not respond to RA. We have generated PML-RARA and PLZF-RARA transgenic mice and show here that these fusion proteins play a critical role in leukaemogenesis and in determining responses to RA in APL, because PLZF-RARA transgenic mice develop RA-resistant leukaemia, while PML-RARA mice are responsive to RA treatment. We demonstrate that both PML-RARalpha and PLZF-RARalpha fusion proteins can act as transcriptional repressors and are able to interact with nuclear receptor transcriptional co-repressors, such as SMRT. PLZF-RARalpha, but not PML-RARalpha, can form, via its PLZF moiety, co-repressor complexes which are insensitive to RA. Histone deacetylase inhibitors such as Trichostatin A (TSA), in combination with RA, can overcome the transcriptional repressor activity of PML-RARalpha and PLZF-RARalpha as well as the unresponsiveness of PLZF-RARalpha-expressing leukaemic cells to RA. Thus, our findings unravel a crucial role for transcriptional silencing in APL pathogenesis and resistance to RA in APL.

Gene rearrangements in the molecular pathogenesis of acute promyelocytic leukemia.

Acute Promyelocytic Leukemia (APL) is a distinct subtype of myeloid leukemia that in the USA alone affects more than 3,000 individuals every year. APL is characterized by three distinct and unique features: i) the accumulation in the bone marrow of tumor cells with promyelocytic features; ii) the invariable association with specific translocations which always involve chromosome 17 and the Retinoic Acid Receptor alpha (RAR alpha) locus; iii) the exquisite sensitivity of APL blasts to the differentiating action of Retinoic Acid (RA). These features have led APL to become the paradigm for therapeutic approaches utilizing differentiating agents. The last 5 years have provided crucial insights into the molecular basis of APL. RAR alpha translocates in 99% of cases to a gene located on chromosome 15 that we initially named myl and subsequently has been called PML. In a few cases, RAR alpha variably translocates to chromosome 11 where it fuses to the PLZF gene or to a newly described partner, NuMA. In addition, RAR alpha is also found translocated to chromosome 5 where it fuses to the NPM gene. The cloning of variant translocations in APL and the comparative analysis of their associated products is crucial for the understanding of the molecular etiopathogenesis of the disease. The generation of animal models, i.e., transgenic mice expressing the fusion genes, will be instrumental in determining the precise contribution of these fusion genes to leukemogenesis. In fact, mice harboring a PML/RAR alpha transgene whose expression is specifically targeted to the myeloid-promyelocytic lineage develop acute myeloid leukemia with promyelocytic features. Moreover, the functional analysis of the various fusion proteins, as well as RAR alpha partners, is revealing striking common features beneath a misleading structural heterogeneity which unravels a possible unifying molecular mechanism towards APL leukemogenesis.

Bapx1: an evolutionary conserved homologue of the Drosophila bagpipe homeobox gene is expressed in splanchnic mesoderm and the embryonic skeleton.

In Drosophila, the visceral mesoderm giving rise to gut musculature is specified by the bagpipe homeobox gene. We have isolated, from both mouse and human, homologues of the bagpipe gene designated Bapx1 and BAPX1, respectively. Bapx1 encodes a predicted protein of 333 amino acids, and has significant regions of homology outside the homeodomain with members of the NK homeobox gene superfamily. Bapx1 maps to the proximal end of chromosome 5 in mouse, near the Msx1 gene. The syntenic region in human corresponds to a chromosomal region containing loci for several skeletal disorders. Bapx1 is first detectable in embryos just prior to axis rotation in lateral plate mesoderm (splanchnic mesoderm) adjacent to the endodermal lining of the prospective gut, and in the most newly formed somites in the region corresponding to the presclerotome, the precursor of the vertebrae. Thus, Bapx1 is one of the earliest developmental markers for the sclerotome portion of the somite and the gut mesentery. Bapx1 continues to be expressed well into organogenesis in lateral plate mesoderm surrounding the mid- and hindgut, and in essentially all cartilaginous condensations which will subsequently undergo endochondral bone formation. The expression pattern of Bapx1 in murine embryos suggests that there are evolutionary conserved mechanisms of visceral mesoderm development across the animal kingdom, and that the mammalian Bapx1 gene may have recently acquired an additional developmental role in skeletal patterning.

Acute leukemia with promyelocytic features in PML/RARalpha transgenic mice.

Acute promyelocytic leukemia (APL) is associated with reciprocal chromosomal translocations involving the retinoic acid receptor alpha (RARalpha) locus on chromosome 17. In the majority of cases, RARalpha translocates and fuses with the promyelocytic leukemia (PML) gene located on chromosome 15. The resulting fusion genes encode the two structurally unique PML/RARalpha and RARalpha/PML fusion proteins as well as aberrant PML gene products, the respective pathogenetic roles of which have not been elucidated. We have generated transgenic mice in which the PML/RARalpha fusion protein is specifically expressed in the myeloid-promyelocytic lineage. During their first year of life, all the PML/RARalpha transgenic mice have an abnormal hematopoiesis that can best be described as a myeloproliferative disorder. Between 12 and 14 months of age, 10% of them develop a form of acute leukemia with a differentiation block at the promyelocytic stage that closely mimics human APL even in its response to retinoic acid. Our results are conclusive in vivo evidence that PML/RARalpha plays a crucial role in the pathogenesis of APL.

Molecular cloning, chromosomal mapping and developmental expression of BAPX1, a novel human homeobox-containing gene homologous to Drosophila bagpipe.

We describe here the cloning of the human BAPX1 gene, a homologue of the Drosophila bagpipe gene which has 87% aa identity within the homeodomain relative to the fly gene. We recently have identified the murine bagpipe homolog. The predicted aa sequence of the human gene has 85% overall identity to the murine gene, with 100% identity in the homeodomain. In mouse, this gene maps to the proximal portion of chromosome 5. We show that the human gene maps to 4p16.1, the human region syntenic with mouse chromosome 5. Expression of BAPX1 was evaluated during human embryonic development by RT-PCR analysis and by RNA in situ hybridization. RT-PCR analysis showed that BAPX1 is expressed in embryo tissues, particularly the limb, and at a lower level in an embryonic lung cell line. RNA in situ hybridization revealed that BAPX1 is predominantly expressed in mesenchymal condensations of the fetal limb and axial skeleton, and in lateral plate mesoderm giving rise to visceral muscle. The expression pattern of BAPX1 combined with the chromosomal localization to 4p16.1, where several human genetic diseases involving dysmorphology of the skeleton have been assigned, raises the potential of it being a candidate gene for one of these disorders. O

An X chromosome-linked gene encoding a protein with characteristics of a rhoGAP predominantly expressed in hematopoietic cells.

An increasingly large number of proteins involved in signal transduction have been identified in recent years and shown to control different steps of cell survival, proliferation, and differentiation. Among the genes recently identified at the tip of the long arm of the human X chromosome, a novel gene, C1, encodes a protein that appears to represent a newly discovered member of the group of signaling proteins involved in regulation of the small GTP binding proteins of the ras superfamily. The protein encoded by C1, p115, is synthesized predominantly in cells of hematopoietic origin. It is characterized by two regions of similarity to motifs present in known proteins: GAP and SH3 homologous regions. Its localization in a narrow cytoplasmic region just below the plasma membrane and its inhibitory effect on stress fiber organization indicate that p115 may down regulate rho-like GTPases in hematopoietic cells.

Isolation of new genes in distal Xq28: transcriptional map and identification of a human homologue of the ARD1 N-acetyl transferase of Saccharomyces cerevisiae.

In this paper, we describe the physical and transcriptional organization of a region of 140 kb in Xq28, 5' to the L1CAM gene. By isolation and mapping of CpG islands to the physical map of the region, isolation of cDNAs, determination of partial nucleotide sequences and study of the pattern of expression and of the orientation of the transcripts identified we have established a transcriptional map of this region. In this map, previously identified genes (L1CAM, V2R, HCF1 and RnBP) have been positioned as well as 3 new genes. All genes in the region are rather small, ranging in size from 2 to 30 kb, and very close to one another. With the exception of the V2R gene, they are housekeeping, have a CpG island at their 5' end and the same orientation of transcription. This kind of organization is consistent with the one previously described for the more distal portion of Xq28, between the Color Vision (CV) and the G6PD genes and indicates that genes with housekeeping and tissue specific pattern of expression are interspersed in the genome but they are probably found in different 'transcriptional domains'. Among the new genes, TE2 demonstrated 40% identity with the protein N-acetyl transferase ARD1 of S. cerevisiae: TE2 may be the human homologue of the S. cerevisiae gene.

Transcriptional organization of a 450-kb region of the human X chromosome in Xq28.

In this paper, we report the transcriptional organization of a 450-kb gene cluster in Xq28, flanked by the glucose-6-phosphate dehydrogenase and the color vision genes. CpG islands previously identified and mapped to distal Xq28 have helped in construction of a continuous contig of cosmids and in identification of cDNAs corresponding to eight transcripts. Thirteen to 16 small genes with CpG islands are clustered in a region of 250-300 kb. Many are highly expressed in muscle or brain and may be the genes responsible for muscle or neurological disorders mapped to distal Xq28. Our analysis indicates that, in this region of the genome, genes not related in sequence are organized in transcriptional domains of 100 kb and that this organization may be important for establishing and regulating gene expression in relation to tissue distribution and X chromosome inactivation.

An archipelago of CpG islands in Xq28: identification and fine mapping of 20 new CpG islands of the human X chromosome.

19 probes for CpG islands, mapping to Xq28, have been used as probes to construct a physical map of genes of this band of the human X chromosome. A total of 22 CpG islands have been precisely mapped in respect to known loci along the 9-10 Mb of Xq28. The fine mapping of such a large number of CpG islands has demonstrated that also in gene rich Giemsa light bands, like Xq28, gene distribution is non uniform: the CpG islands are clustered in the distal portion of the band in a 2 Mb region between the G6PD gene and the DXS15 locus. Moreover, 16 CpG islands were found between the G6PD and the RCP/GCP genes, a region of DNA of only about 300 kb. If this structural organization has a biological function it has yet to be determined. However, the isolation of large genomic regions enriched in gene sequences and the availability of cosmid or YAC contigs will provide the means to test the significance of such gene organization, as well as the material for large sequencing projects and gene search, for the identification of candidate genes for inherited disorders mapped to Xq28 and for comparative mapping.

Methylation and sequence analysis around EagI sites: identification of 28 new CpG islands in XQ24-XQ28.

Thirty-two probes for CpG islands of the distal long arm of the human X chromosome have been identified. From a genomic library of DNA of the hamster-human cell hybrid X3000.1 digested with the rare cutter restriction enzyme EagI, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5' end of genes of the X chromosome has been used to distinguish between EagI sites in CpG islands versus isolated EagI sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5' end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated EagI site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylation of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.

Identification of novel RFLPs in the vicinity of CpG islands in Xq28: application to the analysis of the pattern of X chromosome inactivation.

Probes for CpG islands were cloned from the distal long arm of the human X chromosome; three of them were found to be polymorphic. A HindIII RFLP was identified by the probe 2-25 (DXS606), and it was mapped to the Xq27-Xq28 boundary. Probes 2-19 (DXS605) and 2-55 (DXS707), which identify EcoRI and MspI polymorphisms, respectively, have been mapped to the distal part of Xq28, in the G6PD-RCP/GCP gene region. Probe 2-19 has been further localized about 16 kb from the 3' end of the G6PD gene. The new RFLPs may be useful for the precise mapping of the many disease genes localized in this part of the human X chromosome. Probe 2-19 is highly informative, and it has been studied in greater detail. Using the methylation-sensitive rare-cutter enzyme EagI in conjunction with the polymorphic EcoRI site, we were able to demonstrate that the RFLP may be used both to study randomness of X chromosome inactivation and for carrier detection in X-linked syndromes where nonrandom X inactivation occurs. It is conceivable that the combined use of 2-19 and of the probes described so far (pSPT-PGK and M27 beta) will make analysis of X inactivation feasible in virtually every female.

The nucleotide sequence of a CpG island demonstrates the presence of the first exon of the gene encoding the human lysosomal membrane protein lamp2 and assigns the gene to Xq24.

An EagI-EcoRI clone of human genomic DNA, p2-7, mapped to Xq24 has been sequenced. This analysis has confirmed the presence of a CpG island and has identified the first exon of the human LAMP2 gene, encoding a glycoprotein of the lysosomal membrane. Since the p2-7 clone corresponds to single-copy DNA, we can assign the human LAMP2 gene to Xq24.

Probes for CpG islands on the distal long arm of the human X chromosome are clustered in Xq24 and Xq28.

We have isolated and characterized 55 EagI-containing genomic DNA clones from the distal long arm of the human X chromosome. The presence of additional sites for rare-cutter restriction enzymes and the demethylation of the corresponding genomic DNA demonstrate that at least 30 clones correspond to CpG islands of the Xq24-Xqter region. All clones were regionally mapped with a hybrid panel. The majority are in Xq28 and Xq24 (18 and 14 clones, respectively), 15 are in the Xq26-Xq27 interval, and none is in Xq25. This analysis demonstrates a nonuniform distribution of CpG islands that may reflect the distribution of coding regions in this part of the genome.

Linkage and sequence conservation of the X-linked genes DXS253E (P3) and DXS254E (GdX) in mouse and man.

Lambda G28, a mouse genomic clone homologous to the human P3 gene and associated with a CpG island, also hybridizes to human probes for the neighboring GdX gene. The two genes, P3 and GdX (DXS253E and DXS254E), physically linked on the human X chromosome, lie within a similar physical distance on the mouse X chromosome. The CpG island corresponds to that at the 5' of the human GdX gene. The relative orientation of the two genes is the same. The DNA sequence in coding and noncoding regions is very conserved.

Presence of transcription signals in two putative DNA replication origins of human cells.

We describe the purification and cloning of human DNA replicated at the onset of S phase in HL60 cells synchronized with aphidicolin. A survey of the overall structural properties of these sequences did not show any distinctive features except for an enrichment in Cot0 DNA. The two longer fragments were completely sequenced and studied in more detail. Both were shown to contain transcriptional signals associated with promoters and/or enhancers, such as the binding sites of Sp1, T antigen and nuclear factor III. In one instance, a binding site for a known cellular transcription factor (USF/MLTF) was located inside the sequence by footprinting. Accordingly, by CAT assay and Northern blot, the same sequence was shown to contain an active promoter. The significance of these findings with respect to the role of transcription in initiation of DNA replication at the origin is discussed. None of the tested fragments exhibited autonomously replicating sequence (ARS) activity in transfected cells. The problems connected with the detection of ARS activity in human cells are critically examined.

Characterization of human DNA sequences synthesized at the onset of S-phase.

We developed a method of enrichment for DNA replicated at the onset of S-phase in synchronized human HL60 cells. About 200 such sequences were cloned. The analysis of this selected DNA sample showed that: 1) the cloned DNA fragments derive from a limited number (750-1500) of replicons; 2) there is no extensive homology between different DNA fragments; 3) they are not significantly enriched in highly repeated sequences; 4) they are enriched in snap-back (Cot = o) DNA. The sequence of the longest fragment revealed the presence of numerous signals collected in a few hundred nucleotides: 1) homology with the origin of replication of human Papovaviruses usually associated with potential stem-loop structures; 2) binding sites for known transcription factors and for another nuclear factor; 3) potential binding sites for the chromosome "scaffold".