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C Largman

Publications and source records attributed to C Largman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia.

Multiple members of the A, B, and C clusters of Hox genes are expressed in hematopoietic cells. Several of these Hox genes have been found to display distinctive expression patterns, with genes located at the 3' side of the clusters being expressed at their highest levels in the most primitive subpopulation of human CD34+ bone marrow cells and genes located at the 5' end having a broader range of expression, with downregulation at later stages of hematopoietic differentiation. To explore if these patterns reflect different functional activities, we have retrovirally engineered the overexpression of a 5'-located gene, HOXA10, in murine bone marrow cells and demonstrate effects strikingly different from those induced by overexpression of a 3'-located gene, HOXB4. In contrast to HOXB4, which causes selective expansion of primitive hematopoietic cells without altering their differentiation, overexpression of HOXA10 profoundly perturbed myeloid and B-lymphoid differentiation. The bone marrow of mice reconstituted with HOXA10-transduced bone marrow cells contained in high frequency a unique progenitor cell with megakaryocytic colony-forming ability and was virtually devoid of unilineage macrophage and pre-B-lymphoid progenitor cells derived from the transduced cells. Moreover, and again in contrast to HOXB4, a significant proportion of HOXA10 mice developed a transplantable acute myeloid leukemia with a latency of 19 to 50 weeks. These results thus add to recognition of Hox genes as important regulators of hematopoiesis and provide important new evidence of Hox gene-specific functions that may correlate with their normal expression pattern.

Acute Disease↗
PubMed1997

AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.

Animals↗
PubMed1996

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro.

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

Base Sequence↗
PubMed1996

Hox homeodomain proteins exhibit selective complex stabilities with Pbx and DNA.

Eight of the nine homeobox genes of the Hoxb locus encode proteins which contain a conserved hexapeptide motif upstream from the homeodomain. All eight proteins (Hoxb-1-Hoxb-8) bind to a target oligonucleotide in the presence of Pbx1a under conditions where minimal or no binding is detected for the Hox or Pbx1a proteins alone. The stabilities of the Hox-Pbx1a-DNA complexes vary >100-fold, with the proteins from the middle of the locus (Hoxb-5 and Hoxb-6) forming very stable complexes, while Hoxb-4, Hoxb-7 and Hoxb-8 form complexes of intermediate stability and proteins at the 3'-side of the locus (Hoxb-1-Hoxb-3) form complexes which are very unstable. Although Hox-b proteins containing longer linker sequences between the hexapeptide and homeodomains formed unstable complexes, shortening the linker did not confer complex stability. Homeodomain swapping experiments revealed that this motif does not independently determine complex stability. Naturally occurring variations within the hexapeptides of specific Hox proteins also do not explain complex stability differences. However, two core amino acids (tryptophan and methionine) which are absolutely conserved within the hexapeptide domains appear to be required for complex formation. Removal of N- and C-terminal flanking regions did not influence complex stability and the members of paralog group 4 (Hoxa-4, b-4, c-4 and d-4), which share highly conserved hexapeptides, linkers and homeodomains but different flanking regions, form complexes of similar stability. These data suggest that the structural features of Hox proteins which determine Hox-Pbx1a-DNA complex stability reside within the precise structural relationships between the homeodomain, hexapeptide and linker regions.

Amino Acid Sequence↗
PubMed1996

The role of HOX homeobox genes in normal and leukemic hematopoiesis.

A sizable amount of new data points to a role for the HOX family of homeobox genes in hematopoiesis. Recent studies have demonstrated that HOXA and HOXB genes are expressed in human CD34+ cells, and are downregulated as cells leave the CD34+ compartment. In addition, expression of certain genes, including HOXB3 and HOXB4, is largely restricted to the long-term culture-initiating cell enriched pool, containing the putative stem cell population. Studies have also shown that HOX genes appear to be important for normal T lymphocyte and activated natural killer cell function. Overexpression of Hox-b4 in transplanted murine marrow cell results in a dramatic expansion of stem cells, while maintaining normal peripheral blood counts. In contrast, overexpression of Hox-a10 resulted in expansion of progenitor pools, accompanied by unique changes in the differentiation patterns of committed progenitors. Overexpression of Hox-a10 or Hox-b8 led to the development of myeloid leukemias, while animals transfected with marrow cells overexpressing Hox-b4 do not appear to develop malignancies. Blockade of HOX gene function using antisense oligonucleotides has revealed that several HOX genes appear to influence either myeloid or erythroid colony formation. Mice homozygous for a targeted disruption of the HOX-a9 gene show reduced numbers of granulocytes and lymphocytes, smaller spleens and thymuses, and reduced numbers of committed progenitors. These studies demonstrate that HOX homeobox genes play a role in both the early stem cell function as well as in later stages of hematopoietic differentiation, and that perturbations of HOX gene expression can be leukemogenic.

Animals↗
PubMed1996

Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus.

Pbx cofactors are implicated to play important roles in modulating the DNA-binding properties of heterologous homeodomain proteins, including class I Hox proteins. To assess how Pbx proteins influence Hox DNA-binding specificity, we used a binding-site selection approach to determine high-affinity target sites recognized by various Pbx-Hox homeoprotein complexes. Pbx-Hox heterodimers preferred to bind a bipartite sequence 5'-ATGATTNATNN-3' consisting of two adjacent half sites in which the Pbx component of the heterodimer contacted the 5' half (ATGAT) and the Hox component contacted the more variable 3' half (TNATNN). Binding sites matching the consensus were also obtained for Pbx1 complexed with HoxA10, which lacks a hexapeptide but requires a conserved tryptophan-containing motif for cooperativity with Pbx. Interactions with Pbx were found to play an essential role in modulating Hox homeodomain amino-terminal arm contact with DNA in the core of the Hox half site such that heterodimers of different compositions could distinguish single nucleotide alterations in the Hox half site both in vitro and in cellular assays measuring transactivation. When complexed with Pbx, Hox proteins B1 through B9 and A10 showed stepwise differences in their preferences for nucleotides in the Hox half site core (TTAT to TGAT, 5' to 3') that correlated with the locations of their respective genes in the Hox cluster. These observations demonstrate previously undetected DNA-binding specificity for the amino-terminal arm of the Hox homeodomain and suggest that different binding activities of Pbx-Hox complexes are at least part of the position-specific activities of the Hox genes.

Animals↗
PubMed1995

Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo.

Hox genes were first recognized for their role in embryonic development and may also play important lineage-specific functions in a variety of somatic tissues including the hematopoietic system. We have recently shown that certain members of the Hox A and B clusters, such as HOXB3 and HOXB4, are preferentially expressed in subpopulations of human bone marrow that are highly enriched for the most primitive hematopoietic cell types. To assess the role these genes may play in regulating the proliferation and/or differentiation of such cells, we engineered the overexpression of HOXB4 in murine bone marrow cells by retroviral gene transfer and analyzed subsequent effects on the behavior of various hematopoietic stem and progenitor cell populations both in vitro and in vivo. Serial transplantation studies revealed a greatly enhanced ability of HOXB4-transduced bone marrow cells to regenerate the most primitive hematopoietic stem cell compartment resulting in 50-fold higher numbers of transplantable totipotent hematopoietic stem cells in primary and secondary recipients, compared with serially passaged neo-infected control cells. This heightened expansion in vivo of HOXB4-transduced hematopoietic stem cells was not accompanied by identifiable anomalies in the peripheral blood of these mice. Enhanced proliferation in vitro of day-12 CFU-S and clonogenic progenitors was also documented. These results indicate HOXB4 to be an important regulator of very early but not late hematopoietic cell proliferation and suggest a new approach to the controlled amplification of genetically modified hematopoietic stem cell populations.

Animals↗
PubMed1995

Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins.

The human proto-oncogene PBX1 codes for a homolog of Drosophila extradenticle, a divergent homeo domain protein that modulates the developmental and DNA-binding specificity of select HOM proteins. We demonstrate that wild-type Pbx proteins and chimeric E2a-Pbx1 oncoproteins cooperatively bind a consensus DNA probe with HoxB4, B6, and B7 of the Antennapedia class of Hox/HOM proteins. Specificity of Hox-Pbx interactions was suggested by the inability of Pbx proteins to cooperatively bind the synthetic DNA target with HoxA10 or Drosophila even-skipped. Site-directed mutagenesis showed that the hexapeptide motif (IYPWMK) upstream of the Hox homeo domain was essential for HoxB6 and B7 to cooperatively bind DNA with Pbx proteins. Engraftment of the HoxB7 hexapeptide onto HoxA10 endowed it with robust cooperative properties, demonstrating a functional role for the highly conserved hexapeptide element as one of the molecular determinants delimiting Hox-Pbx cooperativity. The Pbx homeo domain was necessary but not sufficient for cooperativity, which required conserved amino acids carboxy-terminal of the homeo domain. These findings demonstrate that interactions between Hox and Pbx proteins modulate their DNA-binding properties, suggesting that Pbx and Hox proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes.

Amino Acid Sequence↗
PubMed1995

Stage- and lineage-specific expression of the HOXA10 homeobox gene in normal and leukemic hematopoietic cells.

There is growing evidence that the HOX homeobox-containing transcription factors are differentially expressed during hematopoiesis. We have previously demonstrated that the HOXA10 gene is expressed in unfractionated normal marrow and in immortalized leukemic cell lines with myelomonocytic features, but not in cell lines with lymphoid or erythroid features. To gain insights into the patterns of activation of this gene during hematopoietic differentiation, we have examined HOXA10 expression in CD34+ and CD34- subfractions of normal marrow and normal peripheral blood, as well as samples from patients with a variety of acute and chronic leukemias. HOXA10 is strongly expressed in CD34+ normal marrow cells, markedly downregulated in CD34- marrow cells, and inactive in mature neutrophils, monocytes, and lymphocytes. HOXA10 is expressed in all types of acute myelogenous leukemia (AML) with the notable exception of acute promyelocytic leukemia (AML-M3). HOXA10 message is observed in chronic myelogenous leukemia (CML) but appears to be reduced in accelerated phase and blast crisis, particularly lymphoid blast crisis. With rare exception, HOXA10 expression is not observed in samples of acute or chronic lymphoid leukemias. Normal marrow and patient samples appear to contain a single transcript which encodes a full-length homeobox-containing protein, while immortalized cell lines contain an additional alternatively spliced transcript. These studies indicate that HOXA10 expression is restricted to early stages of myeloid differentiation.

Base Sequence↗
PubMed1994

Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells.

Class I homeobox (Hox) genes encode a major group of transcription factors controlling embryonic development and have been implicated in the continuing process of hematopoietic cell differentiation. They are clustered on four chromosomes and, in early development, exhibit spatially restricted expression with respect to their 3'-->5' chromosomal position. By using an improved PCR-based method for amplifying total cDNA derived from limited cell numbers, we now describe the expression of class I Hox genes in highly purified CD34+ cell subpopulations isolated from normal human bone marrow that represent functionally distinct stem and progenitor cell compartments. Our data indicate that at least 16 different Hox genes, mainly from the A and the B clusters, are expressed in one or more of these subpopulations of human hematopoietic cells. Moreover, markedly elevated expression of some of the Hox genes found at the 3' end of the A and B clusters (e.g., HoxB3) was a unique feature of the subpopulations that contained the most primitive functionally defined cells, whereas genes located in the 5' region of each cluster (e.g., HoxA10) were found to be expressed at nearly equal levels in the CD34+ subpopulations analyzed. In contrast to the findings for CD34+ cells, expression of two selected Hox genes, HoxB3 and HoxA10, was virtually extinguished in the CD34- fraction of bone marrow cells. These results demonstrate the expression of a broad range of Hox genes in primitive hematopoietic cells and point to the existence of a regulated program of Hox gene expression during their normal development.

Antigens, CD↗
PubMed1993

Expression of class I homeobox genes in fetal and adult murine skin.

We examined the expression patterns of several class I homeobox genes in mouse fetal and adult skin. All the genes of the Hox-B locus, except Hoxb-1, are expressed in skin from murine fetuses of 17 and 18 d gestation, at which time the epidermis is undergoing stratification and differentiation. The amount of individual Hox gene message varies considerably, but expression of all genes is detectable by RNase protection except Hoxb-1, which could not be detected even by the reverse transcription-polymerase chain reaction (RT-PCR) assay. Homeobox gene expression in skin is not confined to the Hox-B locus; the paralogous genes Hoxa-4, -b-4, -c-4, and -d-4 are all expressed. The amount of Hoxb-4, -b-2, and -c-4 message in skin is relatively constant from the earliest gestational day examined (day 16) through birth at day 19. Expression of several homeobox genes is also seen in adult skin.

Aging↗
PubMed1993

Expression of the Hox 2.2 homeobox gene in murine embryonic epidermis.

The expression of the Hox 2.2 gene was studied in mouse fetal skin by in situ hybridization with an antisense RNA probe derived from the homeobox region of this gene. In contrast to the expression of Hox 2.2 in spinal cord, which is strongest in 11-day embryos, and is greatly diminished by day 14 and day 17, the signal for Hox 2.2 in skin could be not be detected in 11-day epidermis, was barely detectable on day 14, became strong on day 17, and decreased in new-born animals (day 19). RNase protection assays using Hox 2.2 homeobox-containing and 3' flanking region probes confirmed that the signals detected in 17-day fetal skin by in situ hybridization represent Hox 2.2 transcripts, and that the message is expressed throughout the day 15 to day 18 period during which the epidermis is undergoing terminal differentiation. RNase protection analysis also revealed two alternatively spliced forms of the Hox 2.2 mRNA are present throughout fetal skin development. Northern gel analysis of 17-day fetal skin using a Hox 2.2 homeobox-containing probe at high stringency showed two bands of 1.6 and 1.9 kb, respectively. The 1.9 kb band was greatly enhanced by hybridization at reduced stringency, suggesting the expression of additional homeobox genes with homology to Hox 2.2. These results suggest that the Hox 2.2 homeobox gene plays a role in epidermal development.

Animals↗
PubMed1993

Expression of HOX C homeobox genes in lymphoid cells.

The class I homeobox genes located in four clusters in mammalian genomes (HOX A, HOX B, HOX C, and HOX D) appear to play a major role in fetal development. Previous surveys of homeobox gene expression in human leukemic cell lines have shown that certain HOX A genes are expressed only in myeloid cell lines, whereas HOX B gene expression is largely restricted to cells with erythroid potential. We now report a survey of the expression patterns of 9 homeobox genes from the HOX C locus in a panel of 24 human and 7 murine leukemic cell lines. The most striking observation is the lymphoid-specific pattern of expression of HOX C4, located at the 3' end of the locus. A major transcript of 1.9 kilobases is observed in both T-cell and B-cell lines. HOX C4 expression is also detected in normal human marrow and peripheral blood lymphocytes, but not in mature granulocytes or monocytes. HOX C8 is also expressed in human lymphoid cells but is expressed in other blood cell types as well. However, the HOX C8 transcript pattern is lineage specific. These data, in conjunction with earlier findings, suggest that homeobox gene expression influences lineage determination during hematopoiesis.

Animals↗
PubMed1992

DNA sequence of the yeast transketolase gene.

Transketolase (EC 2.2.1.1) is the enzyme that, together with aldolase, forms a reversible link between the glycolytic and pentose phosphate pathways. We have cloned and sequenced the transketolase gene from yeast (Saccharomyces cerevisiae). This is the first transketolase gene of the pentose phosphate shunt to be sequenced from any source. The molecular mass of the proposed translated protein is 73,976 daltons, in good agreement with the observed molecular mass of about 75,000 daltons. The 5'-nontranslated region of the gene is similar to other yeast genes. There is no evidence of 5'-splice junctions or branch points in the sequence. The 3'-nontranslated region contains the polyadenylation signal (AATAAA), 80 base pairs downstream from the termination codon. A high degree of homology is found between yeast transketolase and dihydroxyacetone synthase (formaldehyde transketolase) from the yeast Hansenula polymorpha. The overall sequence identity between these two proteins is 37%, with four regions of much greater similarity. The regions from amino acid residues 98-131, 157-182, 410-433, and 474-489 have sequence identities of 74%, 66%, 83%, and 82%, respectively. One of these regions (157-182) includes a possible thiamin pyrophosphate (TPP) binding domain, and another (410-433) may contain the catalytic domain.

Aldehyde-Ketone Transferases↗
PubMed1992

Modulation of homeobox gene expression alters the phenotype of human hematopoietic cell lines.

We have previously reported that certain genes of the HOX2 cluster of homeobox genes on human chromosome 17 are specifically expressed in human leukemic cell lines with erythroid potential, suggesting that these genes are involved in hematopoietic differentiation. We now show that the expression of the HOX 2.2 gene decreases during erythropoietin-induced differentiation of the erythroid cell line MB02. In order to study the role of the HOX 2.2 homeobox gene in hematopoiesis, vectors producing sense or antisense transcripts were introduced into K562 and HEL cells, pluripotent lines with erythroid and myeloid features. Overexpression of HOX 2.2 is associated with loss of erythroid features in both lines and an increase in certain myelomonocytic markers in K562 cells. Expression of antisense HOX 2.2 is associated with an increase in erythroid features in HEL cells and a mild decrease in myeloid characteristics in K562 cells. Overexpression of the adjacent HOX 2.1 gene in K562 cells does not produce similar phenotype changes. These data demonstrate that modulation of a specific HOX 2 homeobox gene can change the phenotype of somatic cells and suggest that certain HOX 2 genes play a role in blood cell differentiation.

Blotting, Northern↗
PubMed1992

Modulation of HOX2 gene expression following differentiation of neuronal cell lines.

The expression of the genes in the human HOX2 locus has been studied during differentiation of two human neuroblastoma (SH-SY5Y and Kelly), a human glioblastoma (251-MG), and the murine F9 embryonal carcinoma cell lines. Cells were differentiated with retinoic acid (RA), or with RA together with dibutyral cyclic AMP (db-cAMP) and nerve growth factor (NGF) in order to assess the changes in the expression patterns of these homeobox genes during neuronal differentiation. We show that the genes of the HOX2 locus are expressed in a complex transcription pattern that varies with cell type. The two uninduced neuroblastoma cell lines show a similar pattern of expression for a number of HOX2 genes although the levels of expression are different for individual cell lines. The embryonal carcinoma cell line F9 expresses low levels of several HOX2 genes which is restricted to the 5' region of the HOX2 cluster. The glioblastoma cell line, 251-MG expresses almost all of the genes of the HOX2 locus. Differentiation of these cells modulates the expression of the HOX2 genes in a manner that is dependent upon the cell type as well as the differentiation factor. Differentiation affects both the level of HOX2 gene expression and the distribution of transcript sizes. In conclusion, our analysis reveals a complex pattern of expression for the genes of the HOX2 locus in neuronal and glial cells and suggests that the cell-specific expression of these genes may be correlated with the phenotypic differences that are observed between different neuronal and glial cell populations within the nervous system.

Animals↗