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Biomedical subjects

J D Licht

Publications and source records attributed to J D Licht.

At least 19 recordsLinked to original sources
PubMed2002

The human promyelocytic leukemia zinc finger gene is regulated by the Evi-1 oncoprotein and a novel guanine-rich site binding protein.

PLZF (promyelocytic leukemia zinc finger ) is a transcription factor disrupted in t(11;17)-associated acute promyelocytic leukemia which is highly expressed in undifferentiated myeloid cells. To address the tissue-specific regulation of the promoter, we isolated sequences 1.2-kb 5' to the transcriptional start site. Sequence analysis demonstrated that this region contains one TATA box and several putative transcription factor binding sites including four G/C-rich sites and one Evi-1-like site. A fragment of the promoter spanning 158-bp upstream of the transcription start site displayed relative specificity for PLZF-expressing myeloid cells. Functional promoter assays revealed that an Evi-1-like site at -140/-130 was essential for full promoter activity in every cell line tested while a G-rich site at -15/-7 was important for tissue specificity. Electrophoretic mobility shift assays showed that Evi-1 binds specifically to -140/-130 Evi-1-like site and overexpression of Evi-1 in K562 cells activated the PLZF promoter. UV cross-linking assays showed that the proximal, tissue specific element at -15/-7 bound a novel 28 kDa protein. These results indicate as with other myeloid genes, a relatively small segment of DNA can direct tissue-specific expression, but unlike other myeloid promoters, no critical PU.1 or C/EBP sites were found.

Base Sequence↗
PubMed2001

The acute promyelocytic leukemia-associated protein, promyelocytic leukemia zinc finger, regulates 1,25-dihydroxyvitamin D(3)-induced monocytic differentiation of U937 cells through a physical interaction with vitamin D(3) receptor.

Monocyte differentiation induced by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is interrupted during the course of acute promyelocytic leukemia (APL). One form of APL is associated with the translocation t(11;17), which joins the promyelocytic leukemia zinc finger (PLZF) and retinoic acid receptor alpha (RARalpha) genes. Because PLZF is coexpressed in the myeloid lineage with the vitamin D(3) receptor (VDR), the interplay between PLZF and VDR was examined. It was found that PLZF interacts directly with VDR. This occurred at least partly through contacts in the DNA-binding domain of VDR and the broad complex, tram-trak, bric-a-brac/pox virus zinc finger (BTB/POZ) domain of PLZF. Moreover, PLZF altered the mobility of VDR derived from nuclear extracts when bound to its cognate binding site, forming a slowly migrating DNA-protein complex. Overexpression of PLZF in a monocytic cell line abrogated 1,25(OH)(2)D(3) activation from both a minimal VDR responsive reporter and the promoter of p21(WAF1/CIP1), a target gene of VDR. Deletion of the BTB/POZ domain significantly relieved PLZF-mediated repression of 1,25(OH)(2)D(3)-dependent activation. In addition, stable, inducible expression of PLZF in U937 cells inhibited the ability of 1,25(OH)(2)D(3) to induce surface expression of the monocytic marker CD14 and morphologic changes associated with differentiation. These results suggest that PLZF may play an important role in regulating the process by which 1,25(OH)(2)D(3) induces monocytic differentiation in hematopoietic cells.

Binding Sites↗
PubMed2001

Translocations of the RARalpha gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) has been recognized as a distinct clinical entity for over 40 years. Although relatively rare among hematopoietic malignancies (approximately 10% of AML cases), this disease has attracted a particularly good share of attention by becoming the first human cancer in which all-trans-retinoic acid (ATRA), a physiologically active derivative of vitamin A, was able to induce complete remission (CR). ATRA induced remission is not associated with rapid cell death, as in the case of conventional chemotherapy, but with a restoration of the 'normal' granulocytic differentiation pathway. With this remarkable medical success story APL has overnight become a paradigm for the differentiation therapy of cancer. A few years later, excitement with APL was further enhanced by the discovery that a cytogenetic marker for this disease, the t(15:17) reciprocal chromosomal translocation, involves a fusion between the retinoic acid receptor alpha (RARalpha) gene and a previously unknown locus named promyelocytic leukemia (PML). Consequence of this gene rearrangement is expression of the PML-RARalpha chimeric oncoprotein, which is responsible for the cellular transformation as well as ATRA response that is observed in APL. Since this initial discovery, a number of different translocation partner genes of RARalpha have been reported in rarer cases of APL, strongly suggesting that disruption of RARalpha underlies its pathogenesis. This article reviews various rearrangements of the RARalpha gene that have so far been described in literature, functions of the proteins encoded by the different RARalpha partner loci, and implications that these may have for the molecular pathogenesis of APL.

Antigens, Nuclear↗
PubMed2001

Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation.

Sprouty was genetically identified as an antagonist of fibroblast growth factor signaling during tracheal branching in Drosophila. In this study, we provide a functional characterization of mammalian Sprouty1 and Sprouty2. Sprouty1 and Sprouty2 inhibited events downstream of multiple receptor tyrosine kinases and regulated both cell proliferation and differentiation. Using NIH3T3 cell lines conditionally expressing Sprouty1 or Sprouty2, we found that these proteins specifically inhibit the Ras/Raf/MAP kinase pathway by preventing Ras activation. In contrast, activation of the phosphatidylinositol 3-kinase pathway was not affected by Sprouty1 or Sprouty2. We further showed that Sprouty1 and Sprouty2 do no prevent the formation of a SNT.Grb2.Sos complex upon fibroblast growth factor stimulation, yet block Ras activation. Taken together, these results establish mammalian Sprouty proteins as important negative regulators of growth factor signaling and suggest that Sprouty proteins act downstream of the Grb2.Sos complex to selectively uncouple growth factor signals from Ras activation and the MAP Kinase pathway.

3T3 Cells↗
PubMed2001

Cloning and characterization of a novel mouse AP-2 transcription factor, AP-2delta, with unique DNA binding and transactivation properties.

AP-2 transcription factors are sequence-specific DNA-binding proteins expressed in neural crest and other tissues during mammalian development. Three mammalian genes, AP-2alpha, AP-2beta, and AP-2gamma, have been reported previously. A partial predicted AP-2 gene was identified in tandem with AP-2beta on human chromosome 6p12-p21.1. The orthologous mouse gene, which we named Ap-2delta, was identified from a fetal mouse head cDNA library. Northern analysis revealed two transcripts in embryonic and newborn mouse brain, with markedly higher steady-state levels in the former. The predicted Ap-2delta protein comprised 452 amino acids and was highly similar to other AP-2 proteins across the DNA-binding and dimerization domains. Ap-2delta formed homodimers and heterodimers in vitro, bound an optimized AP-2 consensus DNA sequence, and transactivated gene expression in eukaryotic cells. Ap-2delta dimers bound poorly to an AP-2 binding sequence from the human metallothionein IIa promoter in vitro, revealing a sequence specificity not previously observed among other AP-2 proteins. The PY motif and critical residues in the transactivation domain, which are highly conserved in the AP-2 family and believed necessary for transactivation, were divergent in Ap-2delta. The unique protein sequence and functional features of Ap-2delta suggest mechanisms, besides tissue-specific AP-2 gene expression, for specific control of target gene activation.

Amino Acid Sequence↗
PubMed2001

The HIV-1 transactivator protein Tat is a potent inducer of the human DNA repair enzyme beta-polymerase.

OBJECTIVE: This study examines the effects of the HIV-1 regulatory proteins, Tat and Rev, on the expression of the DNA polymerase beta (beta-pol) gene, which encodes a key protein in the DNA base-excision repair pathway. The rationale for these experiments is to examine the potential involvement of base-excision repair protein deregulation in HIV-1-related lymphomas. DESIGN: Expression of beta-pol mRNA was examined in AIDS-related lymphomas and non-AIDS-related lymphomas and as a function of HIV-1 infection of B cells in culture. The effect of Tat or Rev over-expression on beta-pol promoter expression was tested by transient co-transfection assays with a beta-pol promoter reporter plasmid and a Tat or Rev over-expression plasmid. METHODS: Northern blot analysis was used to quantitate beta-pol expression in lymphoma and cells. Raji cells were co-transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid and a plasmid over-expressing Tat or Rev. CAT activity was measured in transfected cells. RESULTS: beta-Pol mRNA was > 10-fold higher in AIDS-related than in non-AIDS B-lineage lymphomas. beta-Pol expression was up-regulated in a B-cell line upon infection with HIV-1, and increased in Raji cells upon recombinant expression of the Tat gene. The beta-pol promoter was transactivated (fourfold induction) by Tat, but not by Rev. Tat-dependent transactivation required a binding site for the transcription factor Sp1 in the beta-pol promoter. CONCLUSION: These results suggest that HIV-1 Tat can interact with cellular transcription factors to increase the steady-state level of beta-pol in B cells. Tat-mediated induction of beta-pol may alter DNA stability in AIDS-related lymphomas.

Animals↗
PubMed2000

AML-1/ETO fusion protein is a dominant negative inhibitor of transcriptional repression by the promyelocytic leukemia zinc finger protein.

The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RAR alpha fusion protein and, in a similar manner, inhibits RAR alpha target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RAR alpha and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia. (Blood. 2000;96:3939-3947)

Binding Sites↗
PubMed2000

BRCA1 physically and functionally interacts with ATF1.

BRCA1, a breast and ovarian cancer susceptibility gene, encodes a 220-kDa protein whose precise biochemical function remains unclear. BRCA1 contains an N-terminal RING finger that mediates protein-protein interaction. The C-terminal domain of BRCA1 (BRCT) can activate transcription and interacts with RNA polymerase holoenzyme. Using the yeast two-hybrid system, we identified an interaction between the BRCA1 RING finger and ATF1, a member of the cAMP response element-binding protein/activating transcription factor (CREB/ATF) family. We demonstrate that BRCA1 and ATF1 can physically associate in vitro, in yeast, and in human cells. BRCA1 stimulated transcription from a cAMP response element reporter gene in transient transfections. BRCA1 also stimulated transcription from a natural promoter, that of tumor necrosis factor-alpha, in a manner dependent on the integrity of the cAMP response element. These results implicate BRCA1 in transcriptional activation of ATF1 target genes, some of which are involved in the transcriptional response to DNA damage.

Activating Transcription Factor 1↗
PubMed2000

E-cadherin is a WT1 target gene.

The WT1 tumor suppressor gene encodes a transcription factor that can activate and repress gene expression. Transcriptional targets relevant for the growth suppression functions of WT1 are poorly understood. We found that mesenchymal NIH 3T3 fibroblasts stably expressing WT1 exhibit growth suppression and features of epithelial differentiation including up-regulation of E-cadherin mRNA. Acute expression of WT1 in NIH 3T3 fibroblasts after retroviral infection induced murine E-cadherin expression. In transient transfection experiments, the human and murine E-cadherin promoters were activated by co-expression of WT1. E-cadherin promoter activity was increased in cells overexpressing WT1 and was blocked by a dominant negative form of WT1. WT1 activated the murine E-cadherin promoter through a conserved GC-rich sequence similar to an EGR-1 binding site as well as through a CAAT box sequence. WT1 produced in vitro or derived from nuclear extracts bound to the WT1-response element within the murine E-cadherin promoter, but not the CAAT box. E-cadherin, a gene important in epithelial differentiation and neoplastic transformation, represents a downstream target gene that links the roles of the WT1 in differentiation and growth control.

3T3 Cells↗
PubMed2000

In-depth mutational analysis of the promyelocytic leukemia zinc finger BTB/POZ domain reveals motifs and residues required for biological and transcriptional functions.

The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.

Amino Acid Motifs↗
PubMed2000

The ETO protein disrupted in t(8;21)-associated acute myeloid leukemia is a corepressor for the promyelocytic leukemia zinc finger protein.

The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.

Acute Disease↗
PubMed1999

A novel BTB/POZ transcriptional repressor protein interacts with the Fanconi anemia group C protein and PLZF.

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome. The phenotype includes developmental defects, bone marrow failure, and cell cycle abnormalities. At least eight complementation groups (A-H) exist, and although three of the corresponding complementation group genes have been cloned, they lack recognizable motifs, and their functions are unknown. We have isolated a binding partner for the Fanconi anemia group C protein (FANCC) by yeast two-hybrid screening. We show that the novel gene, FAZF, encodes a 486 amino acid protein containing a conserved amino terminal BTB/POZ protein interaction domain and three C-terminal Krüppel-like zinc fingers. FAZF is homologous to the promyelocytic leukemia zinc finger (PLZF) protein, which has been shown to act as a transcriptional repressor by recruitment of nuclear corepressors (N-CoR, Sin3, and HDAC1 complex). Consistent with a role in FA, BTB/POZ-containing proteins have been implicated in oncogenesis, limb morphogenesis, hematopoiesis, and proliferation. We show that FAZF is a transcriptional repressor that is able to bind to the same DNA target sequences as PLZF. Our data suggest that the FAZF/FANCC interaction maps to a region of FANCC deleted in FA patients with a severe disease phenotype. We also show that FAZF and wild-type FANCC can colocalize in nuclear foci, whereas a patient-derived mutant FANCC that is compromised for nuclear localization cannot. These results suggest that the function of FANCC may be linked to a transcriptional repression pathway involved in chromatin remodeling.

Amino Acid Sequence↗
PubMed1999

The promyelocytic leukemia zinc finger (PLZF) protein binds DNA in a high molecular weight complex associated with cdc2 kinase.

A binding site selection from a CpG island library for the promyelocytic leukemia zinc finger protein (PLZF) identified two high affinity PLZF binding sites. These sequences also bound RARalpha/PLZF, a fusion protein formed in chromosomal translocation t(11;17)(q23;q21) associated with acute promyelocytic leukemia. PLZF bound DNA as a slowly migrating complex with an estimated mol. wt of 600 kDa whose formation was dependent on the POZ/dimerization domain of PLZF. The PLZF-DNA complex was unable to form in the presence of cdc2 antibodies. A PLZF-cdc2 interaction was further demonstrated by co-immunoprecipitation and a biotin-streptavidin pull-down assay. PLZF is a phosphoprotein and immunoprecipi-tates with a cdc2-like kinase activity. The PLZF-DNA complex was abolished with the addition of a phosphatase. These studies suggest that the activity of PLZF, a regulator of the cell cycle, may be modulated by cell cycle proteins. RARalpha/PLZF did not complex with cdc2, this potentially contributing to its aberrant transcriptional properties and potential role in leukemo-genesis.

Animals↗
PubMed1999

Genomic structure of the human PLZF gene.

The human PLZF (promyelocytic leukaemia zinc finger) gene encodes a Krüppel-like zinc finger protein, which was identified via the reciprocal translocation t(11;17)(q23;q21) fusing it to the retinoic acid receptor alpha (RARalpha) gene in promyelocytic leukaemia. To determine its complete genomic organisation, we constructed a cosmid-map fully containing the hPLZF gene. The gene has seven exons, including a novel 5' untranslated exon, varying in size from 87 to 1358bp and spans at least 120kb. Flanking intronic sequences were identified and all splice acceptor and donor sites conformed to the gt/ag rule. Five polymorphic markers could be fine located in its vicinity. These data will facilitate mutation analysis of hPLZF in t(11;17) leukaemia cases, as well as assist mapping and loss-of-heterozygosity analysis. Here we have tested hPLZF as a possible candidate for the PGL1 locus involved in hereditary head and neck paragangliomas. However, mutation analysis revealed no aberration in 12 paraganglioma patients from different families.

Base Sequence↗
PubMed1999

Tumor-associated WT1 missense mutants indicate that transcriptional activation by WT1 is critical for growth control.

The WT1 gene encodes a zinc finger DNA binding transcription factor and is mutated in up to 15% of Wilms tumor cases. The WT1 protein binds to the promoters of many genes through GC- or TC-rich sequences and can function both as a transcriptional repressor and an activator in co-transfection assays depending on the cell type, the structure of the test promoter, and even the expression vectors used. Engineered expression of WT1 can lead to growth suppression by both cell cycle arrest and induction of apoptosis. However, the transcriptional activity of WT1 that is required for growth control was not defined. We found that three N-terminal tumor-associated missense mutations of WT1 were defective for activation of both a synthetic reporter containing WT1-binding sites as well as the promoter of a WT1 responsive gene, p21. These mutants failed to inhibit cell growth but still retain their ability to repress several putative WT1 target promoters. These results indicate that activation and not repression by WT1 is the critical transcriptional activity of the protein responsible for its growth suppressing properties.

3T3 Cells↗