C-terminal Zinc Finger Motifs Research Articles

Sibling rivalry among the ZBTB transcription factor family: homodimers versus heterodimers.

The BTB domain is an oligomerization domain found in over 300 proteins encoded in the human genome. In the family of BTB domain and zinc finger-containing (ZBTB) transcription factors, 49 members share the same protein architecture. The N-terminal BTB domain is structurally conserved among the family members and serves as the dimerization site, whereas the C-terminal zinc finger motifs mediate DNA binding. The available BTB domain structures from this family reveal a natural inclination for homodimerization. In this study, we investigated the potential for heterodimer formation in the cellular environment. We selected five BTB homodimers and four heterodimer structures. We performed cell-based binding assays with fluorescent protein-BTB domain fusions to assess dimer formation. We tested the binding of several BTB pairs, and we were able to confirm the heterodimeric physical interaction between the BTB domains of PATZ1 and PATZ2, previously reported only in an interactome mapping experiment. We also found this pair to be co-expressed in several immune system cell types. Finally, we used the available structures of BTB domain dimers and newly constructed models in extended molecular dynamics simulations (500 ns) to understand the energetic determinants of homo- and heterodimer formation. We conclude that heterodimer formation, although frequently described as less preferred than homodimers, is a possible mechanism to increase the combinatorial specificity of this transcription factor family.

The anaphase-promoting complex/cyclosome co-activator, Cdh1, is a novel target of human papillomavirus 16 E7 oncoprotein in cervical oncogenesis.

The transforming properties of the high-risk human papillomavirus (HPV) E7 oncoprotein are indispensable for driving the virus life cycle and pathogenesis. Besides inactivation of the retinoblastoma family of tumor suppressors as part of its oncogenic endeavors, E7-mediated perturbations of eminent cell cycle regulators, checkpoint proteins and proto-oncogenes are considered to be the tricks of its transformative traits. However, many such critical interactions are still unknown. In the present study, we have identified the anaphase-promoting complex/cyclosome (APC) co-activator, Cdh1, as a novel interacting partner and a degradation target of E7. We found that HPV16 E7-induced inactivation of Cdh1 promoted abnormal accumulation of multiple Cdh1 substrates. Such a mode of deregulation possibly contributes to HPV-mediated cervical oncogenesis. Our mapping studies recognized the C-terminal zinc-finger motif of E7 to associate with Cdh1 and interfere with the timely degradation of FoxM1, a bona fide Cdh1 substrate and a potent oncogene. Importantly, the E7 mutant with impaired interaction with Cdh1 exhibited defects in its ability for overriding typical cell cycle transition and oncogenic transformation, thereby validating the functional and pathological significance of the E7-Cdh1 axis during cervical carcinoma progression. Altogether, the findings from our study discover a unique nexus between E7 and APC/C-Cdh1, thereby adding to our understanding of the mechanism of E7-induced carcinogenesis and provide a promising target for the management of cervical carcinoma.

KLF3 Represses the Inflammatory Response in Macrophages

Krüppel-like factors (KLFs) are a family of transcription factors that play essential roles in the development and differentiation of the hematopoietic system. These transcription factors possess highly conserved C-terminal zinc-finger motifs, which enable their binding to GC-rich, or CACC-box, motifs in promoter and enhancer regions of target genes. The N-terminal domains of these proteins are more varied and mediate the recruitment of various co-factors, which can form a complex with either activator or repressor function. Acting primarily as a gene repressor through its recruitment of CtBPs and histone deacetylases (HDACs) [1], we have recently shown that KLF3 competes with KLF1 bound sites in the genome to repress gene expression during erythropoiesis [2]. However, the function of Klf3 in other lineages has been less well studied. This widely expressed transcription factor has reported roles in the differentiation of marginal zone B cells, eosinophil function and inflammation [3]. We utilised the Klf3-null mouse model [4] to more closely examine the role of Klf3 in innate inflammatory cells. These mice exhibit elevated white cell counts, including monocytes (Figure 1A), and inflammation of the skin. Conditional knockout of Klf4 in myeloid cells leads to a deficiency of inflammatory macrophages [5]. To test our hypothesis KLF3 normally represses inflammation, perhaps by antagonising the action of KLF4, bone-marrow derived macrophages (BMDM) were generated from wild-type or Klf3-null mice and stimulated with the bacterial toxin lipopolysaccharide (LPS). In wild type BMDM, LPS induces Klf3 gene expression and activation then delayed repression of target genes such as Lgals3 (galectin-3) over a 21 hour time course (Figure 1B). Quantitative real-time PCR and mRNA-seq of WT v Klf3-null macrophages identified ~100 differentially expressed genes involved in proliferation, macrophage activation and inflammation. We transduced the monocyte cell line, RAW264.7 (that expresses Klf4, Klf3 and Klf2), with a retroviral vector expressing a tamoxifen-inducible KLF3-ER fusion construct. KLF3 induced cell cycle arrest and macrophage differentiation. We will report on KLF3-induced gene expression changes (repression and activation), and ChIP-seq for KLF3, in RAW cells. The results shed light on the mechanism by which KLF3 normally represses monocyte/macrophage responses to infection. This study highlights the importance of key transcriptional regulators that tightly control gene expression during inflammation. Loss of Klf3 leads to alterations in this process, resulting in hyper-activation of inflammatory macrophages, increased white cell counts and inflammation of the skin. A greater knowledge of the inflammatory process and how it is regulated is important for our understanding of acute infection and inflammatory disease. Further studies are planned to investigate the role of the KLF3 transcription factor in response to inflammation in vivo.

Inhibition of red blood cell development by arsenic-induced disruption of GATA-1

Anemia is a hematological disorder that adversely affects the health of millions of people worldwide. Although many variables influence the development and exacerbation of anemia, one major contributing factor is the impairment of erythropoiesis. Normal erythropoiesis is highly regulated by the zinc finger transcription factor GATA-1. Disruption of the zinc finger motifs in GATA-1, such as produced by germline mutations, compromises the function of this critical transcription factor and causes dyserythropoietic anemia. Herein, we utilize a combination of in vitro and in vivo studies to provide evidence that arsenic, a widespread environmental toxicant, inhibits erythropoiesis likely through replacing zinc within the zinc fingers of the critical transcription factor GATA-1. We found that arsenic interacts with the N- and C-terminal zinc finger motifs of GATA-1, causing zinc loss and inhibition of DNA and protein binding activities, leading to dyserythropoiesis and an imbalance of hematopoietic differentiation. For the first time, we show that exposures to a prevalent environmental contaminant compromises the function of a key regulatory factor in erythropoiesis, producing effects functionally similar to inherited GATA-1 mutations. These findings highlight a novel molecular mechanism by which arsenic exposure may cause anemia and provide critical insights into potential prevention and intervention for arsenic-related anemias.

Investigation of 1-Methylcytosine as a Ligand in Gold(III) Complexes: Synthesis and Protein Interactions

The HIV nucleocapsid protein NCp7 was previously shown to play a number of roles in the viral life cycle and was previously identified as a potential target for small molecule intervention. In this work, the synthesis of the previously unreported complexes [Au(dien)(1MeCyt)]3+, [Au(N-Medien)(1MeCyt)]3+, and [Au(dien)(Cyt)]3+ is detailed, and the interactions of these complexes with the models for NCp7 are described. The affinity for these complexes with the target interaction site, the “essential” tryptophan of the C-terminal zinc finger motif of NCp7, was investigated through the use of a fluorescence quenching assay and by 1H-NMR spectroscopy. The association of [Au(dien)(1MeCyt)]3+ as determined through fluorescence quenching is intermediate between the previously reported DMAP and 9-EtGua analogs, while the associations of [Au(N-Medien)(1MeCyt)]3+ and [Au(dien)(Cyt)]3+ are lower than the previously reported complexes. Additionally, NMR investigation shows that the self-association of relevant compounds is negligible. The specifics of the interaction with the C-terminal zinc finger were investigated by circular dichroism spectroscopy and electrospray-ionization mass spectrometry. The interaction is complete nearly immediately upon mixing, and the formation of AuxFn+ (x = 1, 2, or 4; F = apopeptide) concomitant with the loss of all ligands is observed. Additionally, oxidized dimerized peptide was observed for the first time as a product, indicating a reaction via a charge transfer mechanism.

NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy.

Parkin-mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N-terminal SKICH domain and C-terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly-ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52-mediated mitophagy, and the NDP52-MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria.

VPS18 recruits VPS41 to the human HOPS complex via a RING–RING interaction

Eukaryotic cells use conserved multisubunit membrane tethering complexes, including CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting), to control the fusion of endomembranes. These complexes have been extensively studied in yeast, but to date there have been far fewer studies of metazoan CORVET and HOPS. Both of these complexes comprise six subunits: a common four-subunit core and two unique subunits. Once assembled, these complexes function to recognise specific endosomal membrane markers and facilitate SNARE-mediated membrane fusion. CORVET promotes the homotypic fusion of early endosomes, while HOPS promotes the fusion of lysosomes to late endosomes and autophagosomes. Many of the subunits of both CORVET and HOPS contain putative C-terminal zinc-finger domains. Here, the contribution of these domains to the assembly of the human CORVET and HOPS complexes has been examined. Using biochemical techniques, we demonstrate that the zinc-containing RING (really interesting new gene) domains of human VPS18 and VPS41 interact directly to form a stable heterodimer. In cells, these RING domains are able to integrate into endogenous HOPS, showing that the VPS18 RING domain is required to recruit VPS41 to the core complex subunits. Importantly, this mechanism is not conserved throughout eukaryotes, as yeast Vps41 does not contain a C-terminal zinc-finger motif. The subunit analogous to VPS41 in human CORVET is VPS8, in which the RING domain has an additional C-terminal segment that is predicted to be disordered. Both the RING and disordered C-terminal domains are required for integration of VPS8 into endogenous CORVET complexes, suggesting that HOPS and CORVET recruit VPS41 and VPS8 via distinct molecular interactions.

Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum.

The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions.

Escherichia coli radD (yejH) gene: a novel function involved in radiation resistance and double-strand break repair.

A transposon insertion screen implicated the yejH gene in the repair of ionizing radiation-induced damage. The yejH gene, which exhibits significant homology to the human transcription-coupled DNA repair gene XPB, is involved in the repair of double-strand DNA breaks. Deletion of yejH significantly sensitized cells to agents that cause double-strand breaks (ionizing radiation, UV radiation, ciprofloxacin). In addition, deletion of both yejH and radA hypersensitized the cells to ionizing radiation, UV and ciprofloxacin damage, indicating that these two genes have complementary repair functions. The ΔyejH ΔradA double deletion also showed a substantial decline in viability following an induced double-strand DNA break, of a magnitude comparable with the defect measured when the recA, recB, recG or priA genes are deleted. The ATPase activity and C-terminal zinc finger motif of yejH play an important role in its repair function, as targeted mutant alleles of yejH did not rescue sensitivity. We propose that yejH be renamed radD, reflecting its role in the DNA repair of radiation damage.

Regulation of the immediate-early genes of white spot syndrome virus by Litopenaeus vannamei kruppel-like factor (LvKLF)

Kruppel-like factors (KLFs) belong to a subclass of Cys2/His2 zinc-finger DNA-binding proteins, and act as important regulators with diverse roles in cell growth, proliferation, differentiation, apoptosis and tumorigenesis. Our previous research showed that PmKLF from Penaeus monodon is crucial for white spot syndrome virus (WSSV) infection, yet the mechanisms by which PmKLF influences WSSV infection remain unclear. This study cloned KLF from Litopenaeus vannamei (LvKLF), which had 93% similarity with PmKLF. LvKLF formed a dimer via the C-terminal zinc-finger motif. Knockdown of LvKLF expression by dsRNA injection in WSSV-challenged shrimps was found to significantly inhibit the transcription of two important immediate-early (IE) genes, IE1 and WSSV304, and also reduced WSSV copy numbers. Moreover, reporter assays revealed that the promoter activities of these two WSSV IE genes were substantially enhanced by LvKLF. Mutations introduced in the promoter sequences of IE1 and WSSV304 were shown to abolish LvKLF activation of promoter activities; and an electrophoretic mobility shift assay demonstrated that LvKLF binds to putative KLF-response elements (KRE) in the promoters. Taken together, these results indicate that LvKLF transcriptional regulation of key IE genes is critical to WSSV replication.

Identification Of BCL11A Structure-Function Domains For Fetal Hemoglobin Silencing

Reactivation of fetal hemoglobin (HbF, α2γ2) expression in adults ameliorates the clinical symptoms in patients with the major β-hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. The zinc-finger protein BCL11A is a major modulator of hemoglobin switching and HbF silencing. BCL11A was initially identified by genome-wide association studies (GWAS) as a new HbF-associated gene. Down-regulation of BCL11A in primary human erythroid cells induces HbF expression. Knockout of BCL11A in mice impairs HbF silencing in adult erythroid cells. Most importantly, inactivation of BCL11A alone in humanized SCD mice corrects the hematologic and pathologic defects through high-level HbF induction. These studies established BCL11A as a genetically and functionally validated transcriptional regulator of HbF switching and silencing.In human and mouse erythroid cells, BCL11A is expressed as several isoforms, yet their individual roles in globin gene expression remain unexplored. Furthermore, the functional domains within the BCL11A protein responsible for its activity in HbF repression are largely unknown. To further understand the mechanistic roles of BCL11A in globin expression, we established a functional assay based on a BCL11A-null erythroid cell line generated by transcription activator-like effector nucleases (TALENs)-mediated deletion of an obligate erythroid-specific enhancer of BCL11A in murine erythroleukemia (MEL) cells. In the BCL11A-null cells, the expression of β-like embryonic globin genes is markedly induced (>200-fold), consistent with the role of BCL11A in repression of murine embryonic globin genes. To examine the activity of known BCL11A isoforms in HbF silencing, we expressed various BCL11A isoforms in these engineered BCL11A-null cells. Ectopic expression of full-length BCL11A-XL isoform, but not the alternatively spliced, C-terminally truncated L isoform, restored the full repression of β-like embryonic globins in BCL11A-null cells. Since XL and L differ only by 91 amino acids containing three tandem C2H2-type zinc finger motifs, these results indicate that the C-terminal zinc finger motifs are indispensable for BCL11A-mediated transcriptional repression.To systemically define BCL11A functional domains for globin gene repression, we next generated a panel of BCL11A mutant cDNAs, including deletion of the N-terminal NuRD-interacting motif and one or more C2H2-type zinc finger domains. Analysis of various BCL11A mutants in the functional rescue assay identified several functional domains, including the N-terminal NuRD-interacting motif and five out of the six C2H2 zinc fingers, that are required for BCL11A-mediated repression. These findings provide the foundation for further molecular analysis of BCL11A functional domains in globin gene repression. BCL11A is known to interact with several transcriptional co-repressor complexes including Mi-2β/NuRD/HDAC1/HDAC2, LSD1/CoREST and SWI/SNF complexes, occupy discrete regions within the human β-globin cluster, and promote long-range chromosomal interactions. Our results suggest that BCL11A functional domains may be involved in protein-protein interactions, protein homo-/heterodimerization, and/or chromatin/DNA association that are required for its activity in HbF silencing.In summary, we demonstrate that several functional domains on BCL11A protein are indispensable for its transcriptional activity in HbF silencing. Further focused studies of BCL11A structure-function domains in HbF silencing not only will advance our understanding of the molecular mechanisms by which BCL11A controls the clinically important fetal-to-adult globin switch, but may identify novel cellular targets for therapeutic HbF induction in β-hemoglobinopathies. Disclosures:No relevant conflicts of interest to declare.

Degradation of CREB-binding protein and modulation of type I interferon induction by the zinc finger motif of the porcine reproductive and respiratory syndrome virus nsp1α subunit

Non-structural protein (nsp) 1 of PRRS virus is a viral antagonist for type I interferons (IFNs), and in cells expressing nsp1, CREB-binding protein (CBP) is degraded. nsp1 is auto-processed into nsp1α and nsp1β subunits and in the present study we show that the nsp1α subunit was responsible for CBP degradation. The nsp1α subunit contains three distinct functional motifs; a papain-like cysteine protease α (PCPα) motif, an N-terminal zinc finger motif (ZF1), and a newly reported C-terminal zinc finger motif (ZF2). To study the structure function of nsp1α and its IFN antagonism, these motifs were individually mutated and the mutants were examined for their IFN suppression ability. The mutations that destroyed the PCPα activities (C76S, H146Y, and C76S/H146Y) did not affect the IFN suppressive activity of nsp1α, indicating that the cysteine protease activity did not participate in IFN suppression. The mutations of C70S, C76S, H146Y, and/or M180I which coordinated the ZF2 motif also did not alter IFN suppression. However, the mutations of C8S, C10S, C25S, and/or C28S for the ZF1 motif impaired the IFN antagonism of nsp1α, demonstrating that ZF1 was the essential element of nsp1α for IFN suppression. Wild-type nsp1α localized in the both nucleus and cytoplasm, but the ZF1 mutants that lost the IFN suppressive activity did not localize in the nucleus and remained in the cytoplasm. Consistent with their cytoplasmic distribution, CBP was not degraded by these mutants. Our results indicate that the ZF1 motif of nsp1α plays an important role for IFN regulation and further demonstrate that the CBP degradation is likely the key mechanism for IFN suppression mediated by the nsp1α subunit protein of PRRS virus.

Studies on Human DNA Polymerase ϵ and GINS Complex and Their Role in DNA Replication

Studies on Human DNA Polymerase ϵ and GINS Complex and Their Role in DNA Replication

Functional evaluation of four putative DNA-binding regions in Thermoanaerobacter tengcongensis reverse gyrase

Reverse gyrase (RG) is an ATP-dependent type I DNA topoisomerase that introduces positive supercoils into DNA in thermophiles. Four regions of RG, i.e., the N-terminal zinc-finger motif, the β-hairpin in subdomain H1, the "latch", and the C-terminal zinc-finger motif, were predicted to be involved in DNA binding previously. In this paper, the functions of these regions in the enzymatic activity were evaluated by mutational analysis of the Thermoanaerobacter tengcongensis reverse gyrase (TtRG). We demonstrated that TtRG exhibited positive-supercoiling activity only at high temperature (>50°C) and low salt concentration (~30 mM NaCl), and three of these four regions (except for the "latch") were involved in DNA binding. Notably, mutations in the "latch" and β-hairpin regions of TtRG strongly impaired the ATPase activity, while mutations in the two zinc-finger motifs dramatically affected its thermal stability besides significant impairment of the DNA-binding ability. Accordingly, all of these four regions were found to be indispensable for the positive-supercoiling activity of TtRG. Taken together, we revealed that these putative DNA-contact regions affect the enzymatic activity of RG in different ways, and provided new insights into the structure and function of RG.

The structure of the Klf4 DNA-binding domain links to self-renewal and macrophage differentiation.

Krueppel-like factor 4 (Klf4) belongs to the Sp/Klf family of zinc-finger transcription factors and is indispensable for terminal maturation of epithelial tissues. Furthermore, it is part of a small set of proteins that are used to generate pluripotent embryonic stem cells from differentiated tissues. Herein, we describe that a Klf4 zinc-finger domain mutant induces self-renewal and block of maturation, while wild-type Klf4 induces terminal macrophage differentiation. Moreover, we present the crystal structure of the zinc-finger domain of Klf4 bound to its target DNA, revealing that primarily the two C-terminal zinc-finger motifs are required for site specificity. Lack of those two zinc fingers leads to deficiency of Klf4 to induce macrophage differentiation. The first zinc finger, on the other hand, inhibits the otherwise cryptic self-renewal and block of differentiation activity of Klf4. Our data show that impairing the DNA binding could potentially contribute to a monocytic leukemia.

The A20 Deubiquitinase Activity Negatively Regulates LMP1 Activation of IRF7

A20 possesses both deubiquitinase (DUB) and ubiquitin E3 ligase activities that are required for termination of Toll-like receptor (TLR) signaling leading to NF-kappaB activation and for blockage of tumor necrosis factor (TNF)-induced cytotoxicity and apoptosis. A20 is induced by the Epstein-Barr virus (EBV) oncoprotein LMP1. However, its dual ubiquitin-editing activities have not been investigated in the context of either EBV infection or IRF7 responses. Both A20 and IRF7 have oncogenic properties. We have recently shown that LMP1 activates IRF7 through K63-linked ubiquitination which requires RIP1 and TRAF6, but how this ubiquitination event is regulated has not been studied. Here, we show that A20 negatively regulates IRF7 transcriptional activity induced by LMP1. Deletion or mutation of A20 C-terminal zinc finger motifs had no effect on the inhibition of IRF7 activity, whereas DUB-deficient truncation or point mutation ablated the ability of A20 to inhibit IRF7. Correspondingly, the A20 N-terminal DUB domain, but not the C-terminal E3 ligase domain, interacts physically with IRF7. Transient expression of A20 reduced K63-linked ubiquitination of IRF7 in vivo, but an in vitro deubiquitination assay with purified constituents shows that IRF7 did not act as a substrate for A20 DUB activity. Moreover, A20 interacts with IRF7 endogenously in latently EBV-infected type 3 Raji cells, in which expression of both A20 and IRF7 is constitutively induced by the considerable level of endogenous LMP1. Knockdown of endogenous A20 in Raji cells by expression of A20 short hairpin RNA (shRNA) vectors increases endogenous IRF7 activity and ubiquitination, as well as the protein level of LMP1, a target of IRF7. Thus, A20 negatively regulates LMP1-stimulated IRF7 ubiquitination and activity in EBV latency, and its DUB activity is indispensable for this function. Finally, we discussed the regulation and function of IRFs in EBV latency.

Sequential Order of Genetic Events in NUP214-ABL1 Positive T-Cell Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease comprising several biological and clinical entities. Based on recent studies and in agreement with the paradigm of multistep oncogenesis the notion that multiple genetic hits are cooperating in the generation of T-ALL has emerged. Genetic and functional data suggests that a stepwise alteration of at least four specific pathways is required before thymocytes become fully malignant:cell cycle deregulation,impaired differentiation,proliferation and survival advantage, andunlimited self-renewal capacity.As the sequential order of the genetic events remains widely elusive, we aimed to determine their consecutive acquisition in the model of NUP214-ABL1 positive T-ALL, which is always accompanied by either a TLX3 or TLX1 rearrangement as well as frequent loss of CDKN2A. Array comparative genomic hybridization (aCGH) analysis of 102 childhood T-ALL patients and 12 matched relapse samples revealed a NUP214-ABL1 amplification in 5 samples, two of which were relapse samples and showed the aberration only at this stage of the disease. However, NUP214-ABL1-specific RT-PCR determined that the fusion gene was also present in those two diagnostic specimens in which aCGH failed to detect the amplification and FISH analysis showed ambiguous results with a minor fraction (5%) of positive cells. At relapse in one of these patients the NUP214-ABL1 fusion was found integrated and amplified at 7q, thus in contrast to the episomal form stably transmitted, and present in 65% of the cells. On the other hand, at diagnosis all five patients showed a TLX3 rearrangement by fluorescence in situ hybridization (FISH) in >60% of the cells. The data obtained by aCGH revealed also homozygous or heterozygous loss of CDKN2A (9p21) in four and one of the cases, respectively, and heterozygous deletion of WT1 (11p13) in three cases. FISH analysis confirmed the heterozygous loss of WT1 and inactivating mutations in the C-terminal zinc-finger motifs of the second allele were found in two of the patients. In one of these cases the WT1 mutation was readily detectable at the stage of relapse, but sensitive allele-specific PCR was required to trace the mutation back to the diagnostic sample, suggesting that - as typical for a tumor suppressor function - loss of the first allele was followed by complete inactivation of WT1 by mutation of the second allele during disease progression. Moreover, in this case monoallelic loss of WT1 was observed in 93% of the cells, whereas TLX3 was rearranged in only 63%. In contrast, in a second case exactly the opposite pattern was observed, namely monoallelic loss of WT1 in 42% and TLX3 rearrangement in 73% of the cells. In this sample, preliminary data obtained by consecutive FISH on the single cell level showed two copies of WT1 in TLX3 positive cells. Moreover, based on the aCGH data, which however still requires confirmation by sequential FISH experiments, CDKN2A was deleted in a very high percentage of the cells indicating that loss of this cell cycle regulating tumor suppressor is an early genetic event. Together our data suggest that rearrangement of TLX3 and loss of CDKN2A and WT1 precede the formation of the NUP214-ABL1 fusion and subsequent amplification. On the other hand loss of WT1 and TLX3 rearrangement may possibly occur in a changeable order, whereas the temporal occurrence of WT1 inactivating mutations remains to be determined.

Identification of FAZF as a novel BMP2‐induced transcription factor during osteoblastic differentiation

Bone morphogenetic protein 2 (BMP2) is a key factor in the regulation of osteoblastic differentiation; however, its downstream mediators are not fully understood. Previously, we identified and characterized transcription factor promyelocytic leukemia zinc finger protein (PLZF), composed of an N-terminal BTB/POZ and C-terminal zinc finger motifs, as an upstream factor of CBFA1 (Runx2/core-binding factor 1). PLZF was induced in an osteoblastic differentiation medium, but was not induced by BMP2. Here, we report the identification of transcription factor fanconi anemia zinc finger protein (FAZF), which is closely related to PLZF. FAZF was induced by BMP2 in human mesenchymal stem cells (hMSCs). In addition to the full-length FAZF, we also identified alternatively spliced mRNAs in which the C-terminal zinc finger motifs were deleted (designated BTB/POZ-only FAZF). Both the full-length and BTB/POZ-only FAZF mRNAs were equally expressed in BMP2-treated hMSCs. The full-length FAZF was exclusively detected in the nucleus, whereas the BTB/POZ-only FAZF protein was localized in the cytoplasm of the transfected cells. The full-length FAZF, but not the BTB/POZ-only FAZF, increased the expression of osteoblastic differentiation markers, including CBFA1, collagen 1A1, osteocalcin, and alkaline phosphatase in C2C12 cells. In conclusion, both FAZF and PLZF differentially participate in the regulation of osteoblastic differentiation via the BMP2 and CBFA1 signaling pathways, respectively.

Regulation of transcription of the Dnmt1 gene by Sp1 and Sp3 zinc finger proteins.

The Sp family is a family of transcription factors that bind to cis-elements in the promoter regions of various genes. Regulation of transcription by Sp proteins is based on interactions between a GC-rich binding site (GGGCGG) in DNA and C-terminal zinc finger motifs in the proteins. In this study, we characterized the GC-rich promoter of the gene for the DNA methyltransferase (Dnmt1) that is responsible for methylation of cytosine residues in mammals and plays a role in gene silencing. We found that a cis-element (nucleotides -161 to -147) was essential for the expression of the mouse gene for Dnmt1. DNA-binding assays indicated that transcription factors Sp1 and Sp3 bound to the same cis-element in this region in a dose-dependent manner. In Drosophila SL2 cells, which lack the Sp family of transcription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter. Stimulation by Sp1 and Sp3 were independent phenomena. Furthermore, cotransfection reporter assays with a p300-expression plasmid revealed the activation of the promoter of the Dnmt1 gene in the presence of Sp3. The transcriptional coactivator p300 interacted with Sp3 in vivo and in vitro. Our results indicate that expression of the Dnmt1 gene is controled by Sp1 and Sp3 and that p300 is involved in the activation by Sp3.

Functional analysis of basic transcription element (BTE)-binding protein (BTEB) 3 and BTEB4, a novel Sp1-like protein, reveals a subfamily of transcriptional repressors for the BTE site of the cytochrome P4501A1 gene promoter.

The Sp1-like family of transcription factors is emerging as an integral part of the cellular machinery involved in the control of gene expression. Members of this family of proteins contain three highly homologous C-terminal zinc-finger motifs that bind GC-rich sequences found in the promoters of a diverse number of genes, such as the basic transcription element (BTE) in the promoter of the carcinogen-metabolizing cytochrome P4501A1 (CYP1A1) gene. In the present study, we report the molecular and functional characterization of BTE-binding protein (BTEB) 4, a novel ubiquitously expressed member of the Sp1-like proteins family. This protein represents a new homologue of BTEB1, originally described as a regulator of the BTE site in the CYP1A1 gene promoter. Similarly to the recently described BTEB3, we demonstrate that the N-terminal region of BTEB4 directly represses transcription and binds the co-repressor mSin3A. In addition, we show that the C-terminal zinc-finger domain of BTEB4 binds specifically the BTE site of the CYP1A1 promoter, similar to BTEB1 and BTEB3. Also, we show that both BTEB3 and BTEB4 repress the CYP1A1 gene promoter via the BTE site in HepG2 and BxPC3 cells. Thus the identification of this protein expands the repertoire of BTEB-like members of the Sp1-like protein family involved in transcriptional repression. Furthermore, our results demonstrate that the BTEB subfamily can repress the CYP1A1 gene promoter via the BTE site.