Biomedical subjects

K Khalili

Publications and source records attributed to K Khalili.

At least 91 records · Page 5Linked to original sources

PubMed1998

Cooperative actions of HIV-1 Vpr and p53 modulate viral gene transcription.

Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is controlled by cooperative interaction of viral encoded proteins and host regulatory proteins. In this study, we have examined the capacity of the viral auxiliary protein, Vpr, to modulate transcriptional activity of the HIV-1 promoter sequence located within the long terminal repeat (LTR). We demonstrate that ectopic expression of Vpr in human astrocytic cells, U-87MG, enhances the basal activity of the viral promoter in transfected cells and that the GC-rich sequences, spanning nucleotides -80 to -43, are important for this activity. Since this region serves as the target for p53-induced suppression of LTR activity and interacts with the ubiquitous transcription factor, Sp1, we examined the cooperative activity of Vpr, p53, and Sp1 upon LTR transcription. Results from co-transfection studies indicated that overexpression of wild type p53, but not mutant p53, decreases the level of activation of the LTR by Vpr. Transcriptional activation of the LTR by Vpr required the presence of Sp1 since overexpression of Vpr in cells with no endogenous Sp1 failed to augment LTR activity. Results from protein-protein interaction studies indicated that Vpr is associated with both p53 and Sp1 in cells with ectopic expression of these proteins. Moreover, it was evident that p53 and Sp1 interact with each other in these cells. These functional and structural studies provided a working model on the cooperative interaction of Vpr with cellular proteins Sp1 and p53 and control of viral gene transcription at immediate early stage of infection prior to the participation of other viral regulatory proteins.

Astrocytes↗

PubMed1998

Regulation of TNFalpha and TGFbeta-1 gene transcription by HIV-1 Tat in CNS cells.

Tat is a transcription transactivator produced by the human immunodeficiency virus type 1 (HIV-1) at the early phase of infection and plays a critical role in the expression and replication of the viral genome. This 86 amino acid protein, which can be secreted from the infected cells, has the ability to enter uninfected cells and exert its activity upon the responsive genes. Earlier results indicated that in addition to the HIV-1 promoter, Tat has the capacity to induce transcription of a variety of cellular genes. In this study, we demonstrate that exposure of cells from the central nervous system (U-87MG and SK-N-MC) and the lymphoid T cells (Jurkat) to highly purified Tat increases transcriptional activity of the reporter constructs containing the promoters from the transforming growth factor beta-1 (TGFbeta-1), the tumor necrosis factor alpha (TNFalpha), and the HIV-1 LTR. In addition, Tat treatment results in increased levels of TGFbeta-1 and TNFalpha mRNAs in these cells. Activation of the TGFbeta-1 and TNFalpha promoter constructs by Tat in U-87MG and SK-N-MC cells required amino acid residues 2 to 36 which spans the acidic and the cysteine-rich domains of Tat. In both CNS and lymphoid cells, the level of endogenous TGFbeta-1 mRNA was increased by mutant Tat protein containing amino acids 1 to 48 but not with a mutant Tat protein with a deletion between residues 2 to 36. TNFalpha mRNA level was increased by mutant Tat spanning residues 1 to 48 in U-87MG cells, but not in SK-N-MC and Jurkat cells. These observations suggest that activation of cellular and viral genes by Tat in various cells may be mediated by different pathways as evidenced by the requirements of the different regions of Tat. Activation of the TGFbeta-1 and TNFalpha promoters by wild-type Tat was severely affected by the mutant peptides spanning residues 2 to 36 and 1 to 48 suggesting that both truncated Tat peptides may function as dominant negative mutants over TNFalpha and TGFbeta-1 gene transcription. The importance of these findings in Tat-induced regulation of viral and cellular genes in various cell types is discussed.

Brain↗

PubMed1998

Evaluation of an autoregulatory tetracycline regulated system.

Tetracycline controlled gene expression systems have become powerful tools in the analysis of gene function in mammalian cell culture as well as transgenic animals and plants. The original description of a tetracycline-regulated gene expression system is based on two plasmids, one of which constitutively expresses a tetracycline-controlled transactivator protein (tTA), a fusion protein between the tetracycline repressor of E. coli and the transcriptional activation domain of the VP16 protein of herpes simplex virus. The second plasmid contains the gene to be regulated by tTA under the control of an inducible promoter which consists of seven copies of the tetracycline resistance operator (tetO). Since this original description, many modifications have been described. In this report, we evaluate an autoregulatory tetracycline controlled system, in which the tTA is itself under the control of the tetO. We demonstrate that this autoregulatory tetracycline system produces adverse effects including cellular morphologic changes, growth rate attenuation and alterations in cell cycle distribution.

Animals↗

PubMed1998

HIV-1 Tat elongates the G1 phase and indirectly promotes HIV-1 gene expression in cells of glial origin.

Human immunodeficiency virus type-1 (HIV-1) infection of the central nervous system (CNS) gives rise to many of the neurological complications in patients with AIDS. Infection of microglial cells and astrocytes in the brain promotes the release of HIV-1 Tat and other candidate neurotoxins that may be associated with the widespread neuropathology. To examine the contribution of HIV-1 Tat to the interplay between virus and CNS cells, the human astrocytic cell line, U-87MG, was treated with recombinant Tat protein. Fluorescence-activated cell sorting analysis indicated that Tat induces a G1 arrest in these cells. Consistent with this observation, lower levels of cyclin E-Cdk2 kinase activity and phosphorylated Rb were detected in the Tat-treated cells compared with the control cells. Interestingly, our observations indicate that the underphosphorylated form of Rb that is prevalent in Tat-treated cells promotes HIV-1 transcription by a mechanism involving the NF-kappaB enhancer region. Taken together, the data presented here provide the first evidence that the HIV-1 regulatory protein, Tat, may manipulate the host cell cycle to promote viral gene expression. The significance of these findings relates to the current hypothesis that indirect effects of HIV-1 infection of the CNS may contribute to the neurological complications associated with AIDS dementia complex.

Astrocytes↗

PubMed1998

The single-stranded DNA binding protein, Pur-alpha, binds HIV-1 TAR RNA and activates HIV-1 transcription.

Previous studies indicate that the bulge and loop domains of TAR, the HIV-1 RNA regulatory element, bind viral and cellular factors that are critical for efficient transcription of the HIV-1 genome. In this report, we demonstrate that the cellular protein, Pur-alpha, a previously characterized sequence specific, single-stranded DNA binding protein, binds to HIV-1 TAR RNA in a specific manner as demonstrated by competition analysis. Pur-alpha binds to the greatest extent to wild-type TAR RNA, and it appears the primary sequence, as well as the secondary structure and its overall stability contribute to this binding. Results from gel shift analysis using mutant Pur-alpha proteins indicate that amino acids 55-85, which contain the first of three basic aromatic repeats, are important for its binding to TAR RNA. Overexpression of Pur-alpha in a glial cell line increased transcription of HIV-1 LTR by a TAR dependent mechanism. The potential contribution by Pur-alpha to HIV-1 expression in relation to basal transcription by cellular factors is discussed.

Base Sequence↗

PubMed1998

MyEF-3, a developmentally controlled brain-derived nuclear protein which specifically interacts with myelin basic protein proximal regulatory sequences.

Myelin basic protein (MBP) gene contains the upstream regulatory sequence that confers cell type- and stage-specific transcription to MBP expression in oligodendrocytes during brain development. The MB1 regulatory motif located between -14 to -50 with respect to transcription start site binds to a brain derived nuclear protein and plays an important role in transcriptional activation of the MBP promoter in transfection assay. Here, we report the isolation of a recombinant cDNA clone, termed myelin expression factor-3, (MyEF-3) from a mouse brain expression library that encodes a novel protein which interacts with the MBP MB1 domain. Computer assisted evaluations of the MyEF-3 sequence revealed several interesting features including four sites for phosphorylation by casein kinase II, a transmembrane domain at the N-terminus, a nuclear localization signal and a Zinc finger domain at the carboxyl terminal. Results from Western and band shift assays indicate that MyEF-3 binds efficiently to double-stranded MB1 as well as the single-stranded non-coding strand of MB1. The use of short DNA fragments encompassing the nucleotide base substitutions across the MB1 domain in competition band shift assay revealed that the ten nucleotide sequence, 5'-GCCTGTCTTT-3' is important for binding of MyEF-3 to DNA. Results from Northern blot studies demonstrate that expression of MyEF-3 is restricted to brain and developmentally regulated during brain maturation. The biological importance of MyEF-3 in the cell type- and stage-specific expression of MBP during brain development is discussed.

Amino Acid Sequence↗

PubMed1998

Structure and function of HIV-1 and SIV Tat proteins based on carboxy-terminal truncations, chimeric Tat constructs, and NMR modeling.

To further define the structure and function of the domains in HIV-1 and SIV Tat proteins, chimeric Tat cDNA expression constructs were generated with crossover points at the carboxy-terminal end of the cysteine rich domain. The chimera containing the amino-terminal region of SIV and carboxy-terminal region of HIV exhibited activity similar to HIV-1 Tat and SIV Tat on both the HIV-1 and SIV LTRs. In contrast, the reciprocal chimera functioned poorly. As determined by the activity of carboxy-terminal truncation mutants, the region immediately downstream of the basic domain is critical for efficient transactivation by HIV-1 Tat, but not SIV Tat protein. In this report, we present a model for Tat domains based on NMR data and the known functional properties of Tat protein. According to our modeling two sites for protein : protein interactions are present in HIV-1 and SIV Tat proteins. Site I, which is presumably involved in cyclin T binding, is similar in both HIV-1 and SIV Tat proteins as well as in Tat chimeras. Site II, however appears structurally different in HIV-1 and SIV Tat models, although in both cases is comprised of amino and carboxy-terminal residues. Differences in Site II may thus account for the differential activities of HIV-1 and SIV Tat carboxy-terminal truncations. Site II in the poorly active chimera differs significantly from that found in HIV-1 and SIV Tat proteins. The two site structural model presented here may have important implications for the role of Tat in HIV pathogenesis and may provide insights for the design of Tat vaccines and targeted therapeutics.

Amino Acid Sequence↗

PubMed1998

Transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in central nervous system cells: effect of YB-1 on expression of the HIV-1 long terminal repeat.

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by a series of host DNA-binding proteins which interact with the upstream viral promoter sequences, and the viral regulatory protein Tat which associates with TAR RNA sequences located in the leader of the viral transcript. Here we have examined the ability of the cellular protein YB-1 to modulate transcription of the HIV-1 promoter in a human astrocytic cell line (U-87MG), a neuronal cell line (SK-N-MC) and lymphoid cells (Jurkat) by transfection assay. Ectopic expression of YB-1 in U-87MG and SK-N-MC augments basal transcriptional activity of the viral sequence located between -80 and -43, which encompasses the GC-rich motif. In accord with the previous report, in Jurkat cells YB-1-mediated activation of the HIV-1 promoter required sequences which are located further upstream from the GC-rich motif. Combined overexpression of YB-1 and the GC-rich binding protein, SP1, in the transfected cells decreased the level of activation of the viral promoter, suggesting that YB-1 and SP1 may exert negative effects on each other's function. Results from band shift assay with purified YB-1 and SP1 indicated that SP1 and YB-1 bind to the GC-rich DNA sequence in the double-stranded and single-stranded configurations, respectively. However, efficient binding of SP1 to the double-stranded GC-rich motif corresponding to the HIV-1 long terminal repeat (LTR) is diminished in the presence of YB-1. Similarly, in the presence of SP1, YB-1 loses its ability to become associated with its target single-stranded DNA probe. No evidence for direct association of YB-1 and SP1 either in the presence or in the absence of DNA was observed. These data suggest that while YB-1 stimulates expression of the LTR in central nervous system cells, the level of activity of other cellular proteins, such as SP1, may dictate binding of YB-1 to its target sequence, and therefore affect the regulatory function of this protein.

Astrocytes↗

PubMed1998

T-antigen-dependent transcriptional initiation and its role in the regulation of human neurotropic JC virus late gene expression.

The multifunctional protein of papovaviruses, T-antigen, regulates the virus lytic cycle partly by exerting transcriptional control over viral and cellular gene expression. In this study, the ability of the T-antigen of human neurotropic JC virus (JCV) to enhance expression from the virus late promoter has been further examined. By deletion analysis, a T-antigen-responsive region was mapped within the JCV 98 bp enhancer/promoter between nucleotides 139 and 168. Interestingly, T-antigen appears to mediate transactivation by increasing expression from a basal transcriptional initiation site and through a novel T-antigen-dependent initiation site (TADI). The TADI element contains a region homologous to initiator (Inr) sequences and is sufficient to confer T-antigen responsiveness to a heterologous minimal promoter. Electrophoretic mobility shift and UV crosslinking analyses demonstrate that multiple cellular proteins interact with both single- and double-stranded forms of this sequence. Mutations within the TADI element which abolish T-antigen-mediated transcriptional activation also prevent the formation of specific nucleoprotein complexes. These data suggest that the ability of JCV T-antigen to regulate JCV late gene expression may be partly due to the formation of specific nucleoprotein complexes and transcriptional initiation from the TADI site on the viral promoter.

Antigens, Viral, Tumor↗

PubMed1998

MR imaging of primary lymphoma of bone: variability of T2-weighted signal intensity.

OBJECTIVE: The purpose of this study was to investigate the MR imaging characteristics of primary lymphoma of bone. MATERIALS AND METHODS: Records of 27 patients with histologic and clinical evidence of primary lymphoma of bone were retrospectively reviewed. Nineteen of these patients underwent MR imaging before surgical biopsy and initiation of therapy. Fifteen of the 19 patients underwent conventional spin-echo T1- and T2-weighted imaging, and the other four patients underwent T1-weighted and fast spin-echo T2-weighted imaging with fat saturation. MR images were separately and independently reviewed by two observers for signal intensity characteristics and homogeneity on T1- and T2-weighted sequences. Signal intensity characteristics were correlated with semiquantitative histopathologic assessments of tumor fibrosis, maturity of fibrosis, and vascularity. RESULTS: T1-weighted signal intensity ranged from isointense to hypointense relative to muscle for all lesions. Twelve of 19 cases showed heterogeneity of signal intensity on T2-weighted images. Predominant tumor T2-weighted signal intensities relative to fat for the 19 patients were assessed as hypointense (observer 1, n = 3; observer 2, n = 1), isointense (observer 1, n = 10; observer 2, n = 11), and hyperintense (observer 1, n = 6; observer 2, n = 7). No correlation among intralesional fibrosis, maturity of fibrosis, or intralesional vascularity and T2-weighted signal intensity characteristics was found. CONCLUSION: T2-weighted MR imaging characteristics of primary lymphoma of bone vary and do not seem to be a simple reflection of histologic findings of intralesional vascularity or fibrosis.

Adipose Tissue↗

PubMed1998

Tumor pathogenesis of human neurotropic JC virus in the CNS.

In the last several years, studies have reported the detection of sequences similar to the polyomavirus, Simian Virus 40 (SV40) in human tumors including choroid plexus papillomas and ependymomas. Taken together with well-established evidence that SV40, as well as the human polyomaviruses JC virus (JCV) and BK virus (BKV), are oncogenic in several animal models, new interest has resurfaced regarding a possible role for these viruses in human tumors. In particular, a strong case can be made for re-evaluating the oncogenic potential of JCV in a neurotropic polyomavirus which is the causative agent of the fatal human demyelinating disease, Progressive Multifocal Leukoencephalopathy (PML). In this review, we discuss the transforming capability of JCV in vitro, JCV's ability to induce neural origin tumors in non-human primates as well as in rodents, and several case reports which suggest a possible role for JCV in tumor pathogenesis in the central nervous system.

Animals↗

PubMed1998

The human polyomavirus, JCV, and neurological diseases (review).

JC virus, a human neurotropic polyomavirus, is the established etiologic agent of the fatal demyelinating disease, progressive multifocal leukoencephalopathy, which usually affects individuals with defects in cell-mediated immunity. Cytolytic destruction of oligodendrocytes, the myelin-producing cells of the central nervous system is attributed as the mechanism by which JCV induces demyelination. PML was at one time a rare complication however it is now a much more common disease affecting patients of all ages due to the prevalence of AIDS. Of interest, in vitro evidence points to a cooperative interaction between JCV and HIV-1, via the HIV-1 regulatory protein, Tat. In addition, JCV has been demonstrated to induce tumors of neural origin in several animal models, including rodents and non-human primates, and several clinical reports have suggested a possible association between JCV and glial-origin tumors in humans. The viral regulatory protein, T-antigen, which has been shown to play a key role in orchestrating the events of the viral lytic cycle, is also capable of altering cellular functions, by nature of its direct interaction with cellular regulatory proteins and by its effect on cellular transcription. In this review, we discuss clinical aspects of PML, the ability of JCV to induce tumors in animal models, and the ability of JCV T-antigen to alter cellular function in vitro.

AIDS-Related Opportunistic Infections↗

PubMed1998

Human S mu binding protein-2 binds to the drug response element and transactivates the human apoA-I promoter: role of gemfibrozil.

Previously, we demonstrated that protein-DNA interactions at the drug response element (DRE) in the human apoA-I promoter were important for the induction of apoA-I gene expression by gemfibrozil. We now report the cloning and characterization of a DRE transactivating factor. The cloned protein is identical to the putative helicase and potential transcription factor human S mu binding protein-2 (HSmuBP2). It is also related to glial factor-1 (GF1), an incomplete version of HSmuBP2 lacking the first 494 and the last 128 amino acids. Gel mobility shift assays demonstrated that HSmuBP2 binds apoA-I DRE oligomers and forms a specific protein-DNA complex. Northern blot analysis showed that HSmuBP2 mRNA is expressed at various levels in a wide range of human tissues. Transient cotransfection experiments performed in HepG2 cells demonstrated that overexpression of HSmuBP2 or GF1 induced apoA-I proximal promoter activity by 3-fold and that the apoA-I DRE was necessary for transactivation. Additionally, we demonstrated that transactivation was increased a further 2- to 3-fold by exposing the cells to gemfibrozil. Together these observations indicate that HSmuBP2 acts as a transcription factor that regulates apoA-I gene expression in hepatoma cells and whose activity may be stimulated by gemfibrozil treatment.

Apolipoprotein A-I↗

PubMed1998

Oncogenic potential of human neurotropic papovavirus, JCV, in CNS.

The human polyomavirus, JCV, is the causative agent of Progressive Multifocal Leukoencephalopathy (PML), a fatal human demyelinating disease. PML results from the cytolytic destruction of oligodendrocytes, the myelin-producing cells of the nervous system. JCV has also been shown to be tumorigenic in several animal models. Transgenic mice expressing the JCV early protein, T-antigen, develop poorly differentiated neural crest origin tumours. Intracerebral inoculation of JCV into newborn hamsters induces medulloblastomas, astrocytomas, and primitive neuroectodermal tumours. Further, inoculation of the virus into the brains of non-human primates, owl and squirrel monkeys, results in astrocytomas and glioblastoma multiforme. Several case reports have associated JCV with human CNS tumours in patients with concomitant PML, and one such report has detected JCV in a glial tumour in the absence of PML. The induction of neural origin tumours by JCV has been studied in transgenic mice harbouring the early genome of the virus. Alterations in the level and function of tumour suppressor proteins p53 and Rb, as well as associated cell cycle regulators, have been detected in tumour tissue from JCV T-antigen transgenic mice. Possible mechanisms by which JCV may exert its oncogenic potential by alteration of cellular growth control pathways in both humans and experimental animals are discussed.

Animals↗

PubMed1997

Identification of a cellular protein that binds to Tat-responsive element of TGF beta-1 promoter in glial cells.

Tat is a transcriptional transactivator produced by the human immunodeficiency virus type 1 (HIV-1) and plays a pivotal role in enhancing expression of the viral genome in the infected cells. Although initial studies have suggested that interaction of Tat with the transactivation responsive element (TAR); located within the LTR, is essential for Tat function, subsequent studies indicated that Tat has the ability to augment transcription of viral and cellular genes by a TAR-independent mechanism. In early studies we demonstrated that HIV-1 Tat stimulates transcription of the transforming growth factor, TGF beta-1, gene in glial cells. In this study, we have identified a cellular protein that interacts with the Tat-responsive region located between nucleotides -323 to -453 of the regulatory sequence of the TGF beta-1 promoter. Results from footprinting analysis revealed association of cellular proteins with the 130 nucleotide sequence located in the Tat-responsive region. Analysis of the associated protein by UV-crosslinking suggested the involvement of a protein between 40-45 kDa in size which preferentially interacts with the GC/GA rich sequence of the TGF beta-1 Tat-responsive sequence in a single-stranded configuration. The ability of the previously identified 40 kDa protein, named Pur alpha to bind to the GC/GA sequence in the single-stranded configuration, similar to those from TGF beta-1 promoter prompted us to investigate its binding capacity to the TGF beta-1 sequence and its transcriptional activity on the TGF beta-1 promoter. Results from band shift studies indicated the association of the bacterially produced Pur alpha to the TGF beta-1 DNA sequences positioned within the Tat-responsive region. Overexpression of Pur alpha in glial cells constitutively producing Tat augmented transcription of the TGF beta-1 gene. These results are consistent with previous reports on the cooperative action of Pur alpha and Tat in modulating other eukaryotic promoters. The importance of these findings with regard to deregulation of other cellular genes by HIV-1 Tat is discussed.

Astrocytes↗

PubMed1997

Interaction between cell cycle regulator, E2F-1, and NF-kappaB mediates repression of HIV-1 gene transcription.

The NF-kappaB/Rel family of transcription factors is one of the main targets of cytokines and other agents that induce HIV-1 gene expression. Some of these extracellular stimuli arrest cells in the G1 phase of the mitotic division cycle and modulate the activity of the tumor suppressor protein Rb and its partner E2F-1. Earlier studies indicated that E2F-1, a transcription factor that stimulates expression of S-phase-specific genes, is able to repress transcription directed by the human immunodeficiency virus (HIV-1) type-1 promoter in a variety of cells, including those of glial and lymphocytic origin. Here, we demonstrate that E2F-1 may regulate the activity of the HIV-1 long terminal repeat through its ability to bind sequences in the NF-kappaB enhancer region and to interact with the NF-kappaB subunit, p50. Gel retardation and methylation interference assays show that E2F-1 is able to bind specifically to a site embedded within the two NF-kappaB elements. Gel retardation/immunoblot analysis using purified E2F-1 and p50 homodimers reveals the presence of complexes containing both proteins. Affinity chromatography and co-immunoprecipitation assays provide evidence for direct interaction of E2F-1 and p50 in the absence of their DNA target sequences. In vitro transcription assay demonstrates that E2F-1 represses NF-kappaB mediated transcription in a cell-free system. Functional studies in Jurkat T lymphocytic cells point to the importance of both the E2F and NF-kappaB binding sites in E2F-1 mediated repression of HIV-1 promoter, in vivo. The results of this study suggest that NF-kappaB activity may be regulated by its interaction with the cell cycle regulatory protein, E2F-1.

Binding Sites↗

PubMed1997

Role of cell cycle regulators in tumor formation in transgenic mice expressing the human neurotropic virus, JCV, early protein.

Transgenic mice harboring the early genome from the human neurotropic JC virus, JCV, develop massive abdominal tumors of neural crest origin during 6-8 months after birth and succumb to death a few weeks later. The viral early protein, T-antigen, which possesses the ability to transform cells of neural origin, is highly expressed in the tumor cells. Immunoblot analysis of protein extract from tumor tissue shows high level expression of the tumor suppressor protein, p53, in complex with T-antigen. Expression of p21, a downstream target for p53, which controls cell cycle progression by regulating the activity of cyclins and their associated kinases during the G1 phase, is extremely low in the tumor cells. Whereas the level of expression and activity of cyclin D1 and its associated kinase, cdk6, was modest in tumor cells, both cyclin A and E, and their kinase partners, cdk2 and cdk4, were highly expressed and exhibited significant kinase activity. The retinoblastoma gene product, pRb, which upon phosphorylation by cyclins:cdk induces rapid cell proliferation, was found in the phosphorylated state in tumor cell extracts, and was detected in association with JCV T-antigen. The transcription factor, E2F-1, which dissociates from the pRb-E2F-1 complex and stimulates S phase-specific genes upon phosphorylation of pRb and/or complexation of pRb with the viral transforming protein, was highly expressed in tumor cells. Accordingly, high level expression of the E2F-1-responsive gene, proliferating cell nuclear antigen (PCNA), was detected in the tumor cells. These observations suggest a potential regulating pathway that, upon expression of JCV T-antigen, induces formation and progression of tumors of neural origin in a whole animal system.

Animals↗

PubMed1997

Evidence for inhibition of MyEF-2 binding to MBP promoter by MEF-1/Pur alpha.

Myelin basic protein (MBP) is a major component of the myelin sheath whose production is developmentally controlled during myelinogenesis. Earlier studies have indicated that programmed expression of the MBP gene is regulated at the level of transcription. Evidently, the MB1 regulatory motif located between nucleotides -14 to -50 plays an important role in transcription of the MBP promoter in both in vivo systems. The MB1 element contains binding sites for the activator protein MEF-1/Pur alpha and the repressor protein MyEF-2. In this study we use bandshift assays with purified MEF-1/Pur alpha and MyEF-2 and demonstrate that binding of MyEF-2 to its target sequence is inhibited by MEF-1/Pur alpha. Under similar conditions, MyEF-2 enhances the association of MEF-1/Pur alpha with MB1 DNA. MEF-1/Pur alpha binds to MB1 in mono- and dimeric forms. Inclusion of MyEF-2 in the binding reaction increases the dimeric association of MEF-1/Pur alpha with the MB1 sequence. The use of MEF-1/Pur alpha variants in the bandshift assay suggests that two distinct regions of this protein may be involved in its binding to the MB1 sequences, and its ability to block MyEF-2 interaction with the MB1 sequence. Based on previous studies on the programmed expression of MEF-1/Pur alpha and MyEF-2 during myelination and the current findings on their interplay for binding to the MB1 motif, a model is proposed for their involvement in transcriptional regulation of the MBP gene during the course of brain development.

Adenosine Triphosphate↗