Search PubMed⌕ Search

Biomedical subjects

J M Boss

Publications and source records attributed to J M Boss.

At least 19 recordsLinked to original sources
PubMed2001

The MHC-specific enhanceosome and its role in MHC class I and beta(2)-microglobulin gene transactivation.

The promoter regions of MHC class I and beta(2)-microglobulin (beta(2)m) genes possess a regulatory module consisting of S, X, and Y boxes, which is shared by MHC class II and its accessory genes. In this study we show that, similar to MHC class II, the SXY module in MHC class I and beta(2)m promoters is cooperatively bound by a multiprotein complex containing regulatory factor X, CREB/activating transcription factor, and nuclear factor Y. Together with the coactivator class II transactivator this multiprotein complex drives transactivation of these genes. In contrast to MHC class II, the multiprotein complex has an additional function in the constitutive transactivation of MHC class I and beta(2)m genes. The requirement for all transcription factors in the complex and correct spacing of the binding sites within the SXY regulatory module for complex formation and functioning of this multiprotein complex strongly suggests that this complex can be regarded as a bona fide enhanceosome. The general coactivators CREB binding protein, p300, general control nonderepressible-5, and p300/CREB binding protein-associated factor exert an ancillary function in MHC class I and beta(2)m transactivation, but exclusively through the class II transactivator component of this enhanceosome. Thus, the SXY module is the basis for a specific enhanceosome important for the constitutive and inducible transactivation of MHC class I and beta(2)m genes.

Cyclic AMP Response Element-Binding Protein↗
PubMed2001

CIITA coordinates multiple histone acetylation modifications at the HLA-DRA promoter.

We present here an in vivo view of major histocompatibility complex (MHC) class II promoter assembly, nucleosome modifications and gene expression mediated by the class II transactivator (CIITA). Acetylation and deacetylation of histones H3 and H4 at the HLA-DRA promoter were found to occur during a time-course that depended on CIITA expression and binding. Expression of a CIITA mutant, which lacked the activation domain, induced H4 but not H3 histone acetylation. This suggested that multiple histone acetyltransferase activities are associated with MHC class II expression. H4 acetylation was mapped to Lys8, which implicated several histone acetyltransferases as possible modulators of this activity.

Acetylation↗
PubMed2001

Varying functions of specific major histocompatibility class II transactivator promoter III and IV elements in melanoma cell lines.

Melanoma cells commonly express MHC class II molecules constitutively. This is a rare, or possibly unique, phenotype for a nonprofessional antigen-presenting cell, where MHC class II expression ordinarily occurs only after IFN-gamma treatment. Despite the fact that constitutive expression of MHC class II on melanoma cells has been observed for decades and that the regulation of the MHC class II genes is well understood for many different cell types, there is no data regarding the basis for constitutive MHC class II expression in melanoma cells. Here we report that MHC class II expression in melanoma cells can be traced to constitutive expression of the class II transactivator protein (CIITA), which mediates both IFN-gamma-inducible and -constitutive MHC class II expression in all other cell types. In addition, we determined that constitutive CIITA expression is the result of the activation of both the B cell-specific CIITA promoter III and the IFN-gamma-inducible CIITA promoter IV, the latter of which previously has never been known to function as a constitutive promoter in any cell type. The recently described B cell-related ARE-1 activity is important for promoter III activation in the melanoma cells. Constitutive promoter IV activation involves the IFN regulatory factor element (IRF-E), which binds members of the IRF family of proteins, although the major, IFN-gamma inducible member of this family, IRF-1, is not constitutively expressed in these cells. In cells with constitutively active promoter IV, the promoter IV IRF-E is most likely activated by IRF-2. The relevance of these results to the pathway of melanoma development is discussed.

DNA-Binding Proteins↗
PubMed2000

Methylation of class II trans-activator promoter IV: a novel mechanism of MHC class II gene control.

Inhibition of class II trans-activator (CIITA) expression prevents embryonic trophoblast cells from up-regulating MHC class II genes in response to IFN-gamma. This is thought to be one mechanism of maternal tolerance to the fetal allograft. The CIITA gene is regulated by four distinct promoters; promoter III directs constitutive (B cell) expression, and promoter IV regulates IFN-gamma-inducible expression. Using in vivo genomic footprinting, promoter-reporter analysis, Southern blot analysis, and RT-PCR, we have examined the cause of CIITA silencing in a trophoblast-derived cell line. We report here that methylation of promoter IV DNA at CpG sites in Jar cells prevents promoter occupancy and IFN-gamma-inducible transcription. The inhibition of CpG methylation in Jar cells by treatment with 5-aza-2'-deoxycytidine restores IFN-gamma inducibility to CIITA. This is the first description of an epigenetic mechanism involved in regulation of CIITA and MHC class II gene expression.

Antimetabolites, Antineoplastic↗
PubMed2000

Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

Adolescent↗
PubMed2000

Sp1 binding is critical for promoter assembly and activation of the MCP-1 gene by tumor necrosis factor.

The monocyte chemoattractant protein-1 gene (MCP-1) is induced by the inflammatory cytokine tumor necrosis factor through the coordinate assembly of an NF-kappaB-dependent distal regulatory region and a proximal region that has been suggested to bind Sp1 as well as other factors. To provide a genetic correlation for Sp1 activity in this system, a cell line homozygous for a targeted truncation of the Sp1 gene was derived and examined. We found that the lack of Sp1 binding activity resulted in the inability of both the distal and proximal regions to assemble in vivo even though the binding of NF-kappaB to distal region DNA was unaffected in vitro. We also found that Sp1 and NF-kappaB were the minimal mammalian transcription factors required for efficient activity when transfected into Drosophila Schneider cells. Additionally, Sp3 was able to compensate for Sp1 in the Drosophila tissue cell system but not in the Sp1(-/-) cell line suggesting that Sp1 usage is site-specific and is likely to depend on the context of the binding site. Together, these data provide genetic and biochemical proof for Sp1 in regulating the MCP-1 gene.

3T3 Cells↗
PubMed2000

Site A of the MCP-1 distal regulatory region functions as a transcriptional modulator through the transcription factor NF1.

The monocyte chemoattractant protein-1 (MCP-1) functions to recruit monocytes and macrophages to areas of inflammation and is a prototypic chemokine subjected to coordinate regulation by immunomodulatory agents. TNF mediated regulation of MCP-1 occurs through a distal regulatory region located 2.5 kb upstream of the transcriptional start site. Within this region are two NF-kB motifs that are each critical for function. Site A, located within the distal regulatory region and upstream of the kappaB elements is required for maximal induction by TNF. However, unlike the kappaB elements and other MCP-1 regulatory elements, Site A is constitutively occupied by factors in vivo. To better understand the nature of Site A function, this report identified a Site A binding protein and provides a functional analysis of the element in driving transcription. The results showed that the transcription factor NF1/CTF binds to Site A both in vitro and in vivo. While Site A has no transcriptional activity on its own, it was found to augment the transcriptional activity of a GAL4-VP16 reporter system in an orientation and position independent manner. Because NF1 is known to interact with factors that modify nucleosomes, these results suggest a unique role for Site A in regulating MCP-1 expression.

3T3 Cells↗
PubMed2000

Associations and interactions between bare lymphocyte syndrome factors.

The bare lymphocyte syndrome, a severe combined immunodeficiency due to loss of major histocompatibility complex (MHC) class II gene expression, is caused by inherited mutations in the genes encoding the heterotrimeric transcription factor RFX (RFX-B, RFX5, and RFXAP) and the class II transactivator CIITA. Mutagenesis of the RFX genes was performed, and the properties of the proteins were analyzed with regard to transactivation, DNA binding, and protein-protein interactions. The results identified specific domains within each of the three RFX subunits that were necessary for RFX complex formation, including the ankyrin repeats of RFX-B. DNA binding was dependent on RFX complex formation, and transactivation was dependent on a region of RFX5. RFX5 was found to interact with CIITA, and this interaction was dependent on a proline-rich domain within RFX5. Thus, these studies have defined the protein domains required for the functional regulation of MHC class II genes.

Animals↗
PubMed1999

trans-Retinoic acid blocks platelet-derived growth factor-BB-induced expression of the murine monocyte chemoattractant-1 gene by blocking the assembly of a promoter proximal Sp1 binding site.

Proper regulation of the CC chemokine MCP-1 (monocyte chemoattractant protein-1) is important for normal inflammatory responses. MCP-1 is regulated by a wide variety of agents, including platelet-derived growth factor-BB (PDGF-BB) and tumor necrosis factor-alpha (TNF). Using both in vivo and in vitro assays, the elements required for expression between these two cytokines were compared. In vivo genomic footprinting showed that PDGF-BB induction occurred through the occupancy of the proximal regulatory region, and unlike TNF induction, no changes in the NF-kappaB binding, distal regulatory region occurred. Treatment of cells with trans-retinoic acid, an inhibitor of PDGF-BB activity, resulted in a 50% reduction in PDGF-BB-mediated induction and a concomitant block in the assembly of the proximal regulatory region. trans-Retinoic acid had minimal effect on TNF induction or promoter occupancy. An inhibitor of histone deacetylation was found to stimulate expression of MCP-1 in a manner that correlated with increased accessibility to the proximal regulatory region. These results show that the mechanisms of PDGF-BB and TNF activation of MCP-1 are distinct, although they both require the proximal regulatory region Sp1 binding site. The results also suggest that part of the mechanism used by both of these cytokines involves a process that regulates transcription factor access to the regulatory regions.

3T3 Cells↗
PubMed1999

Orientation and positional mapping of the subunits of the multicomponent transcription factors RFX and X2BP to the major histocompatibility complex class II transcriptional enhancer.

Major histocompatibility complex class II genes contain a common complex enhancer that allows for their coordinate regulation. The X box element of the enhancer cooperatively binds the multisubunit transcription factors RFX and X2BP. RFX is an essential class II transcription factor and contains three distinct proteins: RFX5, RFX-B/Ank and RFXAP. X2BP, a CREB/ATF family transcription factor, most likely binds as a homodimer. A site-specific protein-DNA photocrosslinking assay was used to investigate the interactions of the subunits of RFX and X2BP with X box DNA. Two of the RFX subunits, RFX5 and RFX-B/Ank, were found to bind defined sites within the X1 half of the X box. The third RFX subunit, RFXAP, made extensive X1 box contacts. The subunits of X2BP made contacts with the edges of the X2 half of the X box in a manner consistent with other bZIP transcription factor contact patterns. The resulting map provides specific base pair contacts and subunit orientation with respect to the DNA sequence of the RFX-X2BP-X box complex. Our results suggest possible stoichiometry of the RFX subunits and potential interaction between RFX-B/Ank and RFXAP with one of the subunits of X2BP.

Base Sequence↗
PubMed1999

Nuclear factor-kappa B p65 mediates the assembly and activation of the TNF-responsive element of the murine monocyte chemoattractant-1 gene.

TNF-alpha transcriptionally regulates murine monocyte chemoattractant protein-1 (MCP-1) expression. Three approaches were used to determine the mechanism by which TNF regulates MCP-1. Mutation analysis showed that two distal kappa B sites, a novel dimethylsulfate-hypersensitive sequence, and a promoter proximal SP-1 site were required for TNF induction. Although the kappa B sites and the hypersensitive sequence function as a NF-kappa B-mediated enhancer, regulating induction by TNF, stereospecific alignment of the kappa B sites was not critical. Trans-activation studies conducted by cotransfection of p50 and/or p65 expression vectors with MCP-1 constructions showed that TNF regulates MCP-1 through NF-kappa B. Examination of MCP-1 induction in NF-kappa B-disrupted embryonic fibroblasts showed that p65 was necessary for both the induction and the TNF-induced protein occupancy of the enhancer in vivo. The action of the antioxidant inhibitor of NF-kappa B activation, pyrrolidine dithiocarbamate, in wild-type and NF-kappa B mutant cells was examined. The results suggested that TNF activates NF-kappa B through both pyrrolidine dithiocarbamate-sensitive and -insensitive mechanisms. This study illustrates the crucial role for NF-kappa B p65 in the induction of the MCP-1 gene by TNF and in the assembly of a NF-kappa B dependent enhancer in vivo.

3T3 Cells↗
PubMed1999

CREB regulates MHC class II expression in a CIITA-dependent manner.

The X2 box of MHC class II promoters is homologous to TRE/CRE elements and is required for expression of MHC class II genes. The X2 box-specific DNA binding activity, X2BP, was purified to homogeneity, sequenced, and identified as CREB. Transient transactivation experiments showed that CREB can cooperate with CIITA to enhance activation of transcription from MHC class II promoters in a dose-dependent manner. Binding of CREB to the class II promoter in vivo was demonstrated by a chromatin immunoprecipitation assay. Additionally, ICER, a dominant inhibitor of CREB function, was found to repress class II expression. These results demonstrate that CREB binds to the X2 box in vivo and cooperates with CIITA to direct MHC class II expression.

Amino Acid Sequence↗
PubMed1999

RFX-B is the gene responsible for the most common cause of the bare lymphocyte syndrome, an MHC class II immunodeficiency.

The bare lymphocyte syndrome (BLS) is characterized by the absence of MHC class II transcription and humoral- and cellular-mediated immune responses to foreign antigens. Three of the four BLS genetic complementation groups have defects in the activity of the MHC class II transcription factor RFX. We have purified the RFX complex and sequenced its three subunits. The sequence of the smallest subunit describes a novel gene, termed RFX-B. RFX-B complements the predominant BLS complementation group (group B) and was found to be mutant in cell lines from this BLS group. The protein has no known DNA-binding domain but does contain three ankyrin repeats that are likely to be important in protein-protein interactions.

Amino Acid Sequence↗
PubMed1998

The MHC class II transactivator (CIITA) requires conserved leucine charged domains for interactions with the conserved W box promoter element.

The class II transactivator CIITA is required for transcriptional activation of the major histocompatibility complex (MHC) class II genes. Aside from an N-terminal acidic transcriptional activation domain, little is known about how this factor functions. Extensive mutagenesis of CIITA was undertaken to identify structural motifs required for function. The ability of mutants to activate a reporter gene under the control of MHC class II conserved W-X-Y or X-Y regulatory elements was determined. Two mutants displayed differential activity between the two promoters, activating transcription with the W-X-Y but not the X-Y elements. All mutants were tested for their ability to interfere with wild-type CIITA activity. Five CIITA mutant constructions were able to down-regulate wild-type CIITA activity. Three of these mutants contained targeted disruptions of potential functional motifs: the acidic activation domain, a putative GTP-binding motif and two leucine charged domains (LCD motifs). The other two contained mutations in regions that do not have homology to described proteins. The characterization of CIITA mutants that are able to discriminate between promoters with or without the W box strongly suggests that CIITA requires such interactions for function. The identification of LCD motifs required for CIITA function brings to light a previously undefined role of these motifs in CIITA function.

Amino Acid Substitution↗
PubMed1998

CIITA stimulation of transcription factor binding to major histocompatibility complex class II and associated promoters in vivo.

CIITA is a master transactivator of the major histocompatibility complex class II genes, which are involved in antigen presentation. Defects in CIITA result in fatal immunodeficiencies. CIITA activation is also the control point for the induction of major histocompatibility complex class II and associated genes by interferon-gamma, but CIITA does not bind directly to DNA. Expression of CIITA in G3A cells, which lack endogenous CIITA, followed by in vivo genomic footprinting, now reveals that CIITA is required for the assembly of transcription factor complexes on the promoters of this gene family, including DRA, Ii, and DMB. CIITA-dependent promoter assembly occurs in interferon-gamma-inducible cell types, but not in B lymphocytes. Dissection of the CIITA protein indicates that transactivation and promoter loading are inseparable and reveal a requirement for a GTP binding motif. These findings suggest that CIITA may be a new class of transactivator.

Antigen Presentation↗
PubMed1998

IFN-gamma induction of the human monocyte chemoattractant protein (hMCP)-1 gene in astrocytoma cells: functional interaction between an IFN-gamma-activated site and a GC-rich element.

We characterized regulation of the human monocyte chemoattractant protein-1 (hMCP-1) gene by IFN-gamma in astrocytoma cells, because astroglial cells express chemokines in several central nervous system inflammatory states. It was found that IFN-gamma-induced hMCP-1 transcription was rapid, transient, and mediated by a 213-bp promoter-proximal regulatory region of the gene. Our studies on both in vitro and in vivo states of the hMCP-1 regulatory region established requirement of an IFN-gamma-activated site (GAS) and the presence of IFN-gamma-inducible GAS-binding activity involving at least STAT-1alpha for IFN-gamma-induced hMCP-1 expression. Unexpectedly, in vivo genomic footprinting of the proximal regulatory region of the IFN-gamma-induced gene revealed protection of a GC-rich sequence (GC box) with the same temporal pattern as that seen at the GAS; in vitro, this GC-rich element is associated with nuclear factor Sp1. These observations suggested a cooperative interaction between the GAS and the GC box element. Interestingly, site-specific mutations that abolished GC-box or GAS-element function produced clearly disparate results. Disruption of the GC box did not affect fold induction by IFN-gamma but reduced promoter-reporter expression by half. Conversely, GAS mutation abrogated induction but did not affect the magnitude of expression. These results establish the importance of the GAS element for induction of hMCP-1 and further our understanding of IFN-gamma-mediated transcriptional induction by providing the first evidence in vivo for inducible signaling to the GC box by this cytokine.

Astrocytoma↗