PubMed Health⌕ Search

Biomedical subjects

T K Howcroft

Publications and source records attributed to T K Howcroft.

At least 19 recordsLinked to original sources
PubMed2001

Transcriptional coactivator, CIITA, is an acetyltransferase that bypasses a promoter requirement for TAF(II)250.

The CIITA coactivator is essential for transcriptional activation of MHC class II genes and mediates enhanced MHC class I transcription. We now report that CIITA contains an intrinsic acetyltransferase (AT) activity that maps to a region within the N-terminal segment of CIITA, between amino acids 94 and 132. The AT activity is regulated by the C-terminal GTP-binding domain and is stimulated by GTP. CIITA-mediated transactivation depends on the AT activity. Further, we report that, although constitutive MHC class I transcription depends on TAF(II)250, CIITA activates the promoter in the absence of functional TAF(II)250.

Acetyltransferases↗
PubMed2000

TAF(II)250-independent transcription can be conferred on a TAF(II)250-dependent basal promoter by upstream activators.

TAF(II)250, a component of the general transcription factor, TFIID, is required for the transcription of a subset of genes, including those involved in regulating cell cycle progression. The tsBN462 cell line, with a temperature-sensitive mutation of TAF(II)250, grows normally at 32 degrees C, but when grown at 39.5 degrees C, it differentially arrests transcription of many, but not all, genes. The present studies examine the basis for the requirement for TAF(II)250. We show that the basal promoter of a major histocompatibility complex class I gene requires TAF(II)250. This dependence can be overcome by select upstream regulatory elements but not by basal promoter elements. Thus, the coactivator CIITA rescues the basal promoter from the requirement for TAF(II)250, whereas introduction of a canonical TATAA box does not. Similarly, the SV40 basal promoter is shown to require TAF(II)250, and the presence of the 72-base pair enhancer overcomes this requirement. Furthermore, the SV40 72-base pair enhancer when placed upstream of the basal class I promoter renders it independent of TAF(II)250. These data suggest that the assembly of transcription initiation complexes is dynamic and can be modulated by specific transcription factors.

Animals↗
PubMed1999

Upstream stimulatory factor regulates major histocompatibility complex class I gene expression: the U2DeltaE4 splice variant abrogates E-box activity.

The tissue-specific expression of major histocompatibility complex class I genes is determined by a series of upstream regulatory elements, many of which remain ill defined. We now report that a distal E-box element, located between bp -309 and -314 upstream of transcription initiation, acts as a cell type-specific enhancer of class I promoter activity. The class I E box is very active in a neuroblastoma cell line, CHP-126, but is relatively inactive in the HeLa epithelial cell line. The basic helix-loop-helix leucine zipper proteins upstream stimulatory factor 1 (USF1) and USF2 were shown to specifically recognize the class I E box, resulting in the activation of the downstream promoter. Fine mapping of USF1 and USF2 amino-terminal functional domains revealed differences in their abilities to activate the class I E box. Whereas USF1 contained only an extended activation domain, USF2 contained both an activation domain and a negative regulatory region. Surprisingly, the naturally occurring splice variant of USF2 lacking the exon 4 domain, U2DeltaE4, acted as a dominant-negative regulator of USF-mediated activation of the class I promoter. This latter activity is in sharp contrast to the known ability of U2DeltaE4 to activate the adenovirus major late promoter. Class I E-box function is correlated with the relative amount of U2DeltaE4 in a cell, leading to the proposal that U2DeltaE4 modulates class I E-box activity and may represent one mechanism to fine-tune class I expression in various tissues.

Alternative Splicing↗
PubMed1998

HIV-1 tat binds TAFII250 and represses TAFII250-dependent transcription of major histocompatibility class I genes.

HIV Tat, a transactivator of viral transcription, represses transcription of major histocompatibility (MHC) class I genes. Repression depends exclusively on the C-terminal domain of Tat, although the mechanism of this repression has not been known. We now show that repression results from the interaction of Tat with the TAFII250 component of the general transcription factor, TFIID. The C-terminal domain of Tat binds to a site on TAFII250 that overlaps the histone acetyl transferase domain, inhibiting TAFII250 histone acetyl transferase activity. Furthermore, promoters repressed by Tat, including the MHC class I promoter, are dependent on TAFII250 whereas those that are not repressed by Tat, such as SV40 and MuLV promoters, are independent of functional TAFII250. Thus, Tat repression of MHC class I transcription would be one mechanism by which HIV avoids immune surveillance.

Acetyltransferases↗
PubMed1998

Elf-1 regulates basal expression from the T cell antigen receptor zeta-chain gene promoter.

In mature T cells, limited synthesis of the TCR-zeta subunit is primarily responsible for regulating surface expression of TCRs. Transcription of zeta is directed by a complex promoter that includes two potential binding sites for the Ets family of transcription factors at -52 (zEBS1) and -135 (zEBS2). Mutation of these two sites results in a marked reduction of transcription from this promoter. Using electrophoretic mobility shift analysis, Elf-1 was demonstrated to be the Ets family member that binds to these sites. One site, zEBS1, matches the optimal Elf-1 consensus sequence in eight of nine bases, making it the best match of any known mammalian Elf-1 binding site. A role for Elf-1 in TCR-zeta trans-activation was confirmed by ectopic expression of Elf-1 in COS-7 cells. This resulted in an increase in TCR-zeta promoter activity that mapped to zEBS1 and zEBS2. Additional support for the involvement of Elf-1 in TCR-zeta trans-activation derives from the finding that a GAL4-Elf-1 fusion protein trans-activated TCR-zeta promoter constructs that had been modified to contain GAL4 DNA binding sites. These results demonstrate that Elf-1 plays an essential role in the trans-activation of a constitutively expressed T cell-specific gene, and that trans-activation occurs in the context of the native promoter in both lymphoid and nonlymphoid cells. Taken together with the existing literature, these data also suggest that the requirement for inducible factors in Elf-1-mediated trans-activation may decrease as the affinity and number of Elf-1 sites increase.

Animals↗
PubMed1998

HIV Tat protein requirements for transactivation and repression of transcription are separable.

The HIV Tat protein, primarily characterized as a transcriptional activator of the viral long terminal repeat (LTR), is also a potent repressor of major histocompatibility complex (MHC) class I transcription. In the present study, we demonstrate that these two functional activities are distinct and mediated by discrete, but overlapping, structural domains of Tat. Tat repressor activity depends on C-terminal sequences, whereas transactivation depends on N-terminal sequences; both functions require core sequences. The repressor activity requires a domain encompassing the region encoded by the second exon of the Tat gene, beginning at amino acid 73, with a C-terminal limit between amino acids 80 and 83. Tat repressor function also depends on the presence of a lysine at position 41, located within the core of the protein. Tat repressor activity is independent of two N-terminal domains essential for transactivation: the acidic segment and the cysteine-rich region. Conversely, Tat transactivation is independent of the second exon-encoded region of Tat. As further support for this novel model of separable Tat functions, we show that in murine fibroblasts, Tat represses class I promoter activity, but does not transactivate the HIV LTR. We propose that distinct structural domains mediate the two functionally distinct activities associated with the Tat protein.

3T3 Cells↗
PubMed1998

Characterization of two class I genes from the H2-M region: evidence for a new subfamily.

We cloned, sequenced, and mapped two divergent major histocompatibility class Ib genes from BALB/c mice. M9d and M10d both have the potential to encode full-length class I molecules, but transcripts were not readily detectable. M9 is 86% similar to M1 in its nucleotide sequence and maps next to it on YAC clones. M9 is only 64% similar to M10 and 60% to H2-K k. Probes from M10 define a new subfamily of eight class I genes in C3H mice; five cluster directly distal to H2-T1, and three are located between M9-1-7-8 and M6-4-5 in the H2-M region.

Amino Acid Sequence↗
PubMed1998

HIV Tat represses transcription of the beta 2-microglobulin promoter.

The MHC class I complex, which binds and presents peptide antigen, is composed of a class I heavy chain and the beta2-microglobulin light chain. HIV-1, which induces a profound immunodeficiency in infected individuals, encodes proteins that cause decreased expression of class I heavy chain. We now report that the HIV Tat protein, which is a potent transactivator of viral transcription, is also a potent repressor of the beta2-microglobulin gene. Repression is mediated through the basal promoter of the beta2-microglobulin gene, which is shown to be predominantly regulated by an initiator element. Tat repression is further augmented by the short viral transcript, TAR, which interacts with Tat. Tat-mediated repression of beta2-microglobulin expression, together with its known repression of class I gene transcription, provides an effective mechanism by which HIV could prevent cell surface expression of the MHC class I complex and avoid immune surveillance.

Animals↗
PubMed1997

Measure of transient transfection efficiency using beta-galactosidase protein.

Although the control elements which regulate the transcriptional activity of promoter sequences are largely determined by the use of reporter plasmids in transient transfection analyses, controlling variability in these experiments can often be a vexing problem. Problems arise when the promoter of the internal control plasmid, used to correct for transfection efficiency, either affects test promoter strength or is itself regulated by trans-acting factors or inducing agents used to study the test promoter. Here we report the use of beta-galactosidase protein as an unbiased standard of transfection efficiency. beta-Galactosidase protein is readily internalized by adherent cell lines when incorporated into a calcium phosphate precipitate; significant enzyme activity can be recovered up to 72 h after transfection. Use of beta-galactosidase protein as a control obviates the concerns associated with promoter-dependent reporter plasmids as controls.

Calcium Phosphates↗
PubMed1997

A spontaneously arising mutation in the DLAARN motif of murine ZAP-70 abrogates kinase activity and arrests thymocyte development.

Development of immature CD4+ CD8+ thymocytes into functionally mature CD4+ and CD8+ T cells is driven by selection events that require signals transduced through the T cell antigen receptor (TCR). Transduction of TCR signals in the thymus involves tyrosine phosphorylation of the protein tyrosine kinase ZAP-70 by p56(lck) and results in induction of ZAP-70 enzymatic activity. We have identified a novel, spontaneously arising point mutation within a highly conserved motif (DLAARN) in the kinase domain of murine ZAP-70 that uncouples tyrosine phosphorylation of ZAP-70 from induction of ZAP-70 kinase activity. Mice homozygous for this mutation are devoid of mature T cells because thymocyte development is arrested at the CD4+ CD8+ stage of differentiation. The developmental arrest is due to the inability of CD4+ CD8+ thymocytes to propagate TCR signals in the absence of ZAP-70 kinase activity despite tyrosine phosphorylation of TCR-associated ZAP-70 molecules.

Amino Acid Sequence↗
PubMed1996

Repression of the nonclassical MHC class I gene H2-M1 by cis-acting silencer DNA elements.

H2-M1 is a non-classical major histocompatibility complex (MHC) class I gene that is highly divergent from classical class I genes; M1 was the first gene in the recently classified M region of the mouse MHC to be cloned. Although the M1 DNA sequence contains normal splice sites, open reading frames within its exons, and a recognizable promoter, no M1 transcripts were detected in various healthy mouse tissues. However, M1 transcripts were detected in transfected L cells and in vivo in brains of M1 transgenic mice, albeit at very low levels, and the level of expression is correlated with transgene copy number. Analysis of the M1 promoter region identified a competent promoter capable of directing transcription, but whose expression is repressed by two strong upstream silencer elements, one mapping between -184 base pairs (bp) and -266 bp and the other between -1149 bp and -1702 bp. These studies suggest that M1 expression is highly regulated and restricted either temporally or to a very limited number of cell types.

Animals↗
PubMed1995

HIV Tat represses transcription through Sp1-like elements in the basal promoter.

MHC class I genes are potently repressed by HIV Tat, which transactivates the HIV LTR. Tat represses class I transcription by binding to complexes associated with a novel promoter element, consisting of Sp1-like DNA binding sites. Transcription by other Sp1-dependent promoters, such as MDR1 and the minimal SV40 promoters, is also repressed by Tat, whereas the human beta-actin promoter is neither activated by Sp1 nor repressed by Tat. Tat repression can be overcome by a strong enhancer element. Thus, the SV40 72 bp enhancer element confers protection from Tat-mediated repression on both the minimal SV40 promoter and the class I promoter. Surprisingly, Tat can activate the class I promoter in the presence of both the HIV TAR element and a strong upstream enhancer. These data demonstrate that Tat differentially affects Sp1-responsive promoters, depending on promoter architecture.

Base Sequence↗
PubMed1993

Repression of MHC class I gene promoter activity by two-exon Tat of HIV.

Major histocompatibility complex (MHC) class I molecules are the major receptors for viral peptides and serve as targets for specific cytotoxic T lymphocytes. Human immunodeficiency virus-type 1 (HIV-1) specifically decreased activity of an MHC class I gene promoter up to 12-fold. Repression was effected by the HIV-1 Tat protein derived from a spliced viral transcript (two-exon Tat). These studies define an activity for two-exon Tat distinct from that of one-exon Tat and suggest a mechanism whereby HIV-1-infected cells might be able to avoid immune surveillance, allowing the virus to persist in the infected host.

Exons↗
PubMed1993

MHC class I gene expression is negatively regulated by the proto-oncogene, c-jun.

The trans-acting factor AP-1 is a heterodimeric complex composed of c-Jun and c-Fos family proteins which bind and regulate genes containing a TPA responsive enhancer element. Although AP-1 binding sites have been identified within the regulatory region of major histocompatibility complex (MHC) class I genes in vitro, the role of AP-1 in regulating MHC class I transcription in vivo has not been investigated previously. The present study demonstrates that expression of c-Jun results in decreased MHC class I promoter activity as determined in cotransfection assays of an MHC class I reporter construct with a c-Jun expression construct. The c-Jun responsive element is located between bp -440 and -431 upstream of initiation of transcription as determined both functionally and by direct binding of purified c-Jun. Furthermore, over-expression of c-Jun reduced the steady state levels of endogenous MHC class I RNA in murine L cells by approximately 10-fold. These data indicate that c-Jun/AP-1 acts as a negative trans-acting factor that down-regulates MHC class I gene expression.

Animals↗
PubMed1991

Membranolytic and nucleolytic activities of cytolytic T lymphocyte clones.

In an attempt to see if the nucleolytic and membranolytic activities of cytolytic T lymphocytes (CTL) were totally independent and could be expressed independently, we cloned CTL and determined their membranolytic and nucleolytic activities. If the two lytic mechanisms were completely independent and could be independently expressed by individual CTL, we anticipated that we would find CTL clones exhibiting only one or the other activity. Initial examination of membranolytic and nucleolytic activities in 99 newly established CTL clones revealed a poor correlation (r = 0.4) between the two activities. In addition, some clones expressed membenolytic activity without nucleolytic activity, and others, nucleolytic activity without membenolytic activity. The results suggest that CTL have 2 or more separate and independent mechanisms that lead either to the membranolytic or to the nucleolytic lesions in target cells.

Animals↗
PubMed1991

The protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) inhibits PMA-induced promiscuous cytolytic activity but not specific cytolytic activity by a cloned cytolytic T lymphocyte.

Phorbol 12-myristate 13-acetate (PMA) induces the cytolytic T lymphocyte (CTL) clone 4D (H-2b anti-H-2d) to promiscuously kill the inappropriate target EL-4 (H-2b). The protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) inhibited the PMA-induced promiscuous lympholysis. The concentration of H-7 that inhibited PMA-induced lympholysis by 50% (IC50) was calculated to be 4 microM, which closely approximates the reported IC50 of H-7 of 6 microM for PKC activity in vitro. In striking contrast, specific cytolysis of appropriate P815 (H-2d) target cell by CTL clone 4D was not inhibited by concentrations of H-7 which inhibited PMA-induced promiscuous lympholysis. These results indicate that PMA-induced promiscuous lympholysis of inappropriate target cell is triggered via activation of PKC, whereas PKC activation is not obligatory in triggering CTL clone 4D to specifically kill appropriate target cells. Thus, these data suggest that cloned CTL have two or more triggering mechanisms than may initiate one or more cytolytic pathways.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗