Jeremy M. Boss

Defining the CREB Regulon: A Genome-Wide Analysis of Transcription Factor Regulatory Regions

The CREB transcription factor regulates differentiation, survival, and synaptic plasticity. The complement of CREB targets responsible for these responses has not been identified, however. We developed a novel approach to identify CREB targets, termed serial analysis of chromatin occupancy (SACO), by combining chromatin immunoprecipitation (ChIP) with a modification of SAGE. Using a SACO library derived from rat PC12 cells, we identified approximately 41,000 genomic signature tags (GSTs) that mapped to unique genomic loci. CREB binding was confirmed for all loci supported by multiple GSTs. Of the 6302 loci identified by multiple GSTs, 40% were within 2 kb of the transcriptional start of an annotated gene, 49% were within 1 kb of a CpG island, and 72% were within 1 kb of a putative cAMP-response element (CRE). A large fraction of the SACO loci delineated bidirectional promoters and novel antisense transcripts. This study represents the most comprehensive definition of transcription factor binding sites in a metazoan species.

The NLR gene family: a standard nomenclature.

Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohns disease and Blau syndrome. A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.

CorrespondenceThe NLR Gene Family: A Standard Nomenclature

Iimmune regulatory proteins such as CIITA, NAIP, IPAF, NOD1, NOD2, NALP1, cryopyrin/NALP3 are members of a family characterized by the presence of a nucleotide-binding domain (NBD) and leucine-rich repeats (LRR). Members of this gene family encode a protein structure similar to the NB-LRR subgroup of disease-resistance genes in plants and are involved in the sensing of pathogenic products and the regulation of cell signaling and apoptosis. Several members of this family have been associated with immunologic disorders. NOD2 for instance is associated with both Crohns disease and Blau syndrome. A variety of different names are currently used to describe this gene family, its subfamilies and individual genes, including CATERPILLER (CLR), NOD-LRR, NACHT-LRR, CARD, NALP, NOD, PAN and PYPAF, and this lack of consistency has led to a pressing need to unify the nomenclature. Consequently, we collectively propose the family designation NLR (nucleotide-binding domain and leucine-rich repeat containing) and provide unique and standardized gene designations for all family members.

MTA3 and the Mi-2/NuRD Complex Regulate Cell Fate during B Lymphocyte Differentiation

The transcriptional repressor BCL-6 regulates B lymphocyte cell fate during the germinal center reaction by preventing terminal differentiation of B lymphocytes into plasma cells until appropriate signals are received. Here, we report a cofactor, MTA3, a cell type-specific subunit of the corepressor complex Mi-2/NuRD, for BCL-6-dependent cell fate determination. MTA3 is expressed in the same pattern in germinal centers as BCL-6. BCL-6 physically interacts with Mi-2/NuRD and this interaction is sensitive to BCL-6 acetylation status. Depletion of MTA3 by RNAi impairs BCL-6-dependent repression and alters the cell-specific transcriptional pattern characteristic of the B lymphocyte. Remarkably, exogenous expression of BCL-6 in a plasma cell line leads, in an MTA3-dependent manner, to repression of plasma cell-specific transcripts, reactivation of the B cell transcriptional program, expression of B lymphocyte cell surface markers, and reprogramming of cell fate.

Transcription factor T-bet represses expression of the inhibitory receptor PD-1 and sustains virus-specific CD8 + T cell responses during chronic infection

T cell exhaustion has a major role in failure to control chronic infection. High expression of inhibitory receptors, including PD-1, and the inability to sustain functional T cell responses contribute to exhaustion. However, the transcriptional control of these processes remains unclear. Here we demonstrate that the transcription factor T-bet regulated the exhaustion of CD8+ T cells and the expression of inhibitory receptors. T-bet directly repressed transcription of the gene encoding PD-1 and resulted in lower expression of other inhibitory receptors. Although a greater abundance of T-bet promoted terminal differentiation after acute infection, high T-bet expression sustained exhausted CD8+ T cells and repressed the expression of inhibitory receptors during chronic viral infection. Persistent antigenic stimulation caused downregulation of T-bet, which resulted in more severe exhaustion of CD8+ T cells. Our observations suggest therapeutic opportunities involving higher T-bet expression during chronic infection.

Regulation of transcription of MHC class II genes.

Genetic and biochemical analyses have identified multiple DNA-binding and non-DNA-binding proteins that functionally regulate MHC class II genes. These include RFX, X2BP, NF-Y, CIITA, OCT-2 and Bob1. One of the essential non-DNA-binding proteins, CIITA, appears to function as a limiting molecular switch that is responsible for the control of class II expression and the regulation of expression by interferon-gamma.

Activation of class II MHC genes requires both the X ☐ region and the class II transactivator (CIITA)

CIITA, a gene that can complement a transcriptional mutation of the major histocompatibility complex (MHC) class II genes, was tested for its ability to function as a coactivator, CIITA cDNA clones isolated showed alternative RNA splicing, but only one splice site combination was able to restore class II MHC gene expression. DNA-mediated transfection experiments showed that CIITA directs its activity through the X box element; the presence of CIITA leads to the formation of a higher order complex at the X box region; and CIITA contains a potent activation domain. These findings support the hypothesis that CIITA directly interacts with the MHC class II-specific transcription factors and is required for expression.

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.

Promoter Region Architecture and Transcriptional Regulation of the Genes for the MHC Class I-Related Chain A and B Ligands of NKG2D

Ligands of the NKG2D receptor, which activates NK cells and costimulates effector T cells, are inducibly expressed under harmful conditions, such as malignancies and microbial infections. Moreover, aberrant expression in autoimmune disease lesions may contribute to disease progression. Among these ligands are the closely related human MHC class I-related chains (MIC) A and B, which appear to be regulated by cellular stress. Analyses of MIC gene 5′-end flanking regions in epithelial tumor cells defined minimal core promoters that directed near maximum heat shock- or oxidative stress-induced transcriptional activation. Considerably larger fully functional promoters were required for maximum proliferation-associated activation. These activities were dependent on core promoter sequences that included heat shock elements, which inducibly bound heat shock factor 1, TATA-like elements, and constitutively occupied Sp1 and inverted CCAAT box factor sites. By contrast, MIC gene activation by CMV infection was largely independent of these and upstream promoter sequences, and expression of viral immediate early gene (IE1 or IE2) products was sufficient for induction of transcription and surface protein expression. Altogether, these results reveal distinct modes of activation of the genes for the MIC ligands of NKG2D and provide a molecular framework for analyses of gene regulation under different cellular insult conditions.

NFATc1 Regulates PD-1 Expression upon T Cell Activation

PD-1 is a transmembrane protein involved in the regulation of immunological tolerance. Multiple studies have reported an association between high levels of PD-1 expressed on T cell surfaces and exhaustion in lymphocyte populations when challenged by chronic viral infections, such as HIV. By using model systems consisting of murine EL4 cells, which constitutively express PD-1, and primary murine CD8 T cells that express PD-1 upon T cell stimulation, we have identified two tissue-specific hypersensitive sites at the 5′ CR of the PD-1 locus. Gene reporter assays in CD8 T cells have shown that one of these sites has robust transcriptional activity in response to cell stimulation. Cell treatment with the calcineurin inhibitor cyclosporine A or a NFAT-specific inhibitor led to a sharp reduction in PD-1 expression in the constitutive and inducible systems. Furthermore, analysis of this region by chromatin immunoprecipitation assay revealed NFATc1 binding associated with gene activation in EL4 and primary CD8 T cells. Mutation of the NFATc1 binding site in PD-1 reporter constructs resulted in a complete loss of promoter activity. Together, these results demonstrate that PD-1 gene regulation occurs in part via the recruitment of NFATc1 to a novel regulatory element at the pdcd1 locus and provides the molecular mechanism responsible for the induction of PD-1 in response to T cell stimulation.