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Dinah S. Singer, Ph.D.
Molecular Mechanisms Regulating Gene Expression
Major histocompatiblity complex (MHC) class I molecules are ubiquitously expressed tumor antigens whose expression is regulated primarily at the transcriptional level. Intracellular regulation establishes tissue-specific levels of transcription, which can vary by almost two-orders of magnitude among different tissues. Extracellular signaling pathways, engaged by numerous cytokines and hormones, superimpose on intracellular pathways and dynamically modulate transcription within any given tissue. Failure to appropriately regulate MHC class I expression results in disease: Many tumors are class I-deficient and, as we were the first to demonstrate, over-expression of class I can lead to autoimmune disease.
The long-term goal of our research program is to define the molecular mechanisms regulating gene expression, using the MHC class I genes as a model system. To that end, we focus on three areas: 1) core promoter architecture and function in basal and activated transcription, 2) analyses of the regulatory mechanisms associated with transcription initiation, and 3) the role of epigenetic regulation.
These three components are designed to provide an integrated view of regulatory events that occur at the class I core promoter. Our studies to date have yielded novel findings that generate new models which challenge prevailing dogma and have provided new insights into the regulation of a gene family critical to maintaining immunocompetence and which serves as a prototype for a novel subclass of non-TATAA promoters.
1) We demonstrated that the class I core promoter is a complex structure that spans approximately 70 bp and is the prototype of a novel subclass of non-TATAA-containing promoters that use multiple transcription start sites (TSS) and are imbedded in an 'ATG desert' (a DNA segment devoid of ATG codons). TSS usage is regulated and selective: constitutive and activated (as modeled by interferon stimulation) class I transcription targets different start sites. Further, differential TSS usage is mediated by distinct general transcription factors (GTFs) recruited to form the preinitiation complex (PIC): Basal transcription is dependent on the TFIID-associated factor TAF1 whereas activated transcription is TAF1-independent. We have discovered that the class I core promoter contains two regions that each support transcription; these two promoter regions also mutually regulate each other. Lastly, although the core promoter region contains elements homologous to canonical promoter elements, none of these elements are essential for gene expression in vivo. These studies represent a unique intensive analysis of core promoter architecture and demonstrate the complexity of core promoter function.
2) The complexity of core promoter architecture is reflected by the complexity of events associated with transcription. We have identified previously unrecognized checkpoints in the transitions from PIC recruitment to initiation to elongation. Our studies revealed that each step is regulated by the TFIID component, TAF7. In basal transcription, TAF7 binds to TAF1, inhibits the acetyltransferase (AT) activity necessary for class I transcription, and remains associated with TFIID until PIC assembly is complete, thereby preventing premature initiation. Subsequently, TAF7 is released and transcription initiates. Significantly, TAF7 also interacts with both the GTF TFIIH and the elongation factor P-TEFb, inhibiting their respective kinase activities. Kinetic in vitro transcription studies documented that TAF7 regulates PIC assembly, pausing, and initiation. Thus, we propose a model in which TAF7 functions as a quality control, check-point regulator of basal transcription. We have extended these studies of TAF7 to activated TAF1-independent transcription. In gamma-interferon-activated transcription, the co-activator CIITA bypasses the requirement for TAF1 by providing its own intrinsic AT activity. Importantly, we discovered that TAF7 binds to CIITA and inhibits its AT activity, suggesting that TAF7 is a check-point regulator of activated, as well as basal, transcription. Another important conclusion from these studies is that TFIID is a functionally dynamic complex whose components can assemble into various transcription complexes.
3) TAF7, a component of the TFIID complex that nucleates the assembly of transcription preinitiation complexes, also independently interacts with and regulates the enzymatic activities of other transcription factors, including P-TEFb, TFIIH and CIITA, ensuring an orderly progression in transcription initiation. Since not all TAFs are required in terminally differentiated cells, we examined the essentiality of TAF7 in cells at different developmental stages in vivo. Germ-line disruption of the TAF7 gene is embryonic lethal between 3.5 and 5.5 days post coitus. TAF7-deleted mouse embryonic fibroblasts cease transcription globally and stop proliferating. In contrast, whereas TAF7 is essential for the differentiation and proliferation of immature thymocytes, it is not required for subsequent, proliferation-independent differentiation of lineage committed thymocytes or for their egress into the periphery. TAF7 deletion in peripheral CD4 T cells affects only a small number of transcripts. However, TAF7-deleted T cells are not able to undergo activation and expansion in response to antigenic stimuli. These findings suggest that TAF7 is essential for proliferation but not for proliferation-independent differentiation.
3) We have also demonstrated that epigenetic mechanisms and chromatin structure maintain a transcriptionally competent class I gene. The class I gene is packaged into euchromatin and protected from heterochromatin by a barrier element in the immediate 3' intergenic segment. The barrier element is composed of two separable activities: one that is necessary for expression and another that maintains euchromatin. In contrast to many other genes, chromatin remodeling is not required to modulate class I transcription rates: nucleosome occupancy across the gene is indistinguishable in spleen, kidney and brain, despite their enormously different transcription rates. Remarkably, neither activation nor inhibition of transcription altered nucleosome positioning. Only histone modifications correlated with tissue-specific transcription rates.
We continue to study, and integrate, each of these three areas to achieve a comprehensive understanding of MHC class I transcriptional regulation.
The role of MHC Class I in Disease
Major histocompatiblity complex (MHC) class I molecules are ubiquitously expressed cell surface molecules that provide immune surveillance against intracellular pathogens and tumor antigens. Their expression is regulated primarily at the transcriptional level; intracellular pathways establish tissue-specific levels, which can vary by almost two-orders of magnitude among different tissues. Extracellular signaling pathways, engaged by numerous cytokines and hormones, superimpose on intracellular pathways and dynamically modulate transcription within any given tissue. Our studies have demonstrated that class I-deficient mice are resistant to the experimentally induced autoimmune diseases, SLE, and blepharitis and to the spontaneous SLE-like disease that occurs in NZBxNZW mice. Pharmacological treatment with an agent that reduces class I expression also reduces the incidence and severity of both experimental and spontaneous autoimmune SLE. Conversely, we found that 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 have led to the model that failure to appropriately regulate class I levels is predicted to result in disease: over-expression associated with autoimmunity and under-expression associated with an ability to avoid immune surveillance.
Methimazole prevents induction of experimental systemic lupus erythematosus in mice. Singer DS, Kohn LD, Zinger H, Mozes E. J Immunol. 1994 Jul 15;153(2):873-80.
Resistance of MHC class I-deficient mice to experimental systemic lupus erythematosus. Mozes E, Kohn LD, Hakim F, Singer DS.,Science. 1993 Jul 2;261(5117):91-3.58.
Repression of MHC class I gene promoter activity by two-exon Tat of HIV. Howcroft TK, Strebel K, Martin MA, Singer DS. Science. 1993 May 28;260(5112):1320-2.
Regulation of Transcription Initiation
Transcription consists of a series of highly regulated steps: assembly of the pre-initiation complex (PIC) at the promoter, initiation, elongation and termination. We have identified previously unrecognized checkpoints in the transitions from PIC recruitment to initiation to elongation. Our studies revealed that each step is regulated by the TFIID component, TAF7. TAF7 binds to the TFIID component, TAF1, inhibits its acetyltransferase (AT) activity necessary for class I transcription, and remains associated with TFIID until PIC assembly is complete, thereby preventing premature initiation. Then TAF7 is released and transcription initiates. Significantly, TAF7 also interacts with both the GTF TFIIH and the elongation factor P-TEFb, inhibiting their respective kinase activities. We have proposed a model in which TAF7 functions as a quality control, check-point regulator of basal transcription. Another important conclusion from these studies is that TFIID is a functionally dynamic complex whose components can assemble into various transcription complexes. Consistent with a central role in transcription, a TAF7 knockout is an embryonic lethal. TAF7-deleted mouse embryonic fibroblasts cease transcription globally and stop proliferating. In contrast, whereas TAF7 is essential for the differentiation and proliferation of immature thymocytes, it is not required for subsequent, proliferation-independent differentiation of lineage committed thymocytes or for their egress into the periphery. TAF7 deletion in peripheral CD4 T cells affects only a small number of transcripts. These findings suggest that TAF7 is essential for transcription associated with proliferation but not for proliferation-independent differentiation.
TAFII55 binding to TAFII250 inhibits its acetyltransferase activity. Gegonne A, Weissman JD, Singer DS. Proc Natl Acad Sci U S A. 2001 Oct 23;98(22):12432-7. Epub 2001 Oct 9.
TAF7: a possible transcription initiation check-point regulator. Gegonne A, Weissman JD, Zhou M, Brady JN, Singer DS. Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):602-7
TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb. Gegonne A, Weissman JD, Lu H, Zhou M, Dasgupta A, Ribble R, Brady JN, Singer DS. Proc Natl Acad Sci U S A. 2008 Apr 8;105(14):5367-72.
Novel functions for TAF7, a regulator of TAF1-independent transcription. Devaiah BN, Lu H, Gegonne A, Sercan Z, Zhang H, Clifford RJ, Lee MP, Singer DS. J Biol Chem. 2010 Dec 10;285(50):38772-80.
The General Transcription Factor TAF7 is Essential for Embryonic Development but not Essential for the Survival or Differentiation of Mature T cells. Gegonne, A., Tai, X., Zhang, J., Wu, G., Xhu, J., Yoshimoto, A., Hanson, J., Cultraro, C., Chen, A., Guinter, T., Yang, Z., Hathcokc, K., Singer, A., Rodriguez-Canales, J., Tessarollo, L., Mackem, S., Meerzaman, D., Buetow, K., Singer, D.S., Mol. Cell Biol, in press, 2012.
Regulation of Transcription through Alternative Transcription Start Site Usage
Early studies of transcription in bacteria, yeast and differentiated metazoan cells identified two core promoter elements, the TATAA box and Initiator (Inr), as necessary for transcription initiation. However, the characterization of an increasing number of promoters in mammalian cells revealed that less than 10% of all promoters contained TATAA boxes and many did not contain Inr elements. In a genome-wide analysis of promoters, we identified a novel core promoter family that is defined by the presence of an Ã¢â‚¬Å“ATG desertÃ¢â‚¬Â. ATG deserts occur nonrandomly and are significantly associated with non-TATAA promoters that use multiple TSS, independent of the presence of CpG islands (CGI). The presence of the ATG desert supports regulated transcription through selective transcription start site (TSS) usage. We demonstrated that TSS selection is actively regulated and context dependent: Analyzing the transcription from the ATG desert promoter of an MHC class I gene, we further demonstrated that transcription initiates from largely non-overlapping TSS regions during basal transcription, which depends on the canonical TFIID general transcription factor, and during interferon-activated transcription, which depends on the interferon-induced coactivator CIITA and bypasses the requirement for TFIID. We proposed that ATG deserts provide a core promoter platform upon which complex upstream regulatory signals can be integrated, targeting multiple TSS whose products encode a single protein.
ATG deserts define a novel core promoter subclass. Lee MP, Howcroft K, Kotekar A, Yang HH, Buetow KH, Singer DS. Genome Res. 2005 Sep;15(9):1189-97. Epub 2005 Aug 18.
Distinct transcriptional pathways regulate basal and activated major histocompatibility complex
Howcroft TK, Raval A, Weissman JD, Gegonne A, Singer DS. Mol Cell Biol. 2003 May;23(10):3377-91.
Transcriptional coactivator, CIITA, is an acetyltransferase that bypasses a promoter requirement for TAF(II)250. Raval A, Howcroft TK, Weissman JD, Kirshner S, Zhu XS, Yokoyama K, Ting J, Singer DS. Mol Cell. 2001 Jan;7(1):105-15.
This page was last updated on 2/22/2013.