CCAAT Promoter element regulates transgenerational expression of the MHC class I gene.

Transgenerational gene expression depends on both underlying DNA sequences and epigenetic modifications. The latter, which can result in transmission of variegated gene expression patterns across multiple generations without DNA alterations, has been termed epigenetic inheritance and has been documented in plants, worms, flies and mammals. Whereas transcription factors binding to cognate DNA sequence elements regulate gene expression, the molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. Here we report that mutation of the CCAAT box promoter element abrogates NF-Y binding and disrupts the stable transgenerational expression of an MHC class I transgene. Transgenic mice with a mutated CCAAT box in the MHC class I transgene display variegated expression of the transgene among littermates and progeny in multiple independently derived transgenic lines. After 4 generations, CCAAT mutant transgenic lines derived from a single founder stably displayed distinct patterns of expression. Histone modifications and RNA polymerase II binding correlate with expression of CCAAT mutant transgenic lines, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box also results in changes to CTCF binding and DNA looping patterns across the transgene that correlate with expression status. These studies identify the CCAAT promoter element as a regulator of stable transgenerational gene expression such that mutation of the CCAAT box results in variegated transgenerational inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study provides insights into how fidelity of gene expression patterns is maintained through multiple generations.

MYC protein stability is negatively regulated by BRD4.

The protooncogene MYC regulates a variety of cellular processes, including proliferation and metabolism. Maintaining MYC at homeostatic levels is critical to normal cell function; overexpression drives many cancers. MYC stability is regulated through phosphorylation: phosphorylation at Thr58 signals degradation while Ser62 phosphorylation leads to its stabilization and functional activation. The bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator with intrinsic kinase and histone acetyltransferase (HAT) activities that activates transcription of key protooncogenes, including MYC We report that BRD4 phosphorylates MYC at Thr58, leading to MYC ubiquitination and degradation, thereby regulating MYC target genes. Importantly, BRD4 degradation, but not inhibition, results in increased levels of MYC protein. Conversely, MYC inhibits BRD4's HAT activity, suggesting that MYC regulates its own transcription by limiting BRD4-mediated chromatin remodeling of its locus. The MYC stabilizing kinase, ERK1, regulates MYC levels directly and indirectly by inhibiting BRD4 kinase activity. These findings demonstrate that BRD4 negatively regulates MYC levels, which is counteracted by ERK1 activation.

The intrinsic kinase activity of BRD4 spans its BD2-B-BID domains.

Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that is a therapeutic target in many cancers and inflammatory diseases. BRD4 plays important roles in transcription as an active kinase, which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II), the proto-oncogene c-MYC, and transcription factors TAF7 and CDK9. BRD4 is also a passive scaffold that recruits transcription factors. Despite these well-established functions, there has been little characterization of BRD4's biophysical properties or its kinase activity. We report here that the 156 kD mouse BRD4 exists in an extended dimeric conformation with a sedimentation coefficient of ∼6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503-548) disrupts BRD4's dimerization. BRD4 kinase activity maps to amino acids 351 to 598, which span bromodomain-2, the B motif, and the BID domain (BD2-B-BID) and contributes to the in vivo phosphorylation of its substrates. As further assessed by analytical ultracentrifugation, BRD4 directly binds purified Pol II CTD. Importantly, the conserved A motif of BRD4 is essential for phosphorylation of Pol II CTD, but not for phosphorylation of TAF7, mapping its binding site to the A motif. Peptides of the viral MLV integrase (MLVIN) protein and cellular histone lysine methyltransferase, NSD3, which have been shown by NMR to bind to the extra-terminal (ET) domain, also are phosphorylated by BRD4. Thus, BRD4 has multiple distinct substrate-binding sites and a common kinase domain. These results provide new insights into the structure and kinase function of BRD4.

Regulation of MHC class I expression by Foxp3 and its effect on regulatory T cell function.

Expression of MHC class I molecules, which provide immune surveillance against intracellular pathogens, is higher on lymphoid cells than on any other cell types. In T cells, this is a result of activation of class I transcription by the T cell enhanceosome consisting of Runx1, CBFß, and LEF1. We now report that MHC class I transcription in T cells also is enhanced by Foxp3, resulting in higher levels of class I in CD4(+)CD25(+) T regulatory cells than in conventional CD4(+)CD25(-) T cells. Interestingly, the effect of Foxp3 regulation of MHC class I transcription is cell type specific: Foxp3 increases MHC class I expression in T cells but represses it in epithelial tumor cells. In both cell types, Foxp3 targets the upstream IFN response element and downstream core promoter of the class I gene. Importantly, expression of MHC class I contributes to the function of CD4(+)CD25(+) T regulatory cells by enhancing immune suppression, both in in vitro and in vivo. These findings identify MHC class I genes as direct targets of Foxp3 whose expression augments regulatory T cell function.

Transgenerational Epigenetic Inheritance of MHC Class I Gene Expression is Regulated by the CCAAT Promoter Element.

Transgenerational epigenetic inheritance is defined as the transmission of traits or gene expression patterns across multiple generations that do not derive from DNA alterations. The effect of multiple stress factors or metabolic changes resulting in such inheritance have been documented in plants, worms and flies and mammals. The molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. In this study, we show that mutation of a promoter element, the CCAAT box, disrupts stable expression of an MHC Class I transgene, resulting in variegated expression among progeny for at least 4 generations in multiple independently derived transgenic lines. Histone modifications and RNA polII binding correlate with expression, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box abrogates NF-Y binding and results in changes to CTCF binding and DNA looping patterns across the gene that correlate with expression status from one generation to the next. These studies identify the CCAAT promoter element as a regulator of stable transgenerational epigenetic inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study could provide important insights into how fidelity of gene expression patterns is maintained through multiple generations.

TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb.

Transcription consists of a series of highly regulated steps: assembly of the preinitiation complex (PIC) at the promoter, initiation, elongation, and termination. PIC assembly is nucleated by TFIID, a complex composed of the TATA-binding protein (TBP) and a series of TBP-associated factors (TAFs). One component, TAF7, is incorporated in the PIC through its interaction with TFIID but is released from TFIID upon transcription initiation. We now report that TAF7 interacts with the transcription factors, TFIIH and P-TEFb, resulting in the inhibition of their Pol II CTD kinase activities. Importantly, in in vitro transcription reactions, TAF7 inhibits steps after PIC assembly and formation of the first phosphodiester bonds. Further, in vivo TAF7 coelongates with P-TEFb and Pol II downstream of the promoter. We propose a model in which TAF7 contributes to the regulation of the transition from PIC assembly to initiation and elongation.

Distinct transcriptional pathways regulate basal and activated major histocompatibility complex class I expression.

Transcription of major histocompatibility complex (MHC) class I genes is regulated by both tissue-specific (basal) and hormone/cytokine (activated) mechanisms. Although promoter-proximal regulatory elements have been characterized extensively, the role of the core promoter in mediating regulation has been largely undefined. We report here that the class I core promoter consists of distinct elements that are differentially utilized in basal and activated transcription pathways. These pathways recruit distinct transcription factor complexes to the core promoter elements and target distinct transcription initiation sites. Class I transcription initiates at four major sites within the core promoter and is clustered in two distinct regions: "upstream" (-14 and -18) and "downstream" (+12 and +1). Basal transcription initiates predominantly from the upstream start site region and is completely dependent upon the general transcription factor TAF1 (TAF(II)250). Activated transcription initiates predominantly from the downstream region and is TAF1 (TAF(II)250) independent. USF1 augments transcription initiating through the upstream start sites and is dependent on TAF1 (TAF(II)250), a finding consistent with its role in regulating basal class I transcription. In contrast, transcription activated by the interferon mediator CIITA is independent of TAF1 (TAF(II)250) and focuses initiation on the downstream start sites. Thus, basal and activated transcriptions of an MHC class I gene target distinct core promoter domains, nucleate distinct transcription initiation complexes and initiate at distinct sites within the promoter. We propose that transcription initiation at the core promoter is a dynamic process in which the mechanisms of core promoter function differ depending on the cellular environment.

Differential context-specific impact of individual core promoter elements on transcriptional dynamics.

Eukaryotic transcription occurs in bursts that vary in size and frequency, but the contribution of individual core promoter elements to transcriptional bursting is not known. Here we analyze the relative contributions to bursting of the individual core promoter elements-CCAAT, TATAA-like, Sp1BS, and Inr-of an MHC class I gene in primary B-cells during both basal and activated transcription. The TATAA-like, Sp1BS, and Inr elements all function as negative regulators of transcription, and each was found to contribute differentially to the overall bursting pattern of the promoter during basal transcription. Whereas the Sp1BS element regulates burst size, the Inr element regulates burst frequency. The TATAA-like element contributes to both. Surprisingly, each element has a distinct role in bursting during transcriptional activation by γ-interferon. The CCAAT element does not contribute significantly to the constitutive transcriptional dynamics of primary B-cells, but modulates both burst size and frequency in response to γ-interferon activation. The ability of core promoter elements to modulate transcriptional bursting individually allows combinatorial fine-tuning of the level of MHC class I gene expression in response to intrinsic and extrinsic signals.

Major histocompatibility complex class I core promoter elements are not essential for transcription in vivo.

The role of core promoter elements in regulating transcription initiation is largely unknown for genes subject to complex regulation. Major histocompatibility complex class I genes are ubiquitously expressed and governed by tissue-specific and hormonal signals. Transcription initiates at multiple sites within the core promoter, which contains elements homologous to the canonical elements CCAAT, TATAA, Sp1 binding site (Sp1BS), and Initiator (Inr). To determine their functions, expression of class I transgenes with individually mutated elements was assessed. Surprisingly, all mutant promoters supported transcription. However, each mutated core promoter element had a distinct effect on expression: CAAT box mutations modulated constitutive expression in nonlymphoid tissues, whereas TATAA-like element mutations dysregulated transcription in lymphoid tissues. Inr mutations aberrantly elevated expression. Sp1BS element mutations resulted in variegated transgene expression. RNA polymerase II binding and histone H3K4me3 patterns correlated with transgene expression; H3K9me3 marks partially correlated. Whereas the wild-type, TATAA-like, and CAAT mutant promoters were activated by gamma interferon, the Sp1 and Inr mutants were repressed, implicating these elements in regulation of hormonal responses. These results lead to the surprising conclusion that no single element is required for promoter activity. Rather, each plays a distinct role in promoter activity, chromatin structure, tissue-specific expression, and extracellular signaling.

Three novel downstream promoter elements regulate MHC class I promoter activity in mammalian cells.

BACKGROUND: MHC CLASS I TRANSCRIPTION IS REGULATED BY TWO DISTINCT TYPES OF REGULATORY PATHWAYS: 1) tissue-specific pathways that establish constitutive levels of expression within a given tissue and 2) dynamically modulated pathways that increase or decrease expression within that tissue in response to hormonal or cytokine mediated stimuli. These sets of pathways target distinct upstream regulatory elements, have distinct basal transcription factor requirements, and utilize discrete sets of transcription start sites within an extended core promoter. METHODOLOGY/PRINCIPAL FINDINGS: We studied regulatory elements within the MHC class I promoter by cellular transfection and in vitro transcription assays in HeLa, HeLa/CIITA, and tsBN462 of various promoter constructs. We have identified three novel MHC class I regulatory elements (GLE, DPE-L1 and DPE-L2), located downstream of the major transcription start sites, that contribute to the regulation of both constitutive and activated MHC class I expression. These elements located at the 3' end of the core promoter preferentially regulate the multiple transcription start sites clustered at the 5' end of the core promoter. CONCLUSIONS/SIGNIFICANCE: Three novel downstream elements (GLE, DPE-L1, DPE-L2), located between +1 and +32 bp, regulate both constitutive and activated MHC class I gene expression by selectively increasing usage of transcription start sites clustered at the 5' end of the core promoter upstream of +1 bp. Results indicate that the downstream elements preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription.

In vivo expression of MHC class I genes depends on the presence of a downstream barrier element.

Regulation of MHC class I gene expression is critical to achieve proper immune surveillance. In this work, we identify elements downstream of the MHC class I promoter that are necessary for appropriate in vivo regulation: a novel barrier element that protects the MHC class I gene from silencing and elements within the first two introns that contribute to tissue specific transcription. The barrier element is located in intergenic sequences 3' to the polyA addition site. It is necessary for stable expression in vivo, but has no effect in transient transfection assays. Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene. Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications. Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.

Histone modifications, but not nucleosomal positioning, correlate with major histocompatibility complex class I promoter activity in different tissues in vivo.

To examine the role of chromatin in transcriptional regulation of the major histocompatibility complex (MHC) class I gene, we determined nucleosome occupancy and positioning, histone modifications, and H2A.Z occupancy across its regulatory region in murine tissues that have widely different expression levels. Surprisingly, nucleosome occupancy and positioning were indistinguishable between the spleen, kidney, and brain. In all three tissues, the 200 bp upstream of the transcription start site had low nucleosome occupancy. In contrast, nuclease hypersensitivity, histone modifications, and H2A.Z occupancy showed tissue-specific differences. Thus, tissue-specific differences in MHC class I transcription correlate with histone modifications and not nucleosomal organization. Further, activation of class I transcription by gamma interferon or its inhibition by alpha-amanitin did not alter nucleosome occupancy, positioning, nuclease hypersensitivity, histone modifications, or H2A.Z occupancy in any of the tissues examined. Thus, chromatin remodeling was not required to dynamically modulate transcriptional levels. These findings suggest that the MHC class I promoter remains poised and accessible to rapidly respond to infection and environmental cues.

TAF7: a possible transcription initiation check-point regulator.

Transcription consists of a series of highly regulated steps: assembly of a preinitiation complex (PIC) at the promoter nucleated by TFIID, followed by initiation, elongation, and termination. The present study has focused on the role of the TFIID component, TAF7, in regulating transcription initiation. In TFIID, TAF7 binds to TAF1 and inhibits its intrinsic acetyl transferase activity. We now report that although TAF7 remains bound to TAF1 and associated with TFIID during the formation of the PIC, TAF7 dissociates from the PIC upon transcription initiation. Entry of polymerase II into the assembling PIC is associated with TAF1 and TAF7 phosphorylation, coincident with TAF7 release. We propose that the TFIID composition is dynamic and that TAF7 functions as a check-point regulator suppressing premature transcription initiation until PIC assembly is complete.

A T lymphocyte-specific transcription complex containing RUNX1 activates MHC class I expression.

MHC class I expression is subject to both tissue-specific and hormonal regulatory mechanisms. Consequently, levels of expression vary widely among tissues, with the highest levels of class I occurring in the lymphoid compartment, in T cells and B cells. Although the high class I expression in B cells is known to involve the B cell enhanceosome, the molecular basis for high constitutive class I expression in T cells has not been explored. T cell-specific genes, such as TCR genes, are regulated by a T cell enhanceosome consisting of RUNX1, CBFbeta, LEF1, and Aly. In this report, we demonstrate that MHC class I gene expression is enhanced by the T cell enhanceosome and results from a direct interaction of the RUNX1-containing complex with the class I gene in vivo. T cell enhanceosome activation of class I transcription is synergistic with CIITA-mediated activation and targets response elements distinct from those targeted by CIITA. These findings provide a molecular basis for the high levels of MHC class I in T cells.

The GTP-binding domain of class II transactivator regulates its nuclear export.

The transcriptional coactivator class II transactivator (CIITA), although predominantly localized in the nucleus, is also present in the cytoplasm. The subcellular distribution of CIITA is actively regulated by the opposing actions of nuclear export and import. In this study, we show that nuclear export is negatively regulated by the GTP-binding domain (GBD; aa 421-561) of CIITA: mutation or deletion of the GBD markedly increased export of CIITA from the nucleus. Remarkably, a CIITA GBD mutant binds CRM1/exportin significantly better than does wild-type CIITA, leading to the conclusion that GTP is a negative regulator of CIITA nuclear export. We also report that, in addition to the previously characterized N- and C-terminal nuclear localization signal elements, there is an additional N-terminal nuclear localization activity, present between aa 209 and 222, which overlaps the proline/serine/threonine-rich domain of CIITA. Thus, fine-tuning of the nucleocytoplasmic distribution of coactivator proteins involved in transcription is an active and dynamic process that defines a novel mechanism for controlling gene regulation.