ELL facilitates RNA polymerase II pause site entry and release.

Transcription is a multi-stage process that coordinates several steps within the transcription cycle including chromatin reorganization, RNA polymerase II recruitment, initiation, promoter clearance and elongation. Recent advances have identified the super elongation complex, containing the eleven-nineteen lysine-rich leukaemia (ELL) protein, as a key regulator of transcriptional elongation. Here we show that ELL has a diverse and kinetically distinct role before its assembly into the super elongation complex by stabilizing Pol II recruitment/initiation and entry into the pause site. Loss of ELL destabilizes the pre-initiation complexes and results in disruption of early elongation and promoter proximal chromatin structure before recruitment of AFF4 and other super elongation complex components. These changes result in significantly reduced transcriptional activation of rapidly induced genes. Thus, ELL has an early and essential role during rapid high-amplitude gene expression that is required for both Pol II pause site entry and release.

Transcriptional autoregulation by BRCA1.

The BRCA1 gene product plays numerous roles in regulating genome integrity. Its role in assembling supermolecular complexes in response to DNA damage has been extensively studied; however, much less is understood about its role as a transcriptional coregulator. Loss or mutation is associated with hereditary breast and ovarian cancers, whereas altered expression occurs frequently in sporadic forms of breast cancer, suggesting that the control of BRCA1 transcription might be important to tumorigenesis. Here, we provide evidence of a striking linkage between the roles for BRCA1 as a transcriptional coregulator with control of its expression via an autoregulatory transcriptional loop. BRCA1 assembles with complexes containing E2F-1 and RB to form a repressive multicomponent transcriptional complex that inhibits BRCA1 promoter transcription. This complex is disrupted by genotoxic stress, resulting in the displacement of BRCA1 protein from the BRCA1 promoter and subsequent upregulation of BRCA1 transcription. Cells depleted of BRCA1 respond by upregulating BRCA1 transcripts, whereas cells overexpressing BRCA1 respond by downregulating BRCA1 transcripts. Tandem chromatin immmunoprecipitation studies show that BRCA1 is regulated by a dynamic coregulatory complex containing BRCA1, E2F1, and Rb at the BRCA1 promoter that is disrupted by DNA-damaging agents to increase its transcription. These results define a novel transcriptional mechanism of autoregulated homeostasis of BRCA1 that selectively titrates its levels to maintain genome integrity in response to genotoxic insult.

Dynamic bookmarking of primary response genes by p300 and RNA polymerase II complexes.

Profiling the dynamic interaction of p300 with proximal promoters of human T cells identified a class of genes that rapidly coassemble p300 and RNA polymerase II (pol II) following mitogen stimulation. Several of these p300 targets are immediate early genes, including FOS, implicating a prominent role for p300 in the control of primary genetic responses. The recruitment of p300 and pol II rapidly transitions to the assembly of several elongation factors, including the positive transcriptional elongation factor (P-TEFb), the bromodomain-containing protein (BRD4), and the elongin-like eleven nineteen lysine-rich leukemia protein (ELL). However, transcription at many of these rapidly induced genes is transient, wherein swift departure of P-TEFb, BRD4, and ELL coincides with termination of transcriptional elongation. Unexpectedly, both p300 and pol II remain accumulated or "bookmarked" at the proximal promoter long after transcription has terminated, demarking a clear mechanistic separation between the recruitment and elongation phases of transcription in vivo. The bookmarked pol II is depleted of both serine-2 and serine-5 phosphorylation of its C-terminal domain and remains proximally positioned at the promoter for hours. Surprisingly, these p300/pol II bookmarked genes can be readily reactivated, and elongation factors can be reassembled by subsequent addition of nonmitogenic agents that, alone, have minimal effects on transcription in the absence of prior preconditioning by mitogen stimulation. These findings suggest that p300 is likely to play an important role in biological processes in which transcriptional bookmarking or preconditioning influences cellular growth and development through the dynamic priming of genes for response to rechallenge by secondary stimuli.

Combinatorial antileukemic disruption of oxidative homeostasis and mitochondrial stability by the redox reactive thalidomide 2-(2,4-difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) and flavopiridol.

2-(2,4-Difluoro-phenyl)-4,5,6,7-tetrafluoro-1H-isoindole-1,3(2H)-dione (CPS49) is a member of a recently identified class of redox-reactive thalidomide analogs that show selective killing of leukemic cells by increasing intracellular reactive oxygen species (ROS) and targeting multiple transcriptional pathways. Flavopiridol is a semisynthetic flavonoid that inhibits cyclin-dependent kinases and also shows selective lethality against leukemic cells. The purpose of this study is to explore the efficacy and mechanism of action of the combinatorial use of the redox-reactive thalidomide CPS49 and the cyclin-dependent kinase inhibitor flavopiridol as a selective antileukemic therapeutic strategy. In combination, CPS49 and flavopiridol were found to induce selective cytotoxicity associated with mitochondrial dysfunction and elevations of ROS in leukemic cells ranging from additive to synergistic activity at low micromolar concentrations. Highest synergy was observed at the level of ROS generation with a strong correlation between cell-specific cytotoxicity and reciprocal coupling of drug-induced ROS elevation with glutathione depletion. Examination of the transcriptional targeting of CPS49 and flavopiridol combinations reveals that the drugs act in concert to initiate a cell specific transcriptional program that manipulates nuclear factor-kappaB (NF-kappaB), E2F-1, and p73 activity to promote enhanced mitochondrial instability by simultaneously elevating the expression of the proapoptotic factors BAX, BAD, p73, and PUMA while depressing expression of the antiapoptotic genes MCL1, XIAP, BCL-xL, SURVIVIN, and MDM2. The coadministration of CPS49 and flavopiridol acts through coordinate targeting of transcriptional pathways that enforce selective mitochondrial dysfunction and ROS elevation and is therefore a promising new therapeutic combination that warrants further preclinical exploration.

Transcriptional networks inferred from molecular signatures of breast cancer.

Global genomic approaches in cancer research have provided new and innovative strategies for the identification of signatures that differentiate various types of human cancers. Computational analysis of the promoter composition of the genes within these signatures may provide a powerful method for deducing the regulatory transcriptional networks that mediate their collective function. In this study we have systematically analyzed the promoter composition of gene classes derived from previously established genetic signatures that recently have been shown to reliably and reproducibly distinguish five molecular subtypes of breast cancer associated with distinct clinical outcomes. Inferences made from the trends of transcription factor binding site enrichment in the promoters of these gene groups led to the identification of regulatory pathways that implicate discrete transcriptional networks associated with specific molecular subtypes of breast cancer. One of these inferred pathways predicted a role for nuclear factor-kappaB in a novel feed-forward, self-amplifying, autoregulatory module regulated by the ERBB family of growth factor receptors. The existence of this pathway was verified in vivo by chromatin immunoprecipitation and shown to be deregulated in breast cancer cells overexpressing ERBB2. This analysis indicates that approaches of this type can provide unique insights into the differential regulatory molecular programs associated with breast cancer and will aid in identifying specific transcriptional networks and pathways as potential targets for tumor subtype-specific therapeutic intervention.

Functional profiling of uncommon VCAM1 promoter polymorphisms prevalent in African American populations.

Multiple variants of the vascular adhesion molecule-1 (VCAM1) promoter show increased nucleotide heterozygosity in the African American population. Using a novel transfection-based transcriptional pathway profiling method, we show that select uncommon variants are functionally hyperactive. Eight candidate VCAM1 promoter haplotypes comprising 13 previously identified SNPs were assessed for response to known mitogens. Activity was correlated with bioinformatic analysis of hyper- and hyporesponsive variants to identify the gain or loss of haplotype-specific transcription factor binding site (TFBS). Using this approach, a low frequency regulatory allele (c.-540A>G; dbSNP rs3783605:A>G), found in a hyperactive VCAM1 promoter haplotype, was shown to create a candidate binding site for ETS2 that was confirmed in vivo by chromatin immunoprecipitation. This report provides the first functional evaluation of VCAM1 promoter polymorphisms and establishes a hypothetical foundation for investigation of their role in the pathogenesis of VCAM1-associated diseases that disproportionately afflict African Americans, including thromboembolic diseases, asthma, and multiple myeloma.

Selective leukemic-cell killing by a novel functional class of thalidomide analogs.

Using a novel cell-based assay to profile transcriptional pathway targeting, we have identified a new functional class of thalidomide analogs with distinct and selective antileukemic activity. These agents activate nuclear factor of activated T cells (NFAT) transcriptional pathways while simultaneously repressing nuclear factor-kappaB (NF-kappaB) via a rapid intracellular amplification of reactive oxygen species (ROS). The elevated ROS is associated with increased intracellular free calcium, rapid dissipation of the mitochondrial membrane potential, disrupted mitochondrial structure, and caspase-independent cell death. This cytotoxicity is highly selective for transformed lymphoid cells, is reversed by free radical scavengers, synergizes with the antileukemic activity of other redox-directed compounds, and preferentially targets cells in the S phase of the cell cycle. Live-cell imaging reveals a rapid drug-induced burst of ROS originating in the endoplasmic reticulum and associated mitochondria just prior to spreading throughout the cell. As members of a novel functional class of "redoxreactive" thalidomides, these compounds provide a new tool through which selective cellular properties of redox status and intracellular bioactivation can be leveraged by rational combinatorial therapeutic strategies and appropriate drug design to exploit cell-specific vulnerabilities for maximum drug efficacy.

Novel cell-specific and dominant negative anti-apoptotic roles of p73 in transformed leukemia cells.

Although extensive homology exists between related genes p53 and p73, recent data suggest that the family members have divergent roles. We demonstrate that the differential regulatory roles of p53 family member p73 are highly cell-context and promoter-specific. Full-length p73 expressed in the transformed leukemia cell line Jurkat behaves as a specific dominant negative transcriptional repressor of the cell cycle inhibitor gene p21 and blocks p53-mediated apoptosis. These findings provide evidence for a new mechanism in oncogenesis through which the functional properties of p73 can be altered in an inheritable and cell-specific fashion independent of transcriptional coding.

Cross-regulation of T cell growth factor expression by p53 and the Tax oncogene.

In this study, we demonstrate that p53 directly inhibits expression of the T cell growth factor (IL-2) in activated T cells. This repression is independent of the intrinsic transcriptional activity of p53 and is mediated by the Tax-responsive CD28RE-3'-12-O-tetradecanoylphorbol-13-acetate response element (AP1) element of the IL-2 promoter. Coexpression of the Tax oncogene causes full reversal of this repression through coordinate targeting of p300, CREB, and the NF-kappaB pathways. Paradoxically, IL-2 repression by p53 is not reversed by mdm2. Instead, mdm2 represses the IL-2 promoter by a mechanism that is synergistic with p53 and resistant to Tax reversal. The p300 structure-function studies show that these effects are linked to competitive associations among p53, Tax, and mdm2 with multiple domains of p300. The functional outcome of these antagonistic associations is revealed further by the observation that Tax and p53 induce apoptosis in activated T cells through separate and mutually exclusive pathways. Interestingly, both pathways are abrogated by mdm2. These results provide evidence that a dynamic interplay, between Tax and specific elements of the p53 network, mediates growth factor expression and programmed cell death in activated T cells.