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Supplementary Figure Legends 1-2 from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-κB kinase β (IKKβ) and confirmed with RNA interference toward IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the nine-gene ovarian cancer–specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion, and adhesion. Downregulating IKKβ activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity. Cancer Res; 70(10); 4005–14. ©2010 AACR.

Supplementary Table 1 from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

Supplementary Figure 1 from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

Supplementary Figure 2 from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

The widely expressed bromodomain and extraterminal motif (BET) proteins bromodomain-containing protein 2 (BRD2), BRD3, and BRD4 are multifunctional transcriptional regulators that bind acetylated chromatin via their conserved tandem bromodomains. Small molecules that target BET bromodomains are being tested for various diseases but typically do not discern between BET family members. Genomic distributions and protein partners of BET proteins have been described, but the basis for differences in BET protein function within a given lineage remains unclear. By establishing a gene knockout-rescue system in a Brd2-null erythroblast cell line, here we compared a series of mutant and chimeric BET proteins for their ability to modulate cell growth, differentiation, and gene expression. We found that the BET N-terminal halves bearing the bromodomains convey marked differences in protein stability but do not account for specificity in BET protein function. Instead, when BET proteins were expressed at comparable levels, their specificity was largely determined by the C-terminal half. Remarkably, a chimeric BET protein comprising the N-terminal half of the structurally similar short BRD4 isoform (BRD4S) and the C-terminal half of BRD2 functioned similarly to intact BRD2. We traced part of the BRD2-specific activity to a previously uncharacterized short segment predicted to harbor a coiled-coil (CC) domain. Deleting the CC segment impaired BRD2's ability to restore growth and differentiation, and the CC region functioned in conjunction with the adjacent ET domain to impart BRD2-like activity onto BRD4S. In summary, our results identify distinct BET protein domains that regulate protein turnover and biological activities.

The widely expressed bromodomain and extraterminal motif (BET) proteins bromodomain-containing protein 2 (BRD2), BRD3, and BRD4 are multifunctional transcriptional regulators that bind acetylated chromatin via their conserved tandem bromodomains. Small molecules that target BET bromodomains are being tested for various diseases but typically do not discern between BET family members. Genomic distributions and protein partners of BET proteins have been described, but the basis for differences in BET protein function within a given lineage remains unclear. By establishing a gene knockout-rescue system in a Brd2-null erythroblast cell line, here we compared a series of mutant and chimeric BET proteins for their ability to modulate cell growth, differentiation, and gene expression. We found that the BET N-terminal halves bearing the bromodomains convey marked differences in protein stability but do not account for specificity in BET protein function. Instead, when BET proteins were expressed at comparable levels, their specificity was largely determined by the C-terminal half. Remarkably, a chimeric BET protein comprising the N-terminal half of the structurally similar short BRD4 isoform (BRD4S) and the C-terminal half of BRD2 functioned similarly to intact BRD2. We traced part of the BRD2-specific activity to a previously uncharacterized short segment predicted to harbor a coiled-coil (CC) domain. Deleting the CC segment impaired BRD2's ability to restore growth and differentiation, and the CC region functioned in conjunction with the adjacent ET domain to impart BRD2-like activity onto BRD4S. In summary, our results identify distinct BET protein domains that regulate protein turnover and biological activities.

Bromodomain and extraterminal motif (BET) proteins have been widely investigated for their roles in gene regulation and their potential as therapeutic targets in cancer. Pharmacologic BET inhibitors target the conserved bromodomain-acetyllysine interaction and do not distinguish between BRD2, BRD3, and BRD4. Thus, comparatively little is known regarding the distinct roles played by individual family members, as well as the underlying mechanisms that drive the transcriptional effects of BET inhibitors. Here we review studies regarding the contributions of BET proteins to genome structure and function, including recent work identifying a role for BRD2 as a component of functional and physical chromatin domain boundaries. We also discuss directions of future studies aimed at providing insights into broader architectural functions of BET proteins and their roles in chromatin domain boundary formation.

Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here we integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we identify a subset of "poorly reprogrammed" interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression.

Mammalian genes do not transcribe RNA continuously but in bursts. Transcriptional output can be modulated by altering burst fraction or burst size, but how regulatory elements control bursting parameters remains unclear. Single-molecule RNA FISH experiments revealed that the β-globin enhancer (LCR) predominantly augments transcriptional burst fraction of the β-globin gene with modest stimulation of burst size. To specifically measure the impact of long range chromatin contacts on transcriptional bursting, we forced an LCR-β-globin promoter chromatin loop. We observed that raising contact frequencies increases burst fraction but not burst size. In cells in which two developmentally distinct LCR-regulated globin genes are cotranscribed in cis, burst sizes of both genes are comparable. However, allelic co-transcription of both genes is statistically disfavored, suggesting mutually exclusive LCR-gene contacts. These results are consistent with competition between the β-type globin genes for LCR contacts and suggest that LCR-promoter loops are formed and released with rapid kinetics.

Bromodomain and extraterminal motif (BET) proteins are pharmacologic targets for the treatment of diverse diseases, yet the roles of individual BET family members remain unclear. We find that BRD2, but not BRD4, co-localizes with the architectural/insulator protein CCCTC-binding factor (CTCF) genome-wide. CTCF recruits BRD2 to co-bound sites whereas BRD2 is dispensable for CTCF occupancy. Disruption of a CTCF/BRD2-occupied element positioned between two unrelated genes enables regulatory influence to spread from one gene to another, suggesting that CTCF and BRD2 form a transcriptional boundary. Accordingly, single-molecule mRNA fluorescence in situ hybridization (FISH) reveals that, upon site-specific CTCF disruption or BRD2 depletion, expression of the two genes becomes increasingly correlated. HiC shows that BRD2 depletion weakens boundaries co-occupied by CTCF and BRD2, but not those that lack BRD2. These findings indicate that BRD2 supports boundary activity, and they raise the possibility that pharmacologic BET inhibitors can influence gene expression in part by perturbing domain boundary function.

Inhibitor of IκB kinases (IKK) are key regulators of NF-κB signaling. Three IKK isoforms—α, β, and ϵ—have been linked to oncogenesis, yet the precise components of NF-κB signaling in ovarian cancer have not yet been dissected. We surveyed 120 ovarian cancer specimens for IKK-ϵ expression. Notably, cytoplasmic expression was elevated in metastatic lesions relative to primary tumors (P = 0.03). Therefore, we hypothesized that IKK-ϵ drives ovarian cancer metastasis. IKK-ϵ was identified previously as a breast cancer oncogene and was associated with poor clinical outcome in ovarian cancer. We now define an ovarian cancer–specific IKK-ϵ–regulated gene expression signature using stably expressed short hairpin RNA targeting IKK-ϵ. Pathway analysis of the signature indicated that IKK-ϵ regulates expression of genes involved in cell motility and inflammation. We further showed that IKK-ϵ depletion in metastatic ovarian cancer cell lines decreased growth, adhesion, and invasion. Consistently, human xenografts depleted of IKK-ϵ in mice showed decreased aggressiveness, whereas overexpression of IKK-ϵ in a less invasive ovarian cancer cell line increased metastasis in vivo. Taken together, these data provide evidence that IKK-ϵ is a key coordinator of invasion and metastasis programs in ovarian cancer. Inhibition of IKK-ϵ signaling thus emerges as a viable therapeutic strategy in women whose ovarian cancer shows aberrant activation of this pathway. Cancer Res; 72(21); 5494–504. ©2012 AACR.

Pharmacologic inhibitors of the bromodomain and extra-terminal motif (BET) protein family are in clinical trials for the treatment of hematologic malignancies, yet the functions of individual BET proteins remain largely uncharacterized. We review the molecular roles of BETs in the context of erythropoiesis. Studies in this lineage have provided valuable insights into their mechanisms of action, and helped define the individual and overlapping functions of BET protein family members BRD2, BRD3, and BRD4. These studies have important ramifications for our understanding of the molecular and physiologic roles of BET proteins, and provide a framework for elucidating some of the beneficial and adverse effects of pharmacologic inhibitors.

Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

Pharmacologic inhibitors of the bromodomain and extraterminal motif (BET) family of proteins have shown promise in the treatment of hematologic and other malignancies and are being developed for clinical use. However, BET inhibitors do not discriminate between the family members BRD2, BRD3, and BRD4, and thus the mechanistic basis for their therapeutic efficacy is not well understood. In addition, BRD2 and BRD4 are individually required for the activation of genes driven by the erythroid transcription factor GATA1 (Stonestrom et al., Blood 2015), yet how BRD2 in particular contributes to this process has not been studied. We examined BRD2 occupancy genome-wide in erythroid cells and find that BRD2 colocalizes extensively with the architectural/insulator protein CCCTC-binding factor (CTCF). We define a functional hierarchy whereby CTCF is required for BRD2 to occupy co-bound sites, while CTCF binding is BRD2-independent. Using CRISPR/Cas9-based genome editing we identify a boundary element occupied by CTCF and BRD2 that is adjacent to a GATA1-driven enhancer and ensures appropriate transcriptional regulation at the locus. Employing single-molecule RNA FISH we show that either site-specific CTCF disruption or BRD2 depletion leads to increased correlation in mature mRNA levels between the genes flanking this boundary, suggesting that they become inappropriately coregulated. Taken together these findings indicate that BRD2 collaborates with CTCF to constrain the activity of an erythroid enhancer and reveal a potential new role for BET proteins in chromatin domain boundary function. Disclosures No relevant conflicts of interest to declare.

The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-κB kinase β (IKKβ) and confirmed with RNA interference toward IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the nine-gene ovarian cancer–specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion, and adhesion. Downregulating IKKβ activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity. Cancer Res; 70(10); 4005–14. ©2010 AACR.

The mammalian Bromodomain and Extra-Terminal motif (BET) proteins associate with acetylated histones and transcription factors including the master erythroid regulator GATA1. Pharmacologic inhibitors that broadly target BET family proteins are being evaluated in clinical trials for hematologic malignancies. Additionally, a rapidly growing number of studies in model organisms and in vitro systems suggests BET modulation is a therapeutic avenue in diverse disorders. However the mechanisms through which BET proteins act as well as the contributions of individual BET family members to biological processes remain mostly uncharacterized. Previously we showed that BET family members Brd3 and Brd4 can bind to GATA1 in an acetylation-dependent manner and that pharmacologic BET inhibition impairs erythroid maturation (Lamonica et al PNAS 2011). Using a combination of genome-wide occupancy analysis, pharmacologic inhibition, genome editing and knockdown we characterized the mechanism and function of BET proteins in the context of GATA1-driven erythroid differentiation. BET inhibitors prevented GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, GATA1 required BETs both for initial chromatin association and for transcriptional activation following the establishment of GATA1 occupancy. As BET proteins associate with chromatin during mitosis when transcription is globally disrupted, they have been implicated as mitotic “bookmarks”. BET inhibition specifically during mitosis failed to elicit a measurable impact on post-mitotic gene activation, calling into question a role of BET proteins as mitotic bookmarks in this system. To determine the BET protein(s) most relevant to GATA1-activated transcription, we genomically disrupted BET family members Brd2, Brd3 and Brd4 using the CRISPR/Cas9 tool. Despite being present at nearly all GATA1-occupied sites genome-wide, Brd3 disruption had little impact on GATA1-dependent transcription. In contrast, both Brd2 and Brd4 were individually required for efficient gene activation by GATA1. A Brd3 requirement was only revealed in the setting of Brd2 deficiency, indicating functional compensation among select BET proteins. These results provide a comprehensive definition of the functions of BET proteins in a model of cellular differentiation. They further suggest that pharmacologic BET inhibition should be viewed in the context of distinct steps in transcriptional activation and overlapping functions among BET family members. Disclosures No relevant conflicts of interest to declare.

The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. To this end, we investigated the biological relevance of NF-κB in ovarian cancer cell lines using a small molecule inhibitor of IKKβ, and confirmed with RNA interference using stably expressed short hairpin RNA molecules towards IKKβ. We demonstrate here that the expression of IKKβ protein itself and the ovarian cancer-specific signature of IKKβ-regulated genes are related to poor outcome in independently collected sets of primary ovarian cancers. IKKβ signaling in ovarian cancer regulates the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion and adhesion in ovarian cancer cell lines. IKKβ was involved in all of these cellular functions, reflecting its modulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggest that therapy targeted to this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C158.