BRD4 Connects Enhancer Remodeling to Senescence Immune Surveillance

“Although initially defined as a phenotype of cultured fibroblasts (3), cellular senescence appears to have a broad impact on human health and disease.”

“Conversely, senescent cells activate the expression of genes known to influence the tissue microenvironment, including matrix remodeling enzymes, growth factors, and immunemodulatory cytokines (22, 23).”

“The Genomic Regions Enrichment of Annotations Tool (GREAT) was used to predict GO terms of ChIP-Seq–enriched regions (77).”

“Following lysis of cross-linked proliferating, senescent, and quiescent IMR90 cells, the enhancer landscape was visualized using chromatin immunoprecipitation coupled with highthroughput sequencing (ChIP-Seq) using the histone modification H3K27Ac as a marker of active enhancers (45).”

“For example, in embryonic stem cells (ESC), exceptionally large stretches of H3K27Ac-marked regulatory elements, termed super-enhancers (SE), are associated with genes that play prominent roles in ESC self-renewal (37, 38).”

“For example, in embryonic stem cells (ESC), exceptionally large stretches of H3K27Ac-marked regulatory elements, termed super-enhancers (SE), are associated with genes that play prominent roles in ESC self-renewal (37, 38).”

“BRD4 Is Recruited to Senescence-Activated SEs Associated with Key SASP Genes One of the proteins that bind acetylated histones at enhancers and are enriched in SEs is the transcriptional coactivator BRD4, which couples enhancer remodeling to RNA Pol II elongation (39).”

“Our data linking BRD4 to a tumor-suppressive program stands in apparent contrast to the established role of BRD4 in tumors as a cancer maintenance gene (39, 53, 68).”

“Accordingly, disruption of senescence regulators can promote cancer or impair wound healing, and the elimination of senescent cells from tissues can enhance lifespan in mice (17, 18).”

“Moreover, senescent cells have been observed in aged or damaged tissues (14) and can contribute to the decline of tissue regeneration capacity with age (15, 16).”

“Accordingly, disruption of senescence regulators can promote cancer or impair wound healing, and the elimination of senescent cells from tissues can enhance lifespan in mice (17, 18).”

“These immune-modulatory activities may be important for the resolution of certain wound-healing responses (9, 10, 13) and underlie a unique form of immune surveillance that can eliminate premalignant or malignant cells undergoing senescence in vivo (33, 35, 36).”

“As additional controls, we inactivated classic tumor suppressor genes, such as p53 and RB, known to direct the gene repression program leading to a permanent cell-cycle arrest (20), as well as the cell-cycle regulators p21 and p16 (CDKN1A and CDKN2A), which when inhibited in combination can bypass senescence in IMR90…”

“For example, IL1A and IL1B, which are induced more than 1,000-fold through BRD4-gained SEs in senescence, are known SASP “amplifiers” (27, 28, 52, 57, 62) and, as such, could induce additional SASP factors through positive feedback loops without the need for direct BRD4 regulation.”

“Cellular senescence is a cell-cycle arrest program that limits proliferation of damaged cells and can be triggered in response to diverse forms of cellular stress (1, 2).”

“These immune-modulatory activities may be important for the resolution of certain wound-healing responses (9, 10, 13) and underlie a unique form of immune surveillance that can eliminate premalignant or malignant cells undergoing senescence in vivo (33, 35, 36).”

“When indicated, cells were cotransduced with vectors encoding validated GFP-linked shRNAs targeting BRD4 (53), p65 (26), p53 (33), p53/RB (49), or p21/p16 (49).”

“Interestingly, motif analysis of all senescence-activated SEs also predicted sites for SMAD3, C/EBPβ, and GATA family of transcription factors (data not shown), previously linked to senescence (31, 51, 52).”

“Cellular senescence is a cell-cycle arrest program that limits proliferation of damaged cells and can be triggered in response to diverse forms of cellular stress (1, 2).”

“These immune-modulatory activities may be important for the resolution of certain wound-healing responses (9, 10, 13) and underlie a unique form of immune surveillance that can eliminate premalignant or malignant cells undergoing senescence in vivo (33, 35, 36).”

“We first assessed the impact of systemic BRD4 inhibition using the BET inhibitor I-BET762 (iBET), a compound analogous to JQ1 but with improved pharmacologic properties in vivo (41).”

“The Senescence-Associated Transcriptional Profile Is Shaped by Enhancer Remodeling Cellular identity and plasticity rely on dynamic remodeling of enhancer landscapes (60).”

“Conversely, senescent cells activate the expression of genes known to influence the tissue microenvironment, including matrix remodeling enzymes, growth factors, and immunemodulatory cytokines (22, 23).”

“Thus, these results indicate that BRD4 inactivation uncouples the SASP from the cell-cycle arrest during senescence, and imply that BET inhibitors may be valuable at modulating SASP activity in vivo, such as has been recently reported for the mTOR inhibitor rapamycin (28, 29).”

“For example, cellular senescence can restrict tumorigenesis (4, 5) and modulate chemotherapy responses (6–8), and may exert a primordial role in wound healing, tissue remodeling, and repair (9–13).”

“Moreover, senescent cells have been observed in aged or damaged tissues (14) and can contribute to the decline of tissue regeneration capacity with age (15, 16).”

“This signature is similar to that produced by our laboratory using microarray technology (26, 49) and to those in previously published reports (54, 55).”

“These immune-modulatory activities may be important for the resolution of certain wound-healing responses (9, 10, 13) and underlie a unique form of immune surveillance that can eliminate premalignant or malignant cells undergoing senescence in vivo (33, 35, 36).”

“Consistent with this view, control of such amplifier genes may underlie the action of GATA4 on SASP during replicative senescence (31).”

“3K), known to be activated by RAS/MAPK signaling (50).”

“Previous work using this model established the importance of heterochromatin formation and transcriptional repression in the senescence-associated cell-cycle arrest program (20, 21) and contributed to the characterization of the SASP (25).”

“First, we examined the ability of BRD4 to affect paracrine senescence, a phenomenon whereby CM from senescent cells can transmit senescence signals to naïve cells (56, 57).”

“This situation contrasts with that of macrophage activation in which a subset of enhancers are newly configured (“latent”) following a proinflammatory stimulus (61).”

“Thus, these results indicate that BRD4 inactivation uncouples the SASP from the cell-cycle arrest during senescence, and imply that BET inhibitors may be valuable at modulating SASP activity in vivo, such as has been recently reported for the mTOR inhibitor rapamycin (28, 29).”

“We also examined the impact of BRD4 on the ability of senescent cells to modulate immune cell function and facilitate senescent cell targeting (26, 33).”

“S3K), the latter process likely reflecting the well-established reduction in extracellular matrix production that accompanies senescence (48).”

“The senescence-induced cell-cycle program involves vast changes in the heterochromatin organization leading to transcriptional repression (20, 21), whereas the gene activation program can involve recruitment of key transcriptional activators (31, 40, 59).”

“For example, IL1A and IL1B, which are induced more than 1,000-fold through BRD4-gained SEs in senescence, are known SASP “amplifiers” (27, 28, 52, 57, 62) and, as such, could induce additional SASP factors through positive feedback loops without the need for direct BRD4 regulation.”

“The senescence-induced cell-cycle program involves vast changes in the heterochromatin organization leading to transcriptional repression (20, 21), whereas the gene activation program can involve recruitment of key transcriptional activators (31, 40, 59).”

“Human macrophages were harvested following an 18-hour incubation with the indicated CM and processed for qRT-PCR analysis of previously described M1 or M2 polarization markers (75, 76).”

“S3E, top plot), which have been previously implicated in senescence (46, 47).”

“Besides their novel role in senescence surveillance as described above, BET proteins are crucial for the inflammatory response of various immune cells (41, 58).”

“In this model, senescence is triggered by the introduction of oncogenic HRASV12, which provokes a proliferative burst followed by induction of p53, p16Ink4a, cellcycle exit, and the production of SA-β-gal and SASP (19, 44).”

“As additional controls, we inactivated classic tumor suppressor genes, such as p53 and RB, known to direct the gene repression program leading to a permanent cell-cycle arrest (20), as well as the cell-cycle regulators p21 and p16 (CDKN1A and CDKN2A), which when inhibited in combination can bypass senescence in IMR90…”

“This signature is similar to that produced by our laboratory using microarray technology (26, 49) and to those in previously published reports (54, 55).”

“Moreover, although BRD4 had no marked impact on p53 transcriptional profiles in our system, phosphorylated BRD4 can act as a transcriptional coactivator for p53 in certain cell types (72).”

“ChIP-Seq and Bioinformatics Analysis ChIP-Seq was performed as previously described (65).”

“Most interestingly, SE activation resulted in induction of genes related to cytokine and growth factor activity, which include essential components of the SASP (24, 26) and also categorically distinguished senescent cells from growth-arrested quiescent counterparts.”

“For example, cellular senescence can restrict tumorigenesis (4, 5) and modulate chemotherapy responses (6–8), and may exert a primordial role in wound healing, tissue remodeling, and repair (9–13).”

“Moreover, analyses of all H3K27Ac-marked SEs identified additional TFs such as SMAD3, C/EBPβ, or the GATA family, previously linked to senescence (31, 51, 52) as well as to liver fibrosis (67), pointing to both specific and common regulatory mechanisms driving enhancer-mediated secretory pathway activation in differen…”

“dedifferentiation in human breast cancer cells and cells from patients with premature aging syndrome (69, 70), and promotes hyperproliferation in the murine epidermis (71).”

“The senescence-induced cell-cycle program involves vast changes in the heterochromatin organization leading to transcriptional repression (20, 21), whereas the gene activation program can involve recruitment of key transcriptional activators (31, 40, 59).”

“Human macrophages were harvested following an 18-hour incubation with the indicated CM and processed for qRT-PCR analysis of previously described M1 or M2 polarization markers (75, 76).”

“This signature is similar to that produced by our laboratory using microarray technology (26, 49) and to those in previously published reports (54, 55).”

“Human macrophages were generated from monocytes isolated from buffy coats from healthy human blood donors (New York Blood Center) and differentiated in Teflon-coated cell culture bags in the presence of human CSF1 (R&D Systems), as previously described (74).”

“dedifferentiation in human breast cancer cells and cells from patients with premature aging syndrome (69, 70), and promotes hyperproliferation in the murine epidermis (71).”

“dedifferentiation in human breast cancer cells and cells from patients with premature aging syndrome (69, 70), and promotes hyperproliferation in the murine epidermis (71).”

“Besides its role in cancer, the SASP response also contributes to tissue repair (10, 13), fibrosis (9, 66), and aging (73), and, in principle, BET inhibitors could provide strategies to modulate the SASP response and these processes at the organismal level.”

“Finally, the 3D arrangement of the chromatin also influences the transcription of SASP genes [64], while chromatin-bound HMGB2 fine tunes SASP expression by avoiding heterochromatin spreading [65].”

“Other epigenetic regulators such as BRD4 (Tasdemir et al., 2016), MLL1 (Capell et al., 2016), HMGB2 (Guerrero and Gil, 2016), histone variant macroH2A1 (Chen et al., 2015), and GATA4 (Kang et al., 2015) are also involved in regulating SASP.”