E2F Target Gene Promoters Research Articles

Maize E2F transcription factors. Expression, association to promoters of S-phase genes and interaction with the RBR1 protein in chromatin during seed germination

For seed germination, it is necessary to restart the cell cycle, a process regulated at multiple levels including transcriptional control, that is executed by the E2F family of transcription factors. We identified 12 genes of the E2F family in maize that are expressed differentially during the first 28 h post imbibition (HAI). E2Fa/b1;1 and E2Fc proteins were characterized as an activator and a putative repressor respectively, both forming heterodimers with DPb2 that bind differentially to consensus E2F response elements in promoters of E2F target genes. Transcripts of target genes for these transcription factors accumulate during germination; in dry seeds E2Fc protein is enriched in the target promoters and is replaced by E2Fa/b1;1 as germination advances. RBR1 is found in the same promoters in non-imbibed and 28 HAI seeds, when DNA replication has concluded, and transcription of the E2F targets should stop. During germination promoters of these target genes seem to be decorated with histone marks related to relaxed chromatin structure. Therefore, E2Fs appear to occupy their target genes in a context of open chromatin, with RBR1 fine tuning the progression between the phases.

Abstract A11: RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1

Abstract The retinoblastoma (RB) tumor suppressor is widely recognized as a master regulator of the transcriptional program that controls entry into the S phase of the cell cycle. Its loss, which is a hallmark of cancer, leads to uncontrolled cell proliferation. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin modifiers and remodelers to the promoters of E2F target genes. Here we show that RB also localizes to DNA double strand breaks (DSBs) dependent on E2F1 and ATM kinase activity. RB promotes DNA end resection and DSB repair through homologous recombination (HR), and its loss results in genome instability. RB is necessary for the recruitment of the BRG1 ATPase to DSBs, which stimulates DNA end resection and HR. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1 and BRG1 to DSBs. This knock-in mutation also impairs DNA repair, increases genomic instability and renders mice hypersensitive to IR. Importantly, depletion of either RB or BRG1 in osteosarcoma and breast cancer cell lines results in sensitivity to DNA damaging agents, thus underscoring the importance of these factors in DNA repair. We uncover a novel, non-transcriptional function for RB in HR, which could contribute to genome instability associated with RB loss. Citation Format: Renier Vélez-Cruz, Swarnalatha Manickavinayaham, Anup K. Biswas, Regina W. Clary, David G. Johnson. RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A11.

RB localizes to DNA double-strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1.

The retinoblastoma (RB) tumor suppressor is recognized as a master regulator that controls entry into the S phase of the cell cycle. Its loss leads to uncontrolled cell proliferation and is a hallmark of cancer. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin modifiers to the promoters of E2F target genes. Here we show that RB also localizes to DNA double-strand breaks (DSBs) dependent on E2F1 and ATM kinase activity and promotes DSB repair through homologous recombination (HR), and its loss results in genome instability. RB is necessary for the recruitment of the BRG1 ATPase to DSBs, which stimulates DNA end resection and HR. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1, and BRG1 to DSBs. This knock-in mutation also impairs DNA repair, increases genomic instability, and renders mice hypersensitive to IR. Importantly, depletion of RB in osteosarcoma and breast cancer cell lines results in sensitivity to DNA-damaging drugs, which is further exacerbated by poly-ADP ribose polymerase (PARP) inhibitors. We uncovered a novel, nontranscriptional function for RB in HR, which could contribute to genome instability associated with RB loss.

Abstract PR05: RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of SWI/SNF complex

Abstract The retinoblastoma (RB) tumor suppressor is widely recognized as a master regulator of the transcriptional program that controls entry into the S phase of the cell cycle. RB works by binding to members of the E2F family of transcription factors and recruiting chromatin-remodelers and modifiers to the promoters of E2F target genes. Here we show that RB localizes to DNA double strand breaks (DSBs) dependent on E2F1 and ATM kinase activity. Moreover, RB promotes DNA end resection and homologous recombination, and its loss results in genome instability. RB mediates this important role in repair by recruiting the SWI/SNF chromatin remodeler BRG1 to DSBs. In agreement with these results, loss of BRG1 also results in impaired DNA end resection and HR. RB is recruited to DSBs through a DNA damage-inducible phospho-specific interaction between E2F1 and TopBP1. A knock-in mutation of the ATM phosphorylation site on E2F1 (S29A) prevents the interaction between E2F1 and TopBP1 and recruitment of RB, E2F1 and BRG1 to DSBs. This E2F1 mutation also impairs DNA repair, increases genomic instability and renders knock-in mice hypersensitive to IR. We uncover a novel, non-transcriptional function for RB in HR, which could contribute to genome instability associated with RB loss in human cancers and explain their sensitivity to DSB-inducing agents. This abstract is also being presented as Poster B09. Citation Format: Renier Velez-Cruz, Swarnalatha Manickavinayaham, Anup K. Biswas, Regina Weaks Clary, David G. Johnson. RB localizes to DNA double strand breaks and promotes DNA end resection and homologous recombination through the recruitment of SWI/SNF complex. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR05.

The retinoblastoma protein and PML collaborate to organize heterochromatin and silence E2F-responsive genes during senescence

Cellular senescence is characterized by silencing of genes involved in DNA replication and cell cycle progression. Stable repression is crucial for preventing inappropriate DNA synthesis and the maintenance of a prolonged senescent state. Many of these genes are targets for E2F transcription factors. The pRB pathway plays a major role in senescence by directly repressing E2Fs and also by regulating chromatin at the promoters of E2F target genes using its LXCXE cleft-dependent interactions. In this study, we sought to investigate the mechanisms by which pRB stably silences E2F target gene transcription during cellular senescence. We report that in mouse embryonic fibroblasts, endogenous promyelocytic leukemia protein (PML) associates with E2F target genes in a pRB LXCXE-dependent manner during HrasV12-induced senescence. Furthermore, using a PML-IV-induced senescence model, we show that the pRB LXCXE binding cleft is essential for PML association with gene promoters, silencing of E2F target genes, and stable cell cycle exit. Binding assays show that pRB can interact with PML specifically during senescence, suggesting that signaling events in senescence regulate assembly of PML and pRB to establish heterochromatin and create a permanent cell cycle arrest.

LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence

Neuronal survival is dependent upon the retinoblastoma family members, Rb1 (Rb) and Rb2 (p130). Rb is thought to regulate gene repression, in part, through direct recruitment of chromatin modifying enzymes to its conserved LXCXE binding domain. We sought to examine the mechanisms that Rb employs to mediate cell cycle gene repression in terminally differentiated cortical neurons. Here, we report that Rb loss converts chromatin at the promoters of E2f-target genes to an activated state. We established a mouse model system in which Rb-LXCXE interactions could be induciblely disabled. Surprisingly, this had no effect on survival or gene silencing in neuronal quiescence. Absence of the Rb LXCXE-binding domain in neurons is compatible with gene repression and long-term survival, unlike Rb deficiency. Finally, we are able to show that chromatin activation following Rb deletion occurs at the level of E2fs. Blocking E2f-mediated transcription downstream of Rb loss is sufficient to maintain chromatin in an inactive state. Taken together our results suggest a model whereby Rb-E2f interactions are sufficient to maintain gene repression irrespective of LXCXE-dependent chromatin remodeling.

ACTR/AIB1 functions as an E2F1 coactivator to promote breast cancer cell proliferation and antiestrogen resistance.

Overexpression or amplification of ACTR (also named AIB1, RAC3, p/CIP, TRAM-1, and SRC-3), a member of the p160 family of coactivators for nuclear hormone receptors, has been frequently detected in multiple types of human tumors, including breast cancer. However, its role in cancer cell proliferation and the underlying mechanism are unclear. Here, we show that overexpression of ACTR not only enhances estrogen-stimulated cell proliferation but also, more strikingly, completely negates the cell cycle arrest effect by tamoxifen and pure antiestrogens. Unexpectedly, we found that ACTR directly interacts, through its N-terminal domain, with E2F1 and is recruited to E2F target gene promoters. Elevation of ACTR in quiescent cells strongly stimulates the transcription of a subset of E2F-responsive genes that are associated with the G(1)/S transition. We also demonstrated, by adenovirus vector-mediated RNA interference, that ACTR is required for E2F1-mediated gene expression and the proliferation of estrogen receptor (ER)-negative breast cancer cells. Moreover, the ability of elevated ACTR to promote estrogen-independent cell proliferation depends on the function of E2F1 and the association between ACTR and E2F1, but not ER. Thus, our results reveal an essential role of ACTR in control of breast cancer cell proliferation and implicate the ACTR-E2F1 pathway as a novel mechanism in antiestrogen resistance.

Differences in stability of repressor complexes at promoters underlie distinct roles for Rb family members.

Oncogenic transformation of cells can induce the cyclin-dependent kinase inhibitor, p16, which leads to hypophosphorylation and activation of retinoblastoma (Rb). Rb is capable of causing permanent growth arrest, which may underlie its role as a tumor suppressor. We show that repression by Rb at E2F target gene promoters involves the establishment of a stable repressor complex that is not displaced by the overexpression of E2F-1. Rather than displacing Rb, excess E2F-1 instead recruits more Rb, leading to direct transcriptional repression. In contrast, the Rb family members, p130 and p107, which have not been demonstrated to be tumor suppressors, bind preferentially to target promoters in the absence of growth factors and in proliferating cells, respectively, and these repressor complexes are displaceable by E2F-1. Heterochromatin protein 1 (HP1), which interacts with Rb, is associated with these distinct repressor complexes and follows a similar pattern of stability/displaceability. Efficient growth arrest by p16/Rb is dependent on histone H3 lysine 9 methylation, which provides a binding site for HP1. We propose that these differences in the stability of repressor complexes at promoters may, in part, underlie the different roles of Rb vs p130 and p107 in cell cycle regulation and tumor suppression.

Che-1 Arrests Human Colon Carcinoma Cell Proliferation by Displacing HDAC1 from the p21WAF1/CIP1 Promoter

Che-1 is a recently identified human RNA polymerase II binding protein involved in the regulation of gene transcription and cell proliferation. We previously demonstrated that Che-1 inhibits the Rb growth-suppressing function by interfering with Rb-mediated HDAC1 recruitment on E2F target gene promoters. By hybridization of cancer profile arrays, we found that Che-1 expression is strongly down-regulated in several tumors, including colon and kidney carcinomas, compared with the relative normal tissues. Consistent with these data, Che-1 overexpression inhibits proliferation of HCT116 and LoVo human colon carcinoma cell lines by activation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 in a p53-independent manner and by promoting growth arrest at the G1 phase of the cell cycle. Che-1 activates p21WAF1/Cip1 by displacing histone deacetylase (HDAC)1 from the Sp1 binding sites of the p21WAF1/Cip1 gene promoter and accumulating acetylated histone H3 on these sites. Accordingly, Che-1-specific RNA interference negatively affects p21WAF1/Cip1 transactivation and increases cell proliferation in HCT116 cells. Taken together, our results indicate that Che-1 can be considered a general HDAC1 competitor and its down-regulation is involved in colon carcinoma cell proliferation.

Identification of novel pRb binding sites using CpG microarrays suggests that E2F recruits pRb to specific genomic sites during S phase.

The retinoblastoma (Rb) tumor suppressor protein is an important regulator of cell proliferation and differentiation. Many studies have shown that pRb can negatively regulate the activity of the E2F family of transcription factors during G(0) and G(1) phases of the cell cycle, perhaps by serving as a bridge between the E2Fs and transcriptional repressors such as histone deacetylases and methylases. However, pRb has also been shown to localize to discrete DNA foci during S phase, a time at which pRb is thought to be dissociated from E2F. Numerous other DNA binding proteins have been shown to interact with pRb, suggesting that pRb may control progression through S phase by binding to sites in the genome distinct from E2F target gene promoters. To test this hypothesis, we have identified novel pRb binding sites within the human genome using an unbiased approach which relies upon a combination of chromatin immunoprecipitation and CpG microarray analysis. To provide the greatest opportunity of finding distinct sets of pRb binding sites, we examined pRb binding in chromatin obtained from human Raji cells synchronized in either G(0)/G(1) phase or S phase. These experiments have allowed us to identify a large set of new genomic binding sites for the pRb protein. We found that some sites are occupied by pRb only during G(0)/G(1) phase, as would be predicted from previous models of pRb function. We also identified sites to which pRb bound only during S phase and other sites which were bound constitutively by pRb. Surprisingly, we found that E2F1 was present at most of the CpG islands bound by pRb, independent of the phase of the cell cycle. Thus, although pRb has the potential to interact with numerous transcription factors, our data suggest that the majority of DNA-bound pRb is recruited to E2F target promoters during both G(0)/G(1) and S phases.

Target gene specificity of E2F and pocket protein family members in living cells.

E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G(0) phase of the cell cycle and release of the pocket protein in late G(1), followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G(0) and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.