Transcription factors are adaptor molecules that detect regulatory sequences in the DNA and target the assembly of protein complexes that control gene expression. Yet much of the DNA in the ...eukaryotic cell is in nucleosomes and thereby occluded by histones, and can be further occluded by higher-order chromatin structures and repressor complexes. Indeed, genome-wide location analyses have revealed that, for all transcription factors tested, the vast majority of potential DNA-binding sites are unoccupied, demonstrating the inaccessibility of most of the nuclear DNA. This raises the question of how target sites at silent genes become bound de novo by transcription factors, thereby initiating regulatory events in chromatin. Binding cooperativity can be sufficient for many kinds of factors to simultaneously engage a target site in chromatin and activate gene expression. However, in cases in which the binding of a series of factors is sequential in time and thus not initially cooperative, special "pioneer transcription factors" can be the first to engage target sites in chromatin. Such initial binding can passively enhance transcription by reducing the number of additional factors that are needed to bind the DNA, culminating in activation. In addition, pioneer factor binding can actively open up the local chromatin and directly make it competent for other factors to bind. Passive and active roles for the pioneer factor FoxA occur in embryonic development, steroid hormone induction, and human cancers. Herein we review the field and describe how pioneer factors may enable cellular reprogramming.
Estrogen receptor α (ER) is the major driver of ∼75% of breast cancers, and multiple ER targeting drugs are routinely used clinically to treat patients with ER
breast cancer. However, many patients ...relapse on these targeted therapies and ultimately develop metastatic and incurable disease, and understanding the mechanisms leading to drug resistance is consequently of utmost importance. It is now clear that, in addition to estrogens, ER function is modulated by other steroid receptors and multiple signaling pathways (e.g., growth factor and cytokine signaling), and many of these pathways affect drug resistance and patient outcome. Here, we review the mechanisms through which these pathways impact ER function and drug resistance as well as discuss the clinical implications.
Antibody-drug conjugates (ADCs) harness the highly specific targeting capabilities of an antibody to deliver a cytotoxic payload to specific cell types. They have garnered widespread interest in drug ...discovery, particularly in oncology, as discrimination between healthy and malignant tissues or cells can be achieved. Nine ADCs have received approval from the US Food and Drug Administration and more than 80 others are currently undergoing clinical investigations for a range of solid tumours and haematological malignancies. Extensive research over the past decade has highlighted the critical nature of the linkage strategy adopted to attach the payload to the antibody. Whilst early generation ADCs were primarily synthesised as heterogeneous mixtures, these were found to have sub-optimal pharmacokinetics, stability, tolerability and/or efficacy. Efforts have now shifted towards generating homogeneous constructs with precise drug loading and predetermined, controlled sites of attachment. Homogeneous ADCs have repeatedly demonstrated superior overall pharmacological profiles compared to their heterogeneous counterparts. A wide range of methods have been developed in the pursuit of homogeneity, comprising chemical or enzymatic methods or a combination thereof to afford precise modification of specific amino acid or sugar residues. In this review, we discuss advances in chemical and enzymatic methods for site-specific antibody modification that result in the generation of homogeneous ADCs.
Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer ...occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen-ESR1-mediated interactions between cis-regulatory elements. Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer.
Most breast cancers are driven by oestrogen receptor-α. Anti-oestrogenic drugs are the standard treatment for these breast cancers; however, treatment resistance is common, necessitating new ...therapeutic strategies. Recent preclinical and historical clinical studies support the use of progestogens to activate the progesterone receptor (PR) in breast cancers. However, widespread controversy exists regarding the role of progestogens in this disease, hindering the clinical implementation of PR-targeted therapies. Herein, we present and discuss data at the root of this controversy and clarify the confusion and misinterpretations that have consequently arisen. We then present our view on how progestogens may be safely and effectively used in treating breast cancer.
Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid ...and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. The method uses formaldehyde fixation to stabilize protein complexes. By using antibodies against the endogenous target, the cross-linked complex is immunoprecipitated, rigorously washed, and then digested into peptides while avoiding antibody contamination (on-bead digestion). By using this method, MS identification of the target protein and several dozen interacting proteins is possible using a 100-min LC-MS/MS run. The protocol does not require substantial proteomics expertise, and it typically takes 2-3 d from the collection of material to results.
FOXA1 is a pioneer factor that binds to enhancer regions that are enriched in H3K4 mono- and dimethylation (H3K4me1 and H3K4me2). We performed a FOXA1 rapid immunoprecipitation mass spectrometry of ...endogenous proteins (RIME) screen in ERα-positive MCF-7 breast cancer cells and found histone-lysine N-methyltransferase (MLL3) as the top FOXA1-interacting protein. MLL3 is typically thought to induce H3K4me3 at promoter regions, but recent findings suggest it may contribute to H3K4me1 deposition. We performed MLL3 chromatin immunoprecipitation sequencing (ChIP-seq) in breast cancer cells, and MLL3 was shown to occupy regions marked by FOXA1 occupancy and H3K4me1 and H3K4me2. MLL3 binding was dependent on FOXA1, indicating that FOXA1 recruits MLL3 to chromatin. MLL3 silencing decreased H3K4me1 at enhancer elements but had no appreciable impact on H3K4me3 at enhancer elements. We propose a mechanism whereby the pioneer factor FOXA1 recruits the chromatin modifier MLL3 to facilitate the deposition of H3K4me1 histone marks, subsequently demarcating active enhancer elements.
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•Mass spectrometry of chromatin-associated proteins with FOXA1 identifies MLL3•FOXA1 recruits MLL3 to deposit H3K4me1 on FOXA1-bound enhancers•MLL3 promotes ERα-dependent gene transcription and proliferation•GRHL2 co-occupies regions of ERα, FOXA1, and MLL3 on enhancers
Jozwik et al. demonstrate that FOXA1 can activate enhancers by recruiting the chromatin-associated histone methyltransferase MLL3 to mediate monomethylation on enhancers. This identifies FOXA1 as an upstream regulatory element in the establishment of enhancers that regulates active chromatin marks.
Considerable work has linked hormone receptors, such as estrogen receptor-alpha (ER), with the pioneer factor FOXA1. Altered FOXA1 levels contribute to endocrine-resistant breast cancer, where it ...maintains ER-chromatin interactions, even in contexts in which cells are refractory to ER-targeted drugs. A recent study controversially suggests that FOXA1 binding can be induced by hormonal pathways, including the estrogen-ER complex. We now show that the vast majority (>99%) of FOXA1 binding events are unaffected by steroid activation. A small number (<1%) of FOXA1 binding sites appear to be induced by estrogen, but these are created from chromatin interactions between ER binding sites and adjacent FOXA1 binding sites and do not represent genuine new FOXA1-pioneering elements. FOXA1 is therefore not regulated by estrogen and remains a bone fide pioneer factor that is entirely upstream of the ER complex.
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•FOXA1 binding events are not regulated by hormones•Chromatin loops create shadow FOXA1 binding sites•FOXA1 is a bone fide pioneer factor independent of hormone stimuli
Glont et al. show that FOXA1 binding sites are not regulated by hormones. A small number (<1%) of FOXA1 binding events appear to be estrogen regulated, but these are shadow peaks that are created via pre-existing binding sites that form chromatin loops.
Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several ...models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.
Abstract
The largest subtype of breast cancer is characterized by the expression and activity of the estrogen receptor alpha (ERalpha/ER). Although several effective therapies have significantly ...improved survival, the adaptability of cancer cells means that patients frequently stop responding or develop resistance to endocrine treatment. ER does not function in isolation and multiple associating factors have been reported to play a role in regulating the estrogen-driven transcriptional program. This review focuses on the dynamic interplay between some of these factors which co-occupy ER-bound regulatory elements, their contribution to estrogen signaling, and their possible therapeutic applications. Furthermore, the review illustrates how some ER association partners can influence and reprogram the genomic distribution of the estrogen receptor. As this dynamic ER activity enables cancer cell adaptability and impacts the clinical outcome, defining how this plasticity is determined is fundamental to our understanding of the mechanisms of disease progression.
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