Abstract:FOXA1 is a transcription factor capable to bind silenced chromatin to direct context-dependent cell fate conversion. Here, we demonstrate that a compact palindromic DNA element (termed ‘DIV’ for its diverging half-sites) induces the homodimerization of FOXA1 with strongly positive cooperativity. Alternative structural models are consistent with either an indirect DNA-mediated cooperativity or a direct protein-protein interaction. The cooperative homodimer formation is strictly constrained by precise half-site … Show more
“…7c-e), suggesting a mechanism by which FOXA1 R219S selectively targets novel genomic loci. Finally, two motifs recently associated with FOXA1 dimers (convergent, divergent) 21 were relatively enriched in cluster 0 versus cluster 1, potentially explaining the novel pioneering activity of FOXA1 F254_E255del (Fig. 4d).…”
Mutations in the FOXA1 transcription factor define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown1-9. By annotating the FOXA1 mutation landscape from 3086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (~50% of all mutations) and R219 (~5%), a highly conserved DNA contact residue. Clinically, Wing2 mutations are seen in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumors with neuroendocrine histology. Interrogation of the biologic properties of FOXA1WT and 14 FOXA1 mutants revealed gain-of-function in mouse prostate organoid proliferation assays. 12 of these mutants, as well as FOXA1WT, promoted an exaggerated pro-luminal differentiation program whereas two different R219 mutants blocked luminal differentiation and activate a mesenchymal and neuroendocrine transcriptional program. ATAC-seq of FOXA1WT and representative Wing2 and R219 mutants revealed dramatic, mutant-specific changes in open chromatin at thousands of genomic loci, together with novel sites of FOXA1 binding and associated increases in gene expression. Of note, peaks in R219 mutant expressing cells lack the canonical core FOXA1 binding motifs (GTAAAC/T) but are enriched for a related, non-canonical motif (GTAAAG/A), which is preferentially activated by R219 mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its normal pioneering function through perturbation of normal luminal epithelial differentiation programs, providing further support to the role of lineage plasticity in cancer progression.
“…7c-e), suggesting a mechanism by which FOXA1 R219S selectively targets novel genomic loci. Finally, two motifs recently associated with FOXA1 dimers (convergent, divergent) 21 were relatively enriched in cluster 0 versus cluster 1, potentially explaining the novel pioneering activity of FOXA1 F254_E255del (Fig. 4d).…”
Mutations in the FOXA1 transcription factor define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown1-9. By annotating the FOXA1 mutation landscape from 3086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (~50% of all mutations) and R219 (~5%), a highly conserved DNA contact residue. Clinically, Wing2 mutations are seen in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumors with neuroendocrine histology. Interrogation of the biologic properties of FOXA1WT and 14 FOXA1 mutants revealed gain-of-function in mouse prostate organoid proliferation assays. 12 of these mutants, as well as FOXA1WT, promoted an exaggerated pro-luminal differentiation program whereas two different R219 mutants blocked luminal differentiation and activate a mesenchymal and neuroendocrine transcriptional program. ATAC-seq of FOXA1WT and representative Wing2 and R219 mutants revealed dramatic, mutant-specific changes in open chromatin at thousands of genomic loci, together with novel sites of FOXA1 binding and associated increases in gene expression. Of note, peaks in R219 mutant expressing cells lack the canonical core FOXA1 binding motifs (GTAAAC/T) but are enriched for a related, non-canonical motif (GTAAAG/A), which is preferentially activated by R219 mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its normal pioneering function through perturbation of normal luminal epithelial differentiation programs, providing further support to the role of lineage plasticity in cancer progression.
“…These experiments performed with a high concentration of purified and tagged proteins could carry artifacts, but the very precise spacing and orientation of the binding sequences selected (overlapping and inverted for FOXM) support the fact that very ordered complexes are formed and might be functional. A decisive recent publication supports this view: Functional DNA-mediated FOXA1 homodimer formation was identified on a palindromic DNA element in the context of chromatin [113].…”
The nematode Caenorhabditis elegans possesses a unique (with various isoforms) FOXO transcription factor DAF-16, which is notorious for its role in aging and its regulation by the insulin-PI3K-AKT pathway. In humans, five genes (including a protein-coding pseudogene) encode for FOXO transcription factors that are targeted by the PI3K-AKT axis, such as in C. elegans. This common regulation and highly conserved DNA-binding domain are the pillars of this family. In this review, I will discuss the possible meaning of possessing a group of very similar proteins and how it can generate additional functionality to more complex organisms. I frame this discussion in relation to the much larger super family of Forkhead proteins to which they belong. FOXO members are very often co-expressed in the same cell type. The overlap of function and expression creates a certain redundancy that might be a safeguard against the accidental loss of FOXO function, which could otherwise lead to disease, particularly, cancer. This is one of the points that will be examined in this “family affair” report.
“…3b). Furthermore, pioneer factors can bind to compacted chromatin to facilitate the binding of other TFs [22, 23]. Based on this, we reasoned that FOXA1 may regulate CYR61 enhancer activity and promote CYR61 expression.Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Moreover, CBP can act as a lysine acetyltransferase to acetylate TFs and histones to increase the accessibility of chromatin [20, 21]. Forkhead box A1 (FOXA1) has been reported to be a pioneer factor in that it can bind to and open compacted chromatin to facilitate the binding of other TFs, and its activity is dysregulated in many physiological and pathological conditions [22, 23]. Other forkhead transcription factors, such as FOXK1 [7] and FOXO3a [24], have been reported to regulate CYR61 expression by binding to the CYR61 promoter in a sequence-dependent manner.…”
Background
High expression of secreted matricellular protein cysteine-rich 61 (
CYR61
) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation has been shown to correlate with expression of key genes involved in cancer progression. However, such mechanisms in
CYR61
transcription regulation remain unexplored.
Methods
Expression of
CYR61
was determined by immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and western blotting (WB) in CRC patients paraffin specimens and colon cell lines. ChIP-seq data of enhancer-characteristic histone modifications, in CRC tissues from the Gene Expression Omnibus (GEO) database, were reanalyzed to search for putative enhancers of
CYR61
. Dual-luciferase reporter assay was used to detected enhancer activity. Physical interactions between putative enhancers and CYR61 promoter were detected by chromosome conformation capture (3C) assay. Histone modification and transcription factors (TFs) enrichment were detected by ChIP-qPCR. Additionally, biological function of enhancers was investigated by transwell migration assays.
Results
CRC tissues and cell lines expressed higher level of
CYR61
than normal colon mucosa. Three putative enhancers located downstream of
CYR61
were found in CRC tissues by ChIP-seq data reanalysis. Consistent with the ChIP-seq analysis results in the GEO database, the normal colon mucosal epithelial cell line NCM460 possessed no active
CYR61
enhancers, whereas colon cancer cells exhibited different patterns of active
CYR61
enhancers. HCT116 cells had an active Enhancer3, whereas RKO cells had both Enhancer1 and Enhancer3 active. Pioneer factor FOXA1 promoted
CYR61
expression by recruiting CBP histone acetyltransferase binding and increasing promoter-enhancer looping frequencies and enhancer activity. CBP knockdown attenuated H3K27ac enrichment, promoter-enhancer looping frequencies, and enhancer activity. Small molecule compound 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment, which stimulated
CYR61
expression, and verteporfin (VP) treatment, which inhibited
CYR61
expression, confirmed that the enhancers regulated
CYR61
expression. Knockdown and ectopic expression of CYR61 rescued cell migration changes induced by over-expressing and knockdown of FOXA1, respectively.
Conclusions
CYR61
enhancer activation, mediated by FOXA1 and CBP, occurs during CRC progression to up-regulate
CYR61
expression and promote cell migration in CRC, suggesting inhibition of recruitment of FOXA1 and/or CBP to
CYR61
enhancers may have therapeutic imp...
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