Pioneer Factor Research Articles

Chromatin Binding Regulates Phase Behavior and Morphology of Condensates Formed by Prion-like Domains.

In vivo differentiation of embryonic cells devoid of key reprogramming factors.

Emerging evidence suggests that lineage-specifying transcription factors control the progression of pancreatic ductal adenocarcinoma (PDAC). We have discovered a transcription factor switching mechanism involving the poorly characterized orphan nuclear receptor HNF4G and the putative pioneer factor FOXA1, which drives PDAC progression. Using our unbiased protein interactome discovery approach, we identified HNF4A and HNF4G as reproducible, FOXA1-associated proteins, in both preclinical models and Whipple surgical samples. In the primary tumor context, we consistently find that the dominant transcription factor is HNF4G, where it functions as the driver. A molecular switch occurs in advanced disease, whereby HNF4G expression or activity decreases, unmasking FOXA1's transcriptional potential. Derepressed FOXA1 drives late-stage disease by orchestrating metastasis-specific enhancer-promoter loops to regulate the expression of metastatic genes. Overall survival is influenced by HNF4G and FOXA1 activity in primary tumor growth and in metastasis, respectively. We suggest that the existence of stage-dependent transcription factor activity, triggered by molecular compartmentalization, mediates the progression of PDAC.

Nuclear receptors (NRs) orchestrate transcriptional programs that regulate cell fate decisions, and when these processes are disrupted, they can drive hormone-dependent cancers. This review summarizes mechanisms by which NRs function collectively, or crosstalk, to bring about the complex transcriptional control of cell fate decisions and indicate where these processes can act as cancer-drivers. These crosstalk mechanisms include the exchange of coregulators between NRs and as well as genomic convergence of NRs. Evidence is also discussed for how NRs potentially pass through a continuum of interactions as part of a biological ratchet mechanism to regulate gene transcription. In this continuum, pioneer factors drive chromatin competence for NRs, and along with mammalian SWI/SNF complexes facilitate transient assisted loading between NRs, as well as more stable crosstalk in the form of mitotic bookmarking, which allows inheritance of transcriptional control. NR crosstalk is also sustained through the function of larger and perhaps more stable interactions, such as through the megatrans complex. Also considered to explain NR crosstalk is the established and emerging understanding of the grammar of motif selection, and this is placed in the context of NR network approaches, for example in breast cancer. Finally, a systems-level framework, a so-called "NuRome", is discussed that combines high dimensional data at the cistrome, transcriptome and proteome levels to provide a predictive understanding of NR crosstalk and transcription in cancer.

Understanding how transcription factors regulate organized cellular diversity in developing tissues remains a major challenge due to their pleiotropic functions. We addressed this by monitoring and genetically modulating the activity of PAX3 and PAX7 during the specification of neural progenitor pools in the embryonic spinal cord. Using mouse models, we show that the balance between the transcriptional activating and repressing functions of these factors is modulated along the dorsoventral axis and is instructive to the patterning of spinal progenitor pools. By combining loss-of-function experiments with functional genomics in spinal organoids, we demonstrate that PAX-mediated repression and activation rely on distinct cis-regulatory genomic modules. This enables both the coexistence of their dual activity in dorsal cell progenitors and the specific control of two major differentiation programs. PAX promote H3K27me3 deposition at silencers to repress ventral identities, while at enhancers, they act as pioneer factors, opening and activating cis-regulatory modules to specify dorsal-most identities. Finally, we show that this pioneer activity is restricted to cells exposed to BMP morphogens, ensuring spatial specificity. These findings reveal how PAX proteins, modulated by morphogen gradients, orchestrate neuronal diversity in the spinal cord, providing a robust framework for neural subtype specification.

Transcription factors represent one of the major groups of proteins, whose suppression leads to tumor growth arrest. Different types of cancer express a specific set of transcription factors that create and maintain unique patterns of gene expression. In prostate cancer cells, one of the key transcriptional regulators is the HOXB13 (Homeobox B13) protein. HOXB13 is known to be an important regulator of embryonic development and terminal cell differentiation. HOXB13 regulates the transcription of many genes in normal and transformed prostate cells and is also capable of acting as a pioneer factor that opens chromatin in the regulatory regions of genes. However, little is known about the protein partners and functions of HOXB13 in prostate cells. In the present study, we searched for protein partners of HOXB13 by immunoaffinity purification followed by high-throughput mass spectrometric analysis (IP/LC-MS) using the PC-3 prostate cancer cell line as a model. The main partners of HOXB13 were found to be transcription factors with different types of DNA-binding domains, including the TBX3, TBX2, ZFHX4, ZFHX3, RUNX1, NFAT5 proteins. Using the DepMap resource, we have shown that one of the identified partners, the TBX3 protein is as critical for the growth and proliferation of prostate cancer cell lines in vitro as HOXB13. Analysis of individual prostate cancer cell lines revealed that knockout of both genes, HOXB13 and TBX3, leads to the death of the same lines: VCaP, LNCaP (clone FGC), PC-3 and 22Rv1. Thus, HOXB13 and TBX3 can be considered together as potential targets for the development of specific inhibitors that suppress prostate cancer cell growth.

Neuroendocrine prostate cancer (NEPC) is a treatment-resistant subtype that arises through lineage plasticity, allowing tumor cells to bypass androgen receptor (AR)–targeted therapies. In a recent study, Lu et al. define a transcriptional and epigenetic hierarchy that drives this transdifferentiation. The pioneer factor FOXA2 initiates enhancer remodeling and regional DNA demethylation, while the neural lineage transcription factor NKX2-1 is required to complete the NE program. Together, these factors reorganize 3D chromatin architecture and activate lineage-specific genes through enhancer–promoter looping. Crucially, the histone acetyltransferase p300/CBP is essential cofactor in this process. Pharmacologic inhibition of p300/CBP with CCS1477 suppresses NE gene expression and impairs tumor growth in NEPC models. These findings offer a mechanistic insight of lineage plasticity and highlight p300/CBP as promising therapeutic targets. The study also raises key questions about the stability and reversibility of chromatin remodeling and sets a framework for understanding enhancer-driven plasticity in other cancers.

Exposure to low levels of environmental challenges, known as hormetic stress, such as nutrient deprivation and heat shock, fosters subsequent stress resistance and promotes healthy aging in later life. However, specific mechanisms governing transcriptional reprogramming upon hormetic nutrient stress remain elusive. In this study, we identified histone H3 lysine 27 acetylation (H3K27ac) as a crucial driver of transcriptomic adaptation to hormetic fasting. Beyond its immediate function of enhancing lipid catabolism for alternative energy sources, stress-induced H3K27ac activates lifelong antioxidant defenses, thereby reducing reactive oxygen species (ROS) produced by stress-induced fatty acid oxidation and their accumulation during aging. The increase in H3K27ac, mediated by pioneer factor PHA-4/FOXA and cooperating transcription factor NHR-49/HNF4, is crucial for lifespan extension under hermetic nutrient stress in Caenorhabditis elegans. Our findings establish H3K27ac as a key transcriptional switch that bridges nutrient status with transcriptomic reprogramming, underpinning the pro-longevity effects of hormetic fasting through orchestrating lipid catabolism and antioxidative defenses.

In brain development, neural stem cells (NSCs) undergo asymmetric cell divisions to replicate themselves and meanwhile produce differentiating siblings. It remains obscure how NSCs preserve their self-renewing fate across mitosis. Even less is known how cell fate memory is differentially propagated to sibling daughter cells adopting distinct cell fates. Here we found that key differentiation genes are dually bookmarked by pioneer factor GAF (GAGA factor) and H3K27ac in asymmetrically-dividing Drosophila central brain NSCs. In daughter cells adopting NSC fate, GAF promotes self-renewal through timely inhibiting differentiation genes via HDAC1-mediated H3K27 deacetylation, whereas in sibling daughter cells adopting neural progenitor fate, GAF occupancy is replaced by competitor SWI/SNF complex, allowing retention of H3K27ac mark and fast activation of differentiation genes. Thus, our study unveils a paradigm by which cell fate memory can be differentially transmitted to sibling daughter cells via dual antagonistic mitotic bookmarking and selective molecular competition mechanism.

Gene expression requires the targeting of transcription factors (TFs) to regulatory sequences often occluded within nucleosomes. To comprehensively examine TF nucleosome binding, we developed Pioneer-Seq. In Pioneer-seq a library of thousands of nucleosomes are formed from sequences containing a TF binding site (TFBS) variant in all possible nucleosome orientations and within the linker regions. Pioneer-seq has the unique ability to simultaneously examine nucleosomes created with various nucleosome positioning sequences and examine binding to in vivo targeted nucleosomes (ITNs). Pioneer-seq can be applied to address various mechanistic models for TF-nucleosome binding directly and can be used to uncover inherent TF-interaction differences. To demonstrate Pioneer-seq, we examined nucleosome binding by OCT4, SOX2, KLF4, and c-MYC. Our results demonstrate that all studied TFs can bind at nucleosome edges and nucleosome sequence is the primary factor regulating TF binding. In addition, KLF4 can bind to a non-canonical TFBS located 20 bp from the nucleosome dyad. Examination of ITNs showed binding differences between the TFs, with KLF4 and SOX2 binding more often near nucleosome centers. Overall, our results demonstrate differences in how TF recognizes their TFBS within a nucleosome and begins to define the mechanistic requirements for pioneer factor binding.

Establishing the anterior-posterior (AP) body axis is a fundamental process during embryogenesis, and the fruit fly, Drosophila melanogaster, provides one of the best-known case studies. But for unknown reasons, different species of flies (Diptera) establish the AP axis through unrelated, structurally distinct anterior determinants (ADs). The AD of Drosophila, Bicoid (Bcd), initiates symmetry-breaking during nuclear cleavage cycles (NCs) when ubiquitous pioneer factors, such as Zelda (Zld), drive zygotic genome activation (ZGA) at the level chromatin accessibility by nucleosome depletion. While Bcd engages in a concentration-dependent competition with nucleosomes at the loci of a small set of transcription factor (TF) genes that are expressed in the anterior embryo, it remains unknown whether unrelated ADs of other fly species function in the same way and target homologous genes. We have examined the symmetry-breaking mechanism of a moth fly, Clogmia albipunctata, in which a maternally expressed transcript isoform of the pair-rule segmentation gene odd-paired serves as AD. We provide a de novo assembly and annotation of the Clogmia genome and describe how Clogmia’s orthologs of zelda (Cal-zld) and odd-paired (Cal-opa) affect chromatin accessibility and gene expression. Our results suggest direct roles of Cal-zld in opening and closing chromatin during nuclear cleavage cycles (NCs) and show that during the early phase of ZGA maternal Cal-opa activity promotes chromatin accessibility and anterior expression at Clogmia’s homeobrain and sloppy-paired loci. These genes are not known as key targets of Bcd but may serve a more widely conserved role in the initiation of anterior pattern formation given their early anterior expression and function in head development in insects. We conclude that the ADs of Drosophila and Clogmia differ in their target genes but share the mechanism of concentration-dependent nucleosome depletion.

Chromatin-dependent motif syntax defines differentiation trajectories

SUMMARYSeed development in Arabidopsis thaliana is largely controlled by a set of transcription factors (TFs) called LAFL, including LEAFY COTYLEDON 2 (LEC2). In this study, we investigated the structure/function relationships of the protein LEC2 outside the well‐described B3 DNA‐binding domain. The results presented here unveil the presence of transcription activation domains (ADs) within the unstructured ends of the protein that are conserved in eudicots. Expression in both yeast and moss protoplasts of deleted and mutated versions of LEC2 confirmed the transcriptional activity of these ADs. Surprisingly, the expression of LEC2 variants lacking their ADs restored a wild‐type seed phenotype in lec2 mutant, showing that these ADs are not essential for LEC2 function in seed development. Moreover, ZmAFL2/ZmABI19, a maize B3 factor related to LEC2 but deprived of N‐ter AD, can also complement lec2 seed phenotype and induce abnormal vegetative development when overexpressed in Arabidopsis, supporting this observation. This work suggests that LEC2 can act both as a classical transcriptional activator or without transactivation activity, probably through its interaction with the pioneer factor LEC1. Taken together, the results provide important insights into the function of the LAFL master regulators during seed development, from cell differentiation to storage accumulation in seed.

The establishment of cell type–specific chromatin landscapes is essential for cellular identity, but how these landscapes are generated remains poorly understood. Here, we demonstrate that the chromatin remodeler SMARCA5 establishes epigenetic priming that is required for retinoic acid (RA)–induced differentiation in the male germline. Germ cell–specific deletion of Smarca5 results in a complete loss of differentiating spermatogonia, phenocopying vitamin A-deficient mice that lack RA signaling. During the perinatal transition from prospermatogonia to undifferentiated spermatogonia, SMARCA5 is recruited to binding sites of the pioneer transcription factor DMRT1, which are located at distal putative enhancers and promoters of germline genes. The SMARCA5–DMRT1 pioneer complex establishes chromatin accessibility at these loci, generating poised enhancers and promoters that serve as RA receptor (RAR)–binding sites. Thus, SMARCA5 licenses transcriptional responses to RA that enable spermatogenic differentiation. Our findings uncover a mechanism linking pioneer factor activity to external signal responsiveness.

During zygotic genome activation (ZGA) in Drosophila, broad domains of Polycomb-modified chromatin are rapidly established across the genome. Here, we investigate the spatial and temporal dynamics by which Polycomb group (PcG) histone modifications, H3K27me3 and H2Aub, emerge during early embryogenesis. Using ChIP-seq and live imaging of CRISPR-engineered GFP-tagged PcG components, we show that PRC2-dependent H3K27me3 accumulates adjacent to a subset of E(z)-bound prospective Polycomb Response Elements (PREs) beginning in nuclear cycle 14 (NC14), with patterns indicative of nucleation followed by spreading. Surprisingly, PRE-binding factors Pho, Combgap, and GAGA-factor are excluded from interphase nuclei prior to NC10 despite nuclear localization of E(z) throughout early interphases. Loss-of-function studies further demonstrate that GAGA-factor is largely dispensable for PcG domain establishment, whereas the pioneer factor Zelda is required for proper deposition of H3K27me3 and H2Aub at a subset of Polycomb domains. The role of Zelda at Polycomb domains is context-dependent; a large subset of targets requires Zelda not for PcG factor recruitment, but instead to license a loaded PRE to deposit H3K27me3 and H2Aub. Our findings support a model where licensing of PcG domains is an initial step in the regulatory processes governing Polycomb-regulated developmental genes.

Age-related macular degeneration (AMD) is a common cause of blindness worldwide, and it is projected to affect several million individuals by 2040. The human retinal pigment epithelium (hRPE) degenerates in dry AMD, prompting the need to develop stem cell therapies to replace the lost tissue by autologous transplantation and restore the visual function. Nevertheless, the molecular factors behind the hRPE cell fate determination have not been elucidated. Here we identify all molecular determinants of the hRPE cell fate identity by comprehensive and unbiased screening of predicted pioneer factors in the human genome: such TFs mediate coordinated transitions in chromatin accessibility and transcriptional outcome along three major stages of the hRPE genesis. Furthermore, we compile a complete census of all transcription factor-specific binding sites by footprinting analysis of the human epigenome along the RPE developmental trajectory. Gene regulatory networks were found to be involved in cellular responses to glucose and hypoxia, RPE nitrosative stress, type II epithelial-to-mesenchymal transition (EMT), and type III tumorigenic EMT, providing routes for therapeutic intervention on pleiotropic targets dysregulated in AMD, diabetic retinopathy, and cancer progression. Genome editing technologies may leverage this repository to devise functional screenings of regulatory elements and pharmacogenomic therapies in complex diseases, paving the way for strategies in precision medicine.

Abstract Breast cancer pathogenesis is closely linked to the mechanisms of cellular reprogramming, the process of converting differentiated cells into undifferentiated or different cell types. This reprogramming is achieved through the orchestrated expression of transcription factors which alter the cell’s existing chromatin landscape and establish a transcriptome corresponding to the reprogrammed cell state. Pioneer factors, a type of transcription factor, initiate cell development and cell fate transition by binding to specific DNA motifs within nucleosomes. This binding leads to the recruitment of co-factors, such as nucleosome remodelers, leading to chromatin opening and subsequent activation of specific genes essential for reprogrammed cell function. Improper engagement of these factors can lead to unanticipated or harmful gene expression, potentially contributing to various human diseases, including cancer. GATA3, a pioneer factor, is essential for normal mammary gland development and differentiation. In breast cancer cells, GATA3 has been shown to suppress tumor growth by inducing mesenchymal-to-epithelial transition (MET), the reverse process of epithelial-to-mesenchymal transition (EMT). Interestingly, although mutations in GATA3 are frequent and considered driver mutations, breast cancer patients carrying GATA3 mutations are generally associated with better patient survival. The roles of these mutations remain largely unexplored. M294R and M294K are hot spot missense mutations found in luminal breast cancer patients. These mutations occur in the DNA-binding domain of GATA3, which is crucial for its function as a transcription factor. For functional characterization of these mutants, we utilized MDA-MB-231 mesenchymal cells, a highly aggressive and invasive breast cancer cell line to model mesenchymal-to-epithelial transition (MET). Since ectopic expression of GATA3 induces MET in this cell line, these cells provide an excellent system to observe phenotypic and molecular alterations caused by its missense mutations M294R/K. To quantitatively measure changes in GATA3 mobility and its DNA binding dynamics in living cells, we utilized Fluorescence Recovery After Photobleaching (FRAP) assay. Additionally, to further elucidate the crucial role of GATA3, genomics analyses and phenotypic assays were conducted. Functional characterization of M294R/K mutants in MDA-MB-231 breast cancer cells demonstrated distinct phenotypic and molecular alterations compared to wild-type GATA3. While wild-type GATA3 induced MET, mutant-expressing cells exhibited differential responses. Remarkably, mutants derived from breast cancer patients displayed increased chromatin binding, leading to a tighter chromatin structure and differential gene expression of mesenchymal or epithelial markers. Altered cellular phenotypes such as cell migration are also observed in M294R/K expressed cells. Our finding underscores the importance of precise regulation of pioneer factor-chromatin interactions in cellular reprogramming processes. Understanding the molecular mechanisms underlying GATA3 mutations and their effects on cellular reprogramming in breast cancer provides valuable insights into the roles of GATA3 in breast cancer progression. Citation Format: Aerica Nagornyuk, Mika Saotome, Nobuki Hida, Alexander Samardzic, Motoki Takaku. Evaluating Functional Impacts of GATA3 Mutations in Breast Cancer Using Mesenchymal-to-Epithelial Transition Model [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-06-09.

Abstract Cellular reprogramming is a fundamental process in specific developmental stages and is also invaluable for disease modeling. It involves the conversion of differentiated cells into undifferentiated or specialized cell types. This transformation relies on coordinated action of transcription factors or chemical stimuli, which reshapes the cell's chromatin structure and establishes a different gene expression profile. Among these transcription factors, pioneer factors play a critical role, initiating cellular reprogramming and guiding cell fate transitions by selectively binding to specific DNA motifs within nucleosomes at inactive chromatin. The GATA3 protein serves as a pioneer transcription factor essential in mammary gland development and is involved in breast tumor development. GATA3 facilitates the cellular reprogramming of mesenchymal breast cancer cells into epithelial breast cancer cells. Despite the established role of pioneer factors in chromatin remodeling and cell fate regulation, the precise molecular mechanisms that govern their selective binding to chromatin are still not fully understood. Using the mesenchymal-to-epithelial transition (MET) model, we aimed to understand how GATA3 selectively binds to genes that are essential for successful cellular reprogramming. Utilizing a combination of machine learning and experimental approaches, we investigated GATA3's binding affinity to DNA and nucleosome substrates. Machine learning analysis identified two distinct zinc-finger domains which are required for effective GATA3 nucleosome binding. The results of this deep learning analysis were used to design a series of GATA3 mutant cell lines and DNA substrates. The experimental approach involved two strategies: first, analyzing the binding affinity of wild-type GATA3 to DNA substrates designed based on machine learning predictions, and second, examining the nucleosome binding affinity of GATA3 mutants, including those predicted by computational modeling and those derived from breast cancer patients. Electrophoretic mobility shift assays confirmed the differential binding affinities of GATA3 mutants, indicating the importance of specific motifs and zinc finger domains. Our findings highlight the significance of DNA sequence specificity in modulating pioneer transcription factor activity and chromatin accessibility. Mutational analysis revealed distinct roles for each zinc finger domain, with mutations affecting DNA binding affinity and chromatin accessibility. This study provides insights into the molecular mechanisms governing GATA3-mediated chromatin remodeling during MET. By characterizing the interaction between DNA/nucleosome binding affinity and chromatin accessibility, we expand our understanding of pioneer transcription factor function in breast cancer progression. Citation Format: Jill Goodman, Mika Saotome, Motoki Takaku. Nucleosome binding affinity of the pioneer factor GATA3 influences chromatin reprogramming in breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-06-15.

Gene regulation by transcription factors (TFs) binding cognate sequences is of paramount importance. For example, the TFs Zelda (Zld) and GAGA factor (GAF) are widely acknowledged for pioneering gene activation during zygotic genome activation (ZGA) in Drosophila. However, quantitative dose/response relationships between bulk TF concentration and DNA binding, an event tied to transcriptional activity, remain elusive. Here, we map these relationships during ZGA: a crucial step in metazoan development. To map the dose/response relationship between nuclear concentration and DNA binding, we performed raster image correlation spectroscopy, a method that can measure biophysical parameters of fluorescent molecules. We found that, although Zld concentration increases during nuclear cycles 10 to 14, its binding in the transcriptionally active regions decreases, consistent with its function as an activator for early genes. In contrast, GAF-DNA binding is nearly linear with its concentration, which sharply increases during the major wave, implicating its involvement in the major wave. This study provides key insights into the properties of the two factors and puts forward a quantitative approach that can be used for other TFs to study transcriptional regulation.

Mechanisms of cell fate specification are central to developmental biology and regenerative medicine. ETV2 is a master regulator for the endothelial cell (EC) lineage specification. Here we study mechanisms by which ETV2 overexpression in human induced pluripotent stem-cell-derived mesodermal progenitors efficiently specifies ECs. We used CUT&RUN, scRNA-seq and scATAC-seq to characterize the molecular features of EC differentiation mediated by ETV2. We defined the scope of ETV2 pioneering activity and identified its direct downstream target genes. Induced ETV2 expression both directed specification of endothelial progenitors and suppressed acquisition of alternative fates. Functional screening and candidate validation revealed cofactors essential for efficient EC specification, including the transcriptional activator GABPA. Notably, the transcriptional repressor REST was also necessary for efficient EC specification. ETV2 recruited REST to repress non-EC lineage genes. Our study provides an unparalleled molecular analysis of EC specification at single-cell resolution and highlights the important role of pioneer factors to recruit repressors that suppress commitment to alternative lineages.