Gene of the month: NKX3.1

NKX3.1 is a multifaceted protein with roles in prostate development and protection from oxidative stress. Acting as a pioneer factor, NKX3.1 interacts with chromatin at enhancers to help integrate androgen regulated signalling. In prostate cancer, NKX3.1 activity is frequently reduced through a combination of mutational and post-translational events. Owing to its specificity for prostate tissue, NKX3.1 has found use as an immunohistochemical marker in routine histopathology practice.

The pioneer factor hypothesis is not necessary to explain ectopic liver gene activation

The Pioneer Factor Hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (nonPFs) that activate batteries of silent genes. The PFH predicts that ectopic gene activation requires the sequential activity of qualitatively different TFs. We tested the PFH by expressing the endodermal PF FOXA1 and nonPF HNF4A in K562 lymphoblast cells. While co-expression of FOXA1 and HNF4A activated a burst of endoderm-specific gene expression, we found no evidence for a functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and 'pioneered' for each other, although FOXA1 required fewer copies of its motif for binding. A subset of targets required both TFs, but the predominant mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results we hypothesize an alternative to the PFH where 'pioneer activity' depends not on categorically different TFs but rather on the affinity of interaction between TF and DNA.

Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila

During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is correlated with differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network driven by cell-type-specific enhancers.

A Test of the Pioneer Factor Hypothesis

The Pioneer Factor Hypothesis (PFH) states that pioneer factors (PFs) are a subclass of transcription factors (TFs) that bind to and open inaccessible sites and then recruit non-pioneer factors (nonPFs) that activate batteries of silent genes. We tested the PFH by expressing the endodermal PF FoxA1 and nonPF Hnf4a in K562 lymphoblast cells. While co-expression of FoxA1 and Hnf4a activated a burst of endoderm-specific gene expression, we found no evidence for functional distinction between these two TFs. When expressed independently, both TFs bound and opened inaccessible sites, activated endodermal genes, and “pioneered” for each other, although FoxA1 required fewer copies of its motif to bind at inaccessible sites. A subset of targets required both TFs, but the mode of action at these targets did not conform to the sequential activity predicted by the PFH. From these results we propose an alternative to the PFH where “pioneer activity” depends not on the existence of discrete TF subclasses, but on TF binding affinity and genomic context.

CLAMP regulates Zygotic Genome Activation in Drosophila embryos

Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. Here we have explored novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, we analyzed transcriptomes of embryos zygotically compromised for either clamp or zelda and found that transcript levels of many zygotically-activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, we propose that CLAMP acts in concert with Zelda to regulate early zygotic transcription.

CLAMP and Zelda function together to promote Drosophila zygotic genome activation

During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, we hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as CLAMP. Here, we demonstrate that Drosophila embryonic development requires maternal CLAMP to: 1) activate zygotic transcription; 2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; 3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor which plays a targeted yet essential role in ZGA.

POU4F3 pioneer activity enables ATOH1 to drive diverse mechanoreceptor differentiation through a feed-forward epigenetic mechanism

During embryonic development, hierarchical cascades of transcription factors interact with lineage-specific chromatin structures to control the sequential steps in the differentiation of specialized cell types. While examples of transcription factor cascades have been well documented, the mechanisms underlying developmental changes in accessibility of cell type–specific enhancers remain poorly understood. Here, we show that the transcriptional “master regulator” ATOH1—which is necessary for the differentiation of two distinct mechanoreceptor cell types, hair cells in the inner ear and Merkel cells of the epidermis—is unable to access much of its target enhancer network in the progenitor populations of either cell type when it first appears, imposing a block to further differentiation. This block is overcome by a feed-forward mechanism in which ATOH1 first stimulates expression of POU4F3, which subsequently acts as a pioneer factor to provide access to closed ATOH1 enhancers, allowing hair cell and Merkel cell differentiation to proceed. Our analysis also indicates the presence of both shared and divergent ATOH1/POU4F3-dependent enhancer networks in hair cells and Merkel cells. These cells share a deep developmental lineage relationship, deriving from their common epidermal origin, and suggesting that this feed-forward mechanism preceded the evolutionary divergence of these very different mechanoreceptive cell types.

LEAFY, a Pioneer Transcription Factor in Plants: A Mini-Review

A subset of eukaryotic transcription factors (TFs) possess the ability to reprogram one cell type into another. Genes important for cellular reprograming are typically located in closed chromatin, which is covered by nucleosomes. Pioneer factors are a special class of TFs that can initially engage their target sites in closed chromatin prior to the engagement with, opening of, or modification of the sites by other factors. Although many pioneer factors are known in animals, a few have been characterized in plants. The TF LEAFY (LFY) acts as a pioneer factor specifying floral fate in Arabidopsis. In response to endogenous and environmental cues, plants produce appropriate floral inducers (florigens). During the vegetative phase, LFY is repressed by the TERMINAL FLOWER 1 (TFL1)–FD complex, which functions as a floral inhibitor, or anti-florigen. The florigen FLOWERING LOCUS T (FT) competes with TFL1 to prevent the binding of the FD TF to the LFY locus. The resulting FT–FD complex functions as a transient stimulus to activate its targets. Once LFY has been transcribed in the appropriate spatiotemporal manner, LFY binds to nucleosomes in closed chromatin regions. Subsequently, LFY opens the chromatin by displacing H1 linker histones and recruiting the SWI/SNF chromatin-remodeling complex. Such local changes permit the binding of other TFs, leading to the expression of the floral meristem identity gene APETALA1. This mini-review describes the latest advances in our understanding of the pioneer TF LFY, providing insight into the establishment of gene expression competence through the shaping of the plant epigenetic landscape.