The PRH (proline-rich homeodomain) [also known as Hex (haematopoietically expressed homeobox)] protein is a transcription factor that functions as an important regulator of vertebrate development and many other processes in the adult including haematopoiesis. The Groucho/TLE (transducin-like enhancer) family of co-repressor proteins also regulate development and modulate the activity of many DNA-binding transcription factors during a range of diverse cellular processes including haematopoiesis. We have shown previously that PRH is a repressor of transcription in haematopoietic cells and that an Eh-1 (Engrailed homology) motif present within the N-terminal transcription repression domain of PRH mediates binding to Groucho/TLE proteins and enables co-repression. In the present study we demonstrate that PRH regulates the nuclear retention of TLE proteins during cellular fractionation. We show that transcriptional repression and the nuclear retention of TLE proteins requires PRH to bind to both TLE and DNA. In addition, we characterize a trans-dominant-negative PRH protein that inhibits wild-type PRH activity by sequestering TLE proteins to specific subnuclear domains. These results demonstrate that transcriptional repression by PRH is dependent on TLE availability and suggest that subnuclear localization of TLE plays an important role in transcriptional repression by PRH.

The PRH/Hhex transcription factor represses multiple genes in the VEGF signalling pathway (VSP) to inhibit myeloid cell survival. Protein kinase CK2 phosphorylates PRH and counteracts the inhibitory effect of this protein on cell survival by blocking the repression of VSP genes. Here we show that the BCR-ABL/Src kinase inhibitor dasatinib decreases PRH phosphorylation and increases PRH-dependent repression of Vegf and Vegfr-1. Moreover in the absence of PRH, dasatinib does not inhibit cell survival as effectively as in PRH expressing cells. Thus the re-establishment of gene control by PRH is in part responsible for the therapeutic effects of dasatinib.

The proline-rich homeodomain protein (PRH/Hex) regulates transcription by binding to specific DNA sequences and regulates mRNA transport by binding to translation initiation factor eIF4E. Protein kinase CK2 plays multiple roles in the regulation of gene expression and cell proliferation. Here, we show that PRH interacts with the beta subunit of CK2 in vitro and in cells and that CK2 phosphorylates PRH. Phosphorylation of PRH by CK2 inhibits the DNA binding activity of this protein and dephosphorylation restores DNA binding indicating that this modification acts as a reversible switch. We show that phosphorylation of the homeodomain is sufficient to block DNA binding and we identify two amino acids within this the domain that are phosphorylated by CK2: S163 and S177. Site-directed mutagenesis demonstrates that mutation of either of these residues to glutamic acid partially mimics phosphorylation but is insufficient to completely block DNA binding whereas an S163E/S177E double mutation severely inhibits DNA binding. Significantly, the S163E and S177E mutations and the S163E/S177E double mutation all inhibit the ability of PRH to regulate transcription in cells. Since these amino acids are conserved between many homeodomain proteins, our results suggest that CK2 may regulate the activity of several homeodomain proteins in this manner.

Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.

Cholangiocarcinoma is a disease with a poor prognosis and increasing incidence and hence there is a pressing unmet clinical need for new adjuvant treatments. Protein kinase CK2 (previously casein kinase II) is a ubiquitously expressed protein kinase that is up-regulated in multiple cancer cell types. The inhibition of CK2 activity using CX-4945 (Silmitasertib) has been proposed as a novel treatment in multiple disease settings including cholangiocarcinoma. Here, we show that CX-4945 inhibited the proliferation of cholangiocarcinoma cell lines in vitro. Moreover, CX-4945 treatment induced the formation of cytosolic vacuoles in cholangiocarcinoma cell lines and other cancer cell lines. The vacuoles contained extracellular fluid and had neutral pH, features characteristic of methuosis. In contrast, simultaneous knockdown of both the α and α' catalytic subunits of protein kinase CK2 using small interfering RNA (siRNA) had little or no effect on the proliferation of cholangiocarcinoma cell lines and failed to induce the vacuole formation. Surprisingly, low doses of CX-4945 increased the invasive properties of cholangiocarcinoma cells due to an upregulation of matrix metallopeptidase 7 (MMP-7), while the knockdown of CK2 inhibited cell invasion. Our data suggest that CX-4945 inhibits cell proliferation and induces cell death via CK2-independent pathways. Moreover, the increase in cell invasion brought about by CX-4945 treatment suggests that this drug might increase tumor invasion in clinical settings.

Protein self-organization is essential for the establishment and maintenance of nuclear architecture and for the regulation of gene expression. We have shown previously that the Proline-Rich Homeodomain protein (PRH/Hex) self-assembles to form oligomeric complexes that bind to arrays of PRH binding sites with high affinity and specificity. We have also shown that many PRH target genes contain suitably spaced arrays of PRH sites that allow this protein to bind and regulate transcription. Here, we use analytical ultracentrifugation and electron microscopy to further characterize PRH oligomers. We use the same techniques to show that PRH oligomers bound to long DNA fragments self-associate to form highly ordered assemblies. Electron microscopy and linear dichroism reveal that PRH oligomers can form protein-DNA fibres and that PRH is able to compact DNA in the absence of other proteins. Finally, we show that DNA compaction is not sufficient for the repression of PRH target genes in cells. We conclude that DNA compaction is a consequence of the binding of large PRH oligomers to arrays of binding sites and that PRH is functionally and structurally related to the Lrp/AsnC family of proteins from bacteria and archaea, a group of proteins formerly thought to be without eukaryotic equivalents.

Protein kinase CK2 promotes cell survival and the activity of this kinase is elevated in several cancers including chronic myeloid leukaemia. We have shown previously that phosphorylation of the Proline-Rich Homeodomain protein (PRH/Hhex) by CK2 inhibits the DNA-binding activity of this transcription factor. Furthermore, PRH represses the transcription of multiple genes encoding components of the VEGF-signalling pathway and thereby influences cell survival. Here we show that the inhibitory effects of PRH on cell proliferation are abrogated by CK2 and that CK2 inhibits the binding of PRH at the Vegfr-1 promoter. Phosphorylation of PRH by CK2 also decreases the nuclear association of PRH and induces its cleavage by the proteasome. Moreover, cleavage of phosphorylated PRH produces a stable truncated cleavage product which we have termed PRHΔC (HhexΔC). PRHΔC acts as a transdominant negative regulator of full-length PRH by sequestering TLE proteins that function as PRH co-repressors. We show that this novel regulatory mechanism results in the alleviation of PRH-mediated repression of Vegfr-1. We suggest that the re-establishment of PRH function through inhibition of CK2 could be of value in treatment of myeloid leukaemias, as well as other tumour types in which PRH is inactivated by phosphorylation.

Cancer cells go through a process known as epithelial-mesenchymal transition (EMT) during which they acquire the ability to migrate and invade extracellular matrix. Some cells also acquire the ability to move across a layer of endothelial cells to enter and exit the bloodstream; intra- and extravasation, respectively. The transcription factor PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) controls cell proliferation and cell migration/invasion in a range of cell types. Our previous work showed that PRH activity is downregulated in prostate cancer cells owing to increased inhibitory PRH phosphorylation and that this increases cell proliferation and invasion. PRH inhibits migration and invasion by prostate and breast epithelial cells in part by activating the transcription of Endoglin, a transforming growth factor β (TGFβ) co-receptor. Here we show that depletion of PRH in immortalised prostate epithelial cells results in increased extravasation in vitro. We show that blood platelets stimulate extravasation of cells with depleted PRH and that inhibition of TGFβ signalling blocks the effects of platelets on these cells. Moreover, TGFβ induces changes characteristic of EMT including decreased E-Cadherin expression and increased Snail expression. We show that in prostate cells PRH regulates multiple genes involved in EMT and TGFβ signalling. However, both platelets and TGFβ increase PRH phosphorylation. In addition, TGFβ increases binding of its effector pSMAD3 to the PRH/HHEX promoter and downregulates PRH protein and mRNA levels. Thus, TGFβ signalling downregulates PRH activity by multiple mechanisms and induces an EMT that facilitates extravasation and sensitises cells to TGFβ.

Quantum biological tunnelling for electron transfer is involved in controlling essential functions for life such as cellular respiration and homoeostasis. Understanding and controlling the quantum effects in biology has the potential to modulate biological functions. Here we merge wireless nano-electrochemical tools with cancer cells for control over electron transfer to trigger cancer cell death. Gold bipolar nanoelectrodes functionalized with redox-active cytochrome c and a redox mediator zinc porphyrin are developed as electric-field-stimulating bio-actuators, termed bio-nanoantennae. We show that a remote electrical input regulates electron transport between these redox molecules, which results in quantum biological tunnelling for electron transfer to trigger apoptosis in patient-derived cancer cells in a selective manner. Transcriptomics data show that the electric-field-induced bio-nanoantenna targets the cancer cells in a unique manner, representing electrically induced control of molecular signalling. The work shows the potential of quantum-based medical diagnostics and treatments.