Sendai virus is robust and consistent in delivering genes into human pancreatic cancer cells.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly intratumorally heterogeneous disease that includes several subtypes and is highly plastic. Effective gene delivery to all PDAC cells is essential for modulating gene expression and identifying potential gene-based therapeutic targets in PDAC. Most current gene delivery systems for pancreatic cells are optimized for islet or acinar cells. Lentiviral vectors are the current main gene delivery vectors for PDAC, but their transduction efficiencies vary depending on pancreatic cell type, and are especially poor for the classical subtype of PDAC cells from both primary tumors and cell lines. Methods: We systemically compare transduction efficiencies of glycoprotein G of vesicular stomatitis virus (VSV-G)-pseudotyped lentiviral and Sendai viral vectors in human normal pancreatic ductal and PDAC cells. Results: We find that the Sendai viral vector gives the most robust gene delivery efficiency regardless of PDAC cell type. Therefore, we propose using Sendai viral vectors to transduce ectopic genes into PDAC cells.

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

Repurposing the lineage-determining transcription factor Atoh1 without redistributing its genomic binding sites.

Although the lineage-determining ability of transcription factors is often modulated according to cellular context, the mechanisms by which such switching occurs are not well known. Using a transcriptional programming model, we found that Atoh1 is repurposed from a neuronal to an inner ear hair cell (HC) determinant by the combined activities of Gfi1 and Pou4f3. In this process, Atoh1 maintains its regulation of neuronal genes but gains ability to regulate HC genes. Pou4f3 enables Atoh1 access to genomic locations controlling the expression of sensory (including HC) genes, but Atoh1 + Pou4f3 are not sufficient for HC differentiation. Gfi1 is key to the Atoh1-induced lineage switch, but surprisingly does not alter Atoh1's binding profile. Gfi1 acts in two divergent ways. It represses the induction by Atoh1 of genes that antagonise HC differentiation, a function in keeping with its well-known repressor role in haematopoiesis. Remarkably, we find that Gfi1 also acts as a co-activator: it binds directly to Atoh1 at existing target genes to enhance its activity. These findings highlight the diversity of mechanisms by which one TF can redirect the activity of another to enable combinatorial control of cell identity.

Dissecting OCT4 defines the role of nucleosome binding in pluripotency.

Pioneer transcription factors such as OCT4 can target silent genes embedded in nucleosome-dense regions. How nucleosome interaction enables transcription factors to target chromatin and determine cell identity remains elusive. Here, we systematically dissect OCT4 to show that nucleosome binding is encoded within the DNA-binding domain and yet can be uncoupled from free-DNA binding. Furthermore, accelerating the binding kinetics of OCT4 to DNA enhances nucleosome binding. In cells, uncoupling nucleosome binding diminishes the ability of OCT4 to individually access closed chromatin, while more dynamic nucleosome binding results in expansive genome scanning within closed chromatin. However, both uncoupling and enhancing nucleosome binding are detrimental to inducing pluripotency from differentiated cells. Remarkably, stable interactions between OCT4 and nucleosomes are continuously required for maintaining the accessibility of pluripotency enhancers in stem cells. Our findings reveal how the affinity and residence time of OCT4-nucleosome complexes modulate chromatin accessibility during cell fate changes and maintenance.

miR-130b and miR-128a are essential lineage-specific codrivers of t(4;11) MLL-AF4 acute leukemia.

t(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in the infant and pediatric population, yet we have little information on the molecular mechanisms responsible for disease progression. This impairs the development of therapeutic regimens that can address the aggressive phenotype and lineage plasticity of MLL-AF4-driven leukemogenesis. This study highlights novel mechanisms of disease development by focusing on 2 microRNAs (miRNAs) upregulated in leukemic blasts from primary patient samples: miR-130b and miR-128a. We show that miR-130b and miR-128a are downstream targets of MLL-AF4 and can individually drive the transition from a pre-leukemic stage to an acute leukemia in an entirely murine Mll-AF4 in vivo model. They are also required to maintain the disease phenotype. Interestingly, miR-130b overexpression led to a mixed/B-cell precursor (BCP)/myeloid leukemia, propagated by the lymphoid-primed multipotent progenitor (LMPP) population, whereas miR-128a overexpression resulted in a pro-B acute lymphoblastic leukemia (ALL), maintained by a highly expanded Il7r+c-Kit+ blast population. Molecular and phenotypic changes induced by these two miRNAs fully recapitulate the human disease, including central nervous system infiltration and activation of an MLL-AF4 expression signature. Furthermore, we identified 2 downstream targets of these miRNAs, NR2F6 and SGMS1, which in extensive validation studies are confirmed as novel tumor suppressors of MLL-AF4+ leukemia. Our integrative approach thus provides a platform for the identification of essential co-drivers of MLL-rearranged leukemias, in which the preleukemia to leukemia transition and lineage plasticity can be dissected and new therapeutic approaches can be tested.

Post-translational modification of SOX family proteins: Key biochemical targets in cancer?

Sox proteins are a family of lineage-associated transcription factors. They regulate expression of genes involved in control of self-renewal and multipotency in both developmental and adult stem cells. Overexpression of Sox proteins is frequently observed in many different human cancers. Despite their importance as therapeutic targets, Sox proteins are difficult to 'drug' using structure-based design. However, Sox protein localisation, activity and interaction partners are regulated by a plethora of post-translational modifications (PTMs), such as: phosphorylation, acetylation, sumoylation, methylation, and ubiquitylation. Here we review the various reported post-translational modifications of Sox proteins and their potential functional importance in guiding cell fate processes. The enzymes that regulate these PTMs could be useful targets for anti-cancer drug discovery.

Ramified rolling circle amplification for synthesis of nucleosomal DNA sequences.

Nucleosomes are a crucial platform for the recruitment and assembly of protein complexes that process the DNA. Mechanistic and structural in vitro studies typically rely on recombinant nucleosomes that are reconstituted using artificial, strong-positioning DNA sequences. To facilitate such studies on native, genomic nucleosomes, there is a need for methods to produce any desired DNA sequence in an efficient manner. The current methods either do not offer much flexibility in choice of sequence or are less efficient in yield and labor. Here, we show that ramified rolling circle amplification (RCA) can be used to produce milligram amounts of a genomic nucleosomal DNA fragment in a scalable, one-pot reaction overnight. The protocol is efficient and flexible in choice of DNA sequence. It yields 10-fold more product than PCR, and rivals production using plasmids. We demonstrate the approach by producing the genomic DNA from the human LIN28B locus and show that it forms functional nucleosomes capable of binding pioneer transcription factor Oct4.

Comparison of reprogramming factor targets reveals both species-specific and conserved mechanisms in early iPSC reprogramming.

BACKGROUND: Both human and mouse fibroblasts can be reprogrammed to pluripotency with Oct4, Sox2, Klf4, and c-Myc (OSKM) transcription factors. While both systems generate pluripotency, human reprogramming takes considerably longer than mouse. RESULTS: To assess additional similarities and differences, we sought to compare the binding of the reprogramming factors between the two systems. In human fibroblasts, the OSK factors initially target many more closed chromatin sites compared to mouse. Despite this difference, the intra- and intergenic distribution of target sites, target genes, primary binding motifs, and combinatorial binding patterns between the reprogramming factors are largely shared. However, while many OSKM binding events in early mouse cell reprogramming occur in syntenic regions, only a limited number is conserved in human. CONCLUSIONS: Our findings suggest similar general effects of OSKM binding across these two species, even though the detailed regulatory networks have diverged significantly.

Cycling through developmental decisions: how cell cycle dynamics control pluripotency, differentiation and reprogramming.

A strong connection exists between the cell cycle and mechanisms required for executing cell fate decisions in a wide-range of developmental contexts. Terminal differentiation is often associated with cell cycle exit, whereas cell fate switches are frequently linked to cell cycle transitions in dividing cells. These phenomena have been investigated in the context of reprogramming, differentiation and trans-differentiation but the underpinning molecular mechanisms remain unclear. Most progress to address the connection between cell fate and the cell cycle has been made in pluripotent stem cells, in which the transition through mitosis and G1 phase is crucial for establishing a window of opportunity for pluripotency exit and the initiation of differentiation. This Review will summarize recent developments in this area and place them in a broader context that has implications for a wide range of developmental scenarios.

Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming.

Pioneer transcription factors (TFs) access silent chromatin and initiate cell-fate changes, using diverse types of DNA binding domains (DBDs). FoxA, the paradigm pioneer TF, has a winged helix DBD that resembles linker histone and thereby binds its target sites on nucleosomes and in compacted chromatin. Herein, we compare the nucleosome and chromatin targeting activities of Oct4 (POU DBD), Sox2 (HMG box DBD), Klf4 (zinc finger DBD), and c-Myc (bHLH DBD), which together reprogram somatic cells to pluripotency. Purified Oct4, Sox2, and Klf4 proteins can bind nucleosomes in vitro, and in vivo they preferentially target silent sites enriched for nucleosomes. Pioneer activity relates simply to the ability of a given DBD to target partial motifs displayed on the nucleosome surface. Such partial motif recognition can occur by coordinate binding between factors. Our findings provide insight into how pioneer factors can target naive chromatin sites.

Mechanisms for enhancing cellular reprogramming.

During development, the genome adopts specific chromatin states to establish and maintain functionally distinct cell types in a well-controlled environment. A select group of transcription factors have the ability to drive the transition of the genome from a pluripotent to a more specialized chromatin state. The same set of factors can be used as reprogramming factors to reset the already established chromatin state back to pluripotency or directly to an alternative cell type. However, under the suboptimal reprogramming conditions, these factors fall short in guiding the majority of cells to their new fate. In this review, we visit the recent findings addressing the manipulation of chromatin structure to enhance the performance of transcription factors in reprogramming. The main emphasis is on the mechanisms underlying the conversion of somatic cells to pluripotency using OSKM. This review is intended to highlight the windows of opportunities for developing mechanistically based approaches to replace the phenotypically guided methods currently employed in reprogramming, in an attempt to move the field of cell conversion towards using next generation technologies.

Understanding impediments to cellular conversion to pluripotency by assessing the earliest events in ectopic transcription factor binding to the genome.

In all known cases of transcription factor (TF)-based reprogramming, the process is relatively slow and inefficient. For example, it takes about a month for the ectopic expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM) to fully reprogram human somatic cells to pluripotency. Furthermore, recent studies indicate that there is an initial stochastic phase, whereby random cells in the converting population begin to express a few genes of the new fate, followed by a so-called deterministic phase, whereby activation of a network for the new fate leads to homogeneous changes in gene expression patterns within a subset of the cell population. We recently mapped the initial interactions between OSKM factors and the human genome during the first 48 h of human fibroblast conversion to pluripotency. Unlike that reported in ES and iPS cells, distal enhancer sites in closed chromatin dominate the initial O, S, K and M binding distribution, showing that promoter occupancy is a later event in reprogramming. O, S and K act as pioneer factors for c-Myc, and c-Myc enhances the engagement of O, S and K. Despite the ability of OSKM to access closed chromatin, megabase-scale chromatin regions in somatic cells, referred to as "differentially bound regions" (DBRs), are remarkably refractory to OSKM binding at 48 h, though they become bound in pluripotent cells. These DBRs are highly enriched for the repressive H3K9me3 mark and span genes at the top of the deterministic hierarchy. Transient knockdown of the relevant chromatin modifiers allows access of OSKM to DBRs and a more rapid and efficient conversion to pluripotency. Thus, overcoming DBR barriers helps explain the conversion from a stochastic to a deterministic phase of transcription factor-mediated cell type conversion.

Facilitators and impediments of the pluripotency reprogramming factors' initial engagement with the genome.

The ectopic expression of transcription factors can reprogram cell fate, yet it is unknown how the initial binding of factors to the genome relates functionally to the binding seen in the minority of cells that become reprogrammed. We report a map of Oct4, Sox2, Klf4, and c-Myc (O, S, K, and M) on the human genome during the first 48 hr of reprogramming fibroblasts to pluripotency. Three striking aspects of the initial chromatin binding events include an unexpected role for c-Myc in facilitating OSK chromatin engagement, the primacy of O, S, and K as pioneer factors at enhancers of genes that promote reprogramming, and megabase-scale chromatin domains spanned by H3K9me3, including many genes required for pluripotency, that prevent initial OSKM binding and impede the efficiency of reprogramming. We find diverse aspects of initial factor binding that must be overcome in the minority of cells that become reprogrammed.

DNA compaction by the higher-order assembly of PRH/Hex homeodomain protein oligomers.

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.

CK2 phosphorylation of the PRH/Hex homeodomain functions as a reversible switch for DNA binding.

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.

PRH/Hex: an oligomeric transcription factor and multifunctional regulator of cell fate.

The PRH (proline-rich homeodomain) [also known as Hex (haematopoietically expressed homeobox)] protein is a critical regulator of vertebrate development. PRH is able to regulate cell proliferation and differentiation and is required for the formation of the vertebrate body axis, the haematopoietic and vascular systems and the formation of many vital organs. PRH is a DNA-binding protein that can repress and activate the transcription of its target genes using multiple mechanisms. In addition, PRH can regulate the nuclear transport of specific mRNAs making PRH a member of a select group of proteins that control gene expression at the transcriptional and translational levels. Recent biophysical analysis of the PRH protein has shown that it forms homo-oligomeric complexes in vivo and in vitro and that the proline-rich region of PRH forms a novel dimerization interface. Here we will review the current literature on PRH and discuss the complex web of interactions centred on this multifunctional protein.

Oligomerisation of the developmental regulator proline rich homeodomain (PRH/Hex) is mediated by a novel proline-rich dimerisation domain.

Homeodomain proteins regulate multiple developmental pathways by altering gene expression temporally and in a tissue-specific fashion. The Proline Rich Homeodomain protein (PRH/Hex) is a transcription factor and an essential regulator of embryonic development and haematopoiesis. Recent discoveries have implicated self-association as an important feature of transcription factor function. Here, we show using a variety of techniques including gel-filtration, analytical ultracentrifugation, electron microscopy and in vitro cross-linking, that purified recombinant PRH is oligomeric and we use in vivo cross-linking to confirm that this protein exists as oligomers in cells. This is the first demonstration that a homeodomain protein can oligomerise in vivo. Consistent with these findings we show that a fraction of endogenous and exogenous PRH appears as discrete foci within the nucleus and at the nuclear periphery. The N-terminal domain of PRH is involved in the regulation of cell proliferation and transcriptional repression and can make multiple protein-protein interactions. We show that this region of PRH contains a novel proline-rich dimerisation domain that mediates oligomerisation. We propose a model that explains how PRH forms oligomers and we discuss how these oligomers might control transcription.

Purification and characterisation of the PRH homeodomain: Removal of the N-terminal domain of PRH increases the PRH homeodomain-DNA interaction.

The Proline-Rich Homeodomain (PRH) protein is a regulator of transcription and translation and plays a key role in the control of cell proliferation and cell differentiation. PRH contains an N-terminal proline-rich domain that can repress transcription when expressed as a fusion protein with an unrelated DNA binding domain, a central homeodomain that binds to specific DNA sequences and an acidic C-terminal domain of no known function. In order to investigate the structure and functions of PRH we have purified the full-length protein and truncated proteins corresponding to different domains of PRH fused to histidine tags. Here we compare the effects of elution conditions and column volume on protein purification and we investigate the DNA binding activity of these proteins. We show that the PRH homeodomain co-purifies with nucleic acids even after nuclease treatment and that a high salt-wash is required to remove bound nucleic acids. In contrast with the full-length PRH protein, the PRH homeodomain binds to DNA with high affinity. We show that a truncated protein comprising the homeodomain and C-terminal domain also binds to DNA with high affinity and we conclude that the N-terminal domain of PRH inhibits the homeodomain-DNA interaction.