FoxA1 and glucocorticoid receptor crosstalk via histone H4K16 acetylation at a hormone regulated enhancer.

The forkhead transcription factor FoxA1 participates in many gene regulatory events with steroid hormone receptors, one example being the integrated mouse mammary tumor virus (MMTV) promoter. Its enhancer harbors several FoxA1 binding sites. FoxA1 promotes glucocorticoid receptor (GR)-DNA binding and transcription. Here we analyze the regulatory capacity of GR, FoxA1 and hormone in quantitative terms when reconstituted with the MMTV enhancer in Xenopus oocytes. By titrating each component we demonstrate that FoxA1 is required for hormone induction at low GR concentration and that FoxA1 is a potent enhancer of GR-induced transcription. Conversely, specific DNA binding of FoxA1 at low intranuclear concentration is highly responsive to minute levels of hormone-activated GR while increased FoxA1 concentration results in constitutive binding. When bound to DNA, FoxA1 induces DNase I hypersensitivity, this is accompanied by increased acetylation, specifically at histone H4K16. Expression of FoxA1 deletion mutants demonstrated its DNA binding domain to be sufficient for DNA binding in vivo. The C-terminal and N-terminal domains both contribute to chromatin remodeling while the latter is more important for GR mediated transcription. Thus FoxA1 is primarily responsible for the chromatin presetting while GR supports chromatin presetting at low hormone concentration and transcriptional induction at high hormone concentration.

Characterization of somatic cell nuclear reprogramming by oocytes in which a linker histone is required for pluripotency gene reactivation.

When transplanted into Xenopus oocytes, the nuclei of mammalian somatic cells are reprogrammed to express stem cell genes such as Oct4, Nanog, and Sox2. We now describe an experimental system in which the pluripotency genes Sox2 and Oct4 are repressed in retinoic acid-treated ES cells but are reprogrammed up to 100% within 24 h by injection of nuclei into the germinal vesicle (GV) of growing Xenopus oocytes. The isolation of GVs in nonaqueous medium allows the reprogramming of individual injected nuclei to be seen in real time. Analysis using fluorescence recovery after photobleaching shows that nuclear transfer is associated with an increase in linker histone mobility. A simultaneous loss of somatic H1 linker histone and incorporation of the oocyte-specific linker histone B4 precede transcriptional reprogramming. The loss of H1 is not required for gene reprogramming. We demonstrate both by antibody injection experiments and by dominant negative interference that the incorporation of B4 linker histone is required for pluripotency gene reactivation during nuclear reprogramming. We suggest that the binding of oocyte-specific B4 linker histone to chromatin is a key primary event in the reprogramming of somatic nuclei transplanted to amphibian oocytes.

Self-Assembly of RGD-Functionalized Recombinant Spider Silk Protein into Microspheres in Physiological Buffer and in the Presence of Hyaluronic Acid.

Biomaterials made of self-assembling protein building blocks are widely explored for biomedical applications, for example, as drug carriers, tissue engineering scaffolds, and functionalized coatings. It has previously been shown that a recombinant spider silk protein functionalized with a cell binding motif from fibronectin, FN-4RepCT (FN-silk), self-assembles into fibrillar structures at interfaces, i.e., membranes, fibers, or foams at liquid/air interfaces, and fibrillar coatings at liquid/solid interfaces. Recently, we observed that FN-silk also assembles into microspheres in the bulk of a physiological buffer (PBS) solution. Herein, we investigate the self-assembly process of FN-silk into microspheres in the bulk and how its progression is affected by the presence of hyaluronic acid (HA), both in solution and in a cross-linked HA hydrogel. Moreover, we characterize the size, morphology, mesostructure, and protein secondary structure of the FN-silk microspheres prepared in PBS and HA. Finally, we examine how the FN-silk microspheres can be used to mediate cell adhesion and spreading of human mesenchymal stem cells (hMSCs) during cell culture. These investigations contribute to our fundamental understanding of the self-assembly of silk protein into materials and demonstrate the use of silk microspheres as additives for cell culture applications.

Scalable Production of Monodisperse Bioactive Spider Silk Nanowires.

Elongated protein-based micro- and nanostructures are of great interest for a wide range of biomedical applications, where they can serve as a backbone for surface functionalization and as vehicles for drug delivery. Current production methods for protein constructs lack precise control of either shape and dimensions or render structures fixed to substrates. This work demonstrates production of recombinant spider silk nanowires suspended in solution, starting with liquid bridge induced assembly (LBIA) on a substrate, followed by release using ultrasonication, and concentration by centrifugation. The significance of this method lies in that it provides i) reproducability (standard deviation of length <13% and of diameter <38%), ii) scalability of fabrication, iii) compatibility with autoclavation with retained shape and function, iv) retention of bioactivity, and v) easy functionalization both pre- and post-formation. This work demonstrates how altering the function and nanotopography of a surface by nanowire coating supports the attachment and growth of human mesenchymal stem cells (hMSCs). Cell compatibility is further studied through integration of nanowires during aggregate formation of hMSCs and the breast cancer cell line MCF7. The herein-presented industrial-compatible process enables silk nanowires for use as functionalizing agents in a variety of cell culture applications and medical research.

Mechanism of histone H1-stimulated glucocorticoid receptor DNA binding in vivo.

Xenopus oocytes lack somatic linker histone H1 but contain an oocyte-specific variant, B4. The glucocorticoid receptor (GR) inducible mouse mammary tumor virus (MMTV) promoter was reconstituted in Xenopus oocytes to address the effects of histone H1. The expression of Xenopus H1o [corrected] (H1) via cytoplasmic mRNA injection resulted in H1 incorporation into in vivo assembled chromatin based on (i) the appearance of a chromatosome stop, (ii) the increased nucleosome repeat length (NRL), and (iii) H1-DNA binding assayed by chromatin immunoprecipitation (ChIP). The H1 effect on the NRL was saturable and hence represents H1-binding to a specific site. A subsaturating level of H1 enhanced the hormone-dependent binding of GR to the glucocorticoid response elements (GREs) and the hormone-dependent MMTV transcription while it reduced the access to DNA as revealed by micrococcal nuclease (MNase) analysis. These H1 effects were lost at higher levels of H1. ChIP and MNase analysis revealed a hormone-dependent dissociation of H1 from the activated chromatin domain. The proposed mechanism of H1-induced GR binding is based on two effects: (i) a GR-induced asymmetric distribution of H1 in favor of inactive chromatin and (ii) an H1-induced reduction in DNA access. These effects results in increased concentration of free GR and, hence, in increased GR-GRE binding.

Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter.

Transcription from the mouse mammary tumor virus (MMTV) promoter is induced by the glucocorticoid receptor (GR). This switch was reconstituted in Xenopus oocytes. Previously, we showed that Nuclear Factor 1 (NF1) and Octamer Transcription Factor 1 (Oct1) bind constitutively to the MMTV promoter and thereby induce translational nucleosome positioning representing an intermediary, i.e. preset, state of nucleosome organization. Here we further characterize this NF1 and Oct1 induced preset chromatin in relation to the inactive and the hormone-activated state. The preset chromatin exhibits increased histone acetylation but does not cause dissociation of histone H1 as oppose to the hormone-activated state. Furthermore, upon hormone induction the preset MMTV chromatin displays an enhanced and prolonged GR binding capacity and transcription during an intrinsic and time-dependent silencing of the injected template. The silencing process correlates with a reduced histone acetylation. However, a histone deacetylase inhibitor, trichostatin A (TSA), does not counteract silencing in spite of its distinct stimulation of GR-DNA binding. The latter indicates the importance of histone acetylation to maintain DNA access for inducible factor binding. We discuss how constitutively bound factors such as NF1 and Oct1 may participate in the maintenance of tissue specificity of hormone responsive genes.

Assembly of FN-silk with laminin-521 to integrate hPSCs into a three-dimensional culture for neural differentiation.

Three-dimensional (3D) neural tissue cultures recapitulate the basic concepts during development and disease better than what can be obtained using conventional two-dimensional cultures. Here, we use a recombinant spider silk protein functionalized with a cell binding motif from fibronectin (FN-silk) in combination with a human recombinant laminin 521 (LN-521) to create a fully defined stem cell niche in 3D. A novel method to assemble silk blended with LN-521 together with human pluripotent stem cells (hPSC) is used to create centimeter-sized foams, which upon cultivation develop into 3D cell constructs supported by a microfibrillar network. After initial cell expansion, neural differentiation was induced to form a homogenous layer of continuous neuroectodermal tissue that allows further differentiation into neuronal subtypes. The silk-supported 3D cell constructs could then be detached from the bottom of the well and cultured as floating entities, where cells appeared in distinctive radial organization resembling early neural tube. This shows that the neural progenitors retain their cellular self-organization ability in the FN-silk/LN-521-supported 3D culture. Calcium imaging demonstrated spontaneous activity, which is important for the formation of neuronal networks. Together, the results show that hPSCs integrated into FN-silk/LN-521 foam develop into neural progenitors and that these stay viable during long-term differentiations. FN-silk/LN-521 also supports morphogenesis mimicking the human brain development and can serve as base for engineering of hPSC-derived neural tissue.

Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers.

Tissues are built of cells integrated in an extracellular matrix (ECM) which provides a three-dimensional (3D) microfiber network with specific sites for cell anchorage. By genetic engineering, motifs from the ECM can be functionally fused to recombinant silk proteins. Such a silk protein, FN-silk, which harbours a motif from fibronectin, has the ability to self-assemble into networks of microfibers under physiological-like conditions. Herein we describe a method by which mammalian cells are added to the silk solution before assembly, and thereby get uniformly integrated between the formed microfibers. In the resulting 3D scaffold, the cells are highly proliferative and spread out more efficiently than when encapsulated in a hydrogel. Elongated cells containing filamentous actin and defined focal adhesion points confirm proper cell attachment to the FN-silk. The cells remain viable in culture for at least 90 days. The method is also scalable to macro-sized 3D cultures. Silk microfibers formed in a bundle with integrated cells are both strong and extendable, with mechanical properties similar to that of artery walls. The described method enables differentiation of stem cells in 3D as well as facile co-culture of several different cell types. We show that inclusion of endothelial cells leads to the formation of vessel-like structures throughout the tissue constructs. Hence, silk-assembly in presence of cells constitutes a viable option for 3D culture of cells integrated in a ECM-like network, with potential as base for engineering of functional tissue.

Human embryonic stem cell dispersion in electrospun PCL fiber scaffolds by coating with laminin-521 and E-cadherin-Fc.

Advances in human pluripotent cell cultivation and differentiation protocols have led to production of stem cell-derived progenitors as a promising cell source for replacement therapy. Three-dimensional (3-D) culture is a better mimic of the natural niche for stem cells and is widely used for disease modeling. Here, we describe a nonaggregate culture system of human embryonic stem cells inside electrospun polycaprolactone (PCL) fiber scaffolds combined with defined extracellular proteins naturally occurring in the stem cell niche. PCL fiber scaffolds coated with recombinant human laminin-521 readily supported initial stem cell attachment and growth from a single-cell suspension. The combination of recombinant E-cadherin-Fc and laminin-521 further improved cell dispersion rendering a uniform cell population. Finally, we showed that the cells cultured in E-cadherin-Fc- and laminin-521-coated PCL scaffolds could differentiate into all three germ layers. Importantly, we provided a chemically defined 3-D system in which pluripotent stem cells grown and differentiated avoiding the formation of cell aggregates. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1226-1236, 2018.

Chromatin-mediated restriction of nuclear factor 1/CTF binding in a repressed and hormone-activated promoter in vivo.

Mouse mammary tumor virus (MMTV) promoter-driven transcription is induced by glucocorticoid hormone via binding of the glucocorticoid receptor (GR). The MMTV promoter also harbors a binding site for nuclear factor 1 (NF1). NF1 and GR were expressed in Xenopus oocytes; this revealed GR-NF1 cooperativity both in terms of DNA binding and chromatin remodeling but not transcription. A fraction of NF1 sites were occupied in a hormone-dependent fashion, but a significant and NF1 concentration-dependent fraction were constitutively bound. Activation of the MMTV promoter resulted in an approximately 50-fold increase in the NF1 accessibility for its DNA site. The hormone-dependent component of NF1 binding was dissociated by addition of a GR antagonist; however, the antagonist RU486, which supports partial GR-DNA binding, also maintained partial NF1 binding. Hence GR-NF1 cooperativity is independent of agonist-driven chromatin remodeling. NF1 induced the formation of a micrococcal-nuclease-resistant protein-DNA complex containing the DNA segment from -185 to -55, the MMTV enhanceosome. Coexpression of NF1 and Oct1 resulted in a significant stimulation of hormone-induced MMTV transcription and also in increased basal transcription. We propose that hormone-independent NF1 binding may be involved in maintaining transcriptional competence and establishment of tissue-specific gene networks.

Apolipoprotein C-I mediates Wnt/Ctnnb1 signaling during neural border formation and is required for neural crest development.

In vertebrates, the neural crest and placodes originate in the neural border, which is located between the neural plate and epidermal ectoderm. The neural crest and placodes give rise to a vast array of cell types. Formation of neural crest is a multi-step process, in which Wnt signals are used reiteratively, but it is currently not clear if a Wnt signal is required for neural border formation. Here, we have identified apolipoprotein C-I (apoc1) in a screen for genes regulated by Wnt/Ctnnb1 signaling in late blastula stage Xenopus tropicalis embryos. We show that Xenopus laevis apoc1 encodes a small, secreted protein, and is induced by Wnt/Ctnnb1 signaling. Depletion of Apoc1 protein results in a neural border formation defect and loss of border fates, including neural crest cells. However, unlike another Wnt/Ctnnb1 target, gbx2.2, apoc1 is not required for patterning of the neural border. We further show that gbx2.2 and apoc1 are independently regulated by Wnt signaling. Our results thus suggest that Wnt regulates border formation and patterning by distinct genetic mechanisms.

HIRA dependent H3.3 deposition is required for transcriptional reprogramming following nuclear transfer to Xenopus oocytes.

BACKGROUND: Nuclear reprogramming is potentially important as a route to cell replacement and drug discovery, but little is known about its mechanism. Nuclear transfer to eggs and oocytes attempts to identify the mechanism of this direct route towards reprogramming by natural components. Here we analyze how the reprogramming of nuclei transplanted to Xenopus oocytes exploits the incorporation of the histone variant H3.3. RESULTS: After nuclear transplantation, oocyte-derived H3.3 but not H3.2, is deposited on several regions of the genome including rDNA, major satellite repeats, and the regulatory regions of Oct4. This major H3.3 deposition occurs in absence of DNA replication, and is HIRA-and transcription-dependent. It is necessary for the shift from a somatic- to an oocyte-type of transcription after nuclear transfer. CONCLUSIONS: This study demonstrates that the incorporation of histone H3.3 is an early and necessary step in the direct reprogramming of somatic cell nuclei by oocyte. It suggests that the incorporation of histone H3.3 is necessary during global changes in transcription that accompany changes in cell fate.

FoxA1 binding directs chromatin structure and the functional response of a glucocorticoid receptor-regulated promoter.

Reconstitution of the glucocorticoid receptor (GR)-regulated mouse mammary tumor virus (MMTV) promoter in Xenopus oocytes was used to monitor the effects of different transcription factor contexts. Three constitutively binding factors, nuclear factor 1 (NF1), octamer transcription factor 1 (Oct1), and the Forkhead box A1 (FoxA1), were shown to act in concert, to direct the chromatin structure, and to enhance the GR response. FoxA1 has a dominant effect in the absence of hormone and induces a cluster of DNase I-hypersensitive sites in the segment comprising bp -400 to +25. This FoxA1-mediated chromatin remodeling does not induce MMTV transcription, as opposed to that of the GR. However, the robust FoxA1-dependent chromatin opening has the following drastic functional consequences on the hormone regulation: (i) GR-DNA binding is facilitated, as revealed by dimethyl sulfate in vivo footprinting, leading to increased hormone-induced transcription, and (ii) the GR antagonist RU486 is converted into a partial agonist in the presence of FoxA1 via ligand-independent GR activation. We conclude that FoxA1 mediates a preset chromatin structure and directs a context-specific response of a nuclear receptor. Furthermore, the alternative nucleosome arrangement induced by GR and FoxA1 implies this to be determined by constitutive binding of transcription factors rather than by the DNA sequence itself.

Nuclear factor 1 and octamer transcription factor 1 binding preset the chromatin structure of the mouse mammary tumor virus promoter for hormone induction.

When the mouse mammary tumor virus (MMTV) is integrated into the genome of a mammalian cell, its long terminal repeat (LTR) harbors six specifically positioned nucleosomes. Transcription from the MMTV promoter is regulated by the glucocorticoid hormone via the glucocorticoid receptor (GR). The mechanism of the apparently constitutive nucleosome arrangement has remained unclear. Previous in vitro reconstitution of nucleosome(s) on small segments of the MMTV LTR suggested that the DNA sequence was decisive for the nucleosome arrangement. However, microinjection of MMTV LTR DNA in Xenopus oocytes rendered randomly distributed nucleosomes. This indicated that oocytes lack factor(s) that induces nucleosome positioning at the MMTV LTR in other cells. Here we demonstrate that specific and concomitant binding of nuclear factor 1 (NF1) and octamer factor 1 (Oct1) to their cognate sites within the MMTV promoter induce a partial nucleosome positioning that is an intermediary state between the randomly organized inactive promoter and the hormone and GR-activated promoter containing distinctly positioned nucleosomes. Oct1 and NF1 reciprocally facilitate each other's binding to the MMTV LTR in vivo. The NF1 and Oct1 binding also facilitate hormone-dependent GR-DNA interaction and result in a faster and stronger hormone response. Since NF1 and Oct1 generate an intermediary state of nucleosome positioning and enhance the hormone-induced response, we refer to this as a preset chromatin structure. We propose that this state of NF1 and Oct1-induced chromatin presetting mimics the early step(s) of chromatin remodeling involved in tissue-specific gene expression.

Trichostatin A reduces hormone-induced transcription of the MMTV promoter and has pleiotropic effects on its chromatin structure.

The deacetylase inhibitor trichostatin A (TSA) has long been used to study the relationship between gene transcription and the acetylation status of chromatin. We have used Xenopus laevis oocytes to study the effects of TSA on glucocorticoid receptor (GR)-dependent transcription and we have related these effects to changes in the chromatin structure of a reporter mouse mammary tumor virus (MMTV) promoter. We show that TSA induces a low level of constitutive transcription. This correlates with a change of acetylation pattern and a more open chromatin structure over the MMTV chromatin, and with specific acetylation and remodeling events in the promoter region. Specifically, a repositioning of initially randomly positioned nucleosomes along the distal MMTV long terminal repeat is seen. This nucleosome rearrangement is similar to the translational nucleosome positioning that occurs upon hormone activation. We also note a reduced hormone response in the presence of TSA. TSA effects have for a long time been associated with transcriptional activation and chromatin opening through inhibition of the deacetylation of histones. However, our results and those of others show that TSA-induced changes in expression and chromatin structure can be quite different in different promoter contexts and, thus, the effects of TSA are more complex than previously believed.