Transcriptional Coactivator BOB1 (OBF1, OCA-B) Modulates the Specificity of DNA Recognition by the POU-Domain Factors OCT1 and OCT2 in a Monomeric Configuration.

BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA-SELEX-Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1-an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies.

Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

Structure and Functions of HMGB2 Protein.

High-Mobility Group (HMG) chromosomal proteins are the most numerous nuclear non-histone proteins. HMGB domain proteins are the most abundant and well-studied HMG proteins. They are involved in variety of biological processes. HMGB1 and HMGB2 were the first members of HMGB-family to be discovered and are found in all studied eukaryotes. Despite the high degree of homology, HMGB1 and HMGB2 proteins differ from each other both in structure and functions. In contrast to HMGB2, there is a large pool of works devoted to the HMGB1 protein whose structure-function properties have been described in detail in our previous review in 2020. In this review, we attempted to bring together diverse data about the structure and functions of the HMGB2 protein. The review also describes post-translational modifications of the HMGB2 protein and its role in the development of a number of diseases. Particular attention is paid to its interaction with various targets, including DNA and protein partners. The influence of the level of HMGB2 expression on various processes associated with cell differentiation and aging and its ability to mediate the differentiation of embryonic and adult stem cells are also discussed.

Tryptophan Hydroxylase-2-Mediated Serotonin Biosynthesis Suppresses Cell Reprogramming into Pluripotent State.

The monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has important functions both in the neural system and during embryonic development in mammals. In this study, we set out to investigate whether and how endogenous serotonin affects reprogramming to pluripotency. As serotonin is synthesized from tryptophan by the rate limiting enzymes tryptophan hydroxylase-1 and -2 (TPH1 and TPH2), we have assessed the reprogramming of TPH1- and/or TPH2-deficient mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs). The reprogramming of the double mutant MEFs showed a dramatic increase in the efficiency of iPSC generation. In contrast, ectopic expression of TPH2 alone or in conjunction with TPH1 reverted the rate of reprogramming of the double mutant MEFs to the wild-type level and besides, TPH2 overexpression significantly suppressed reprogramming of wild-type MEFs. Our data thus suggest a negative role of serotonin biosynthesis in the reprogramming of somatic cells to a pluripotent state.

Structural Characteristics of High-Mobility Group Proteins HMGB1 and HMGB2 and Their Interaction with DNA.

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners.

Metabolic control of induced pluripotency.

Pluripotent stem cells of the mammalian epiblast and their cultured counterparts-embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs)-have the capacity to differentiate in all cell types of adult organisms. An artificial process of reactivation of the pluripotency program in terminally differentiated cells was established in 2006, which allowed for the generation of induced pluripotent stem cells (iPSCs). This iPSC technology has become an invaluable tool in investigating the molecular mechanisms of human diseases and therapeutic drug development, and it also holds tremendous promise for iPSC applications in regenerative medicine. Since the process of induced reprogramming of differentiated cells to a pluripotent state was discovered, many questions about the molecular mechanisms involved in this process have been clarified. Studies conducted over the past 2 decades have established that metabolic pathways and retrograde mitochondrial signals are involved in the regulation of various aspects of stem cell biology, including differentiation, pluripotency acquisition, and maintenance. During the reprogramming process, cells undergo major transformations, progressing through three distinct stages that are regulated by different signaling pathways, transcription factor networks, and inputs from metabolic pathways. Among the main metabolic features of this process, representing a switch from the dominance of oxidative phosphorylation to aerobic glycolysis and anabolic processes, are many critical stage-specific metabolic signals that control the path of differentiated cells toward a pluripotent state. In this review, we discuss the achievements in the current understanding of the molecular mechanisms of processes controlled by metabolic pathways, and vice versa, during the reprogramming process.

Dual Mode of Mitochondrial ROS Action during Reprogramming to Pluripotency.

Essential changes in cell metabolism and redox signaling occur during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this paper, using genetic and pharmacological approaches, we have investigated the role of electron transport chain (ETC) complex-I (CI) of mitochondria in the process of cell reprogramming to pluripotency. Knockdown of NADH-ubiquinone oxidoreductase core subunits S1 (Ndufs1) or subunit B10 (Ndufb10) of the CI or inhibition of this complex with rotenone during mouse embryonic fibroblast (MEF) reprogramming resulted in a significantly decreased number of induced pluripotent stem cells (iPSCs). We have found that mitochondria and ROS levels due course of the reprogramming tightly correlate with each other, both reaching peak by day 3 and significantly declining by day 10 of the process. The transient augmentation of mitochondrial reactive oxygen species (ROS) could be attenuated by antioxidant treatment, which ameliorated overall reprogramming. However, ROS scavenging after day 3 or during the entire course of reprogramming was suppressive for iPSC formation. The ROS scavenging within the CI-deficient iPSC-precursors did not improve, but further suppressed the reprogramming. Our data therefore point to distinct modes of mitochondrial ROS action during the early versus mid and late stages of reprogramming. The data further substantiate the paradigm that balanced levels of oxidative phosphorylation have to be maintained on the route to pluripotency.

The functional diversity of the POUV-class proteins across vertebrates.

POUV is a relatively newly emerged class of POU transcription factors present in jawed vertebrates (Gnathostomata). The function of POUV-class proteins is inextricably linked to zygotic genome activation (ZGA). A large body of evidence now extends the role of these proteins to subsequent developmental stages. While some functions resemble those of other POU-class proteins and are related to neuroectoderm development, others have emerged de novo. The most notable of the latter functions is pluripotency control by Oct4 in mammals. In this review, we focus on these de novo functions in the best-studied species harbouring POUV proteins-zebrafish, Xenopus (anamniotes) and mammals (amniotes). Despite the broad diversity of their biological functions in vertebrates, POUV proteins exert a common feature related to their role in safeguarding the undifferentiated state of cells. Here we summarize numerous pieces of evidence for these specific functions of the POUV-class proteins and recap available loss-of-function data.

Building Blocks of Artificial CRISPR-Based Systems beyond Nucleases.

Tools developed in the fields of genome engineering, precise gene regulation, and synthetic gene networks have an increasing number of applications. When shared with the scientific community, these tools can be used to further unlock the potential of precision medicine and tissue engineering. A large number of different genetic elements, as well as modifications, have been used to create many different systems and to validate some technical concepts. New studies have tended to optimize or improve existing elements or approaches to create complex synthetic systems, especially those based on the relatively new CRISPR technology. In order to maximize the output of newly developed approaches and to move from proof-of-principle experiments to applications in regenerative medicine, it is important to navigate efficiently through the vast number of genetic elements to choose those most suitable for specific needs. In this review, we have collected information regarding the main genetic elements and their modifications, which can be useful in different synthetic systems with an emphasis of those based on CRISPR technology. We have indicated the most suitable elements and approaches to choose or combine in planning experiments, while providing their deeper understanding, and have also stated some pitfalls that should be avoided.

Key features of the POU transcription factor Oct4 from an evolutionary perspective.

Oct4, a class V POU-domain protein that is encoded by the Pou5f1 gene, is thought to be a key transcription factor in the early development of mammals. This transcription factor plays indispensable roles in pluripotent stem cells as well as in the acquisition of pluripotency during somatic cell reprogramming. Oct4 has also been shown to play a role as a pioneer transcription factor during zygotic genome activation (ZGA) from zebrafish to human. However, during the past decade, several studies have brought these conclusions into question. It was clearly shown that the first steps in mouse development are not affected by the loss of Oct4. Subsequently, the role of Oct4 as a genome activator was brought into doubt. It was also found that the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) could proceed without Oct4. In this review, we summarize recent findings, reassess the role of Oct4 in reprogramming and ZGA, and point to structural features that may underlie this role. We speculate that pluripotent stem cells resemble neural stem cells more closely than previously thought. Oct4 orthologs within the POUV class hold key roles in genome activation during early development of species with late ZGA. However, in Placentalia, eutherian-specific proteins such as Dux overtake Oct4 in ZGA and endow them with the formation of an evolutionary new tissue-the placenta.

Physiological Signaling Functions of Reactive Oxygen Species in Stem Cells: From Flies to Man.

Reactive oxygen species (ROS), superoxide anion and hydrogen peroxide, are generated as byproducts of oxidative phosphorylation in the mitochondria or via cell signaling-induced NADPH oxidases in the cytosol. In the recent two decades, a plethora of studies established that elevated ROS levels generated by oxidative eustress are crucial physiological mediators of many cellular and developmental processes. In this review, we discuss the mechanisms of ROS generation and regulation, current understanding of ROS functions in the maintenance of adult and embryonic stem cells, as well as in the process of cell reprogramming to a pluripotent state. Recently discovered cell-non-autonomous ROS functions mediated by growth factors are crucial for controlling cell differentiation and cellular immune response in Drosophila. Importantly, many physiological functions of ROS discovered in Drosophila may allow for deciphering and understanding analogous processes in human, which could potentially lead to the development of novel therapeutic approaches in ROS-associated diseases treatment.

Pluripotent stem cell-based gene therapy approach: human de novo synthesized chromosomes.

A novel approach in gene therapy was introduced 20 years ago since artificial non-integrative chromosome-based vectors containing gene loci size inserts were engineered. To date, different human artificial chromosomes (HAC) were generated with the use of de novo construction or "top-down" engineering approaches. The HAC-based therapeutic approach includes ex vivo gene transferring and correction of pluripotent stem cells (PSCs) or highly proliferative modified stem cells. The current progress in the technology of induced PSCs, integrating with the HAC technology, resulted in a novel platform of stem cell-based tissue replacement therapy for the treatment of genetic disease. Nowadays, the sophisticated and laborious HAC technology has significantly improved and is now closer to clinical studies. In here, we reviewed the achievements in the technology of de novo synthesized HACs for a chromosome transfer for developing gene therapy tissue replacement models of monogenic human diseases.

Post-Translational Modifications of Extracellular Proteasome.

The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits ß2 and ß3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of ß3. Interestingly, succinylation of ß6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the ß3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.

Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice.

Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/- mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/- iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector-the alphoidtetO-HAC.

Human artificial chromosomes for pluripotent stem cell-based tissue replacement therapy.

The gene therapy approach aiming at curing various human diseases began to develop as a technology from early eighties of the last century. To date the delivery of therapeutic genes are mainly mediated by virus-based, predominantly, non-integrated virus vectors. These gene delivery approaches have several fundamental limitations on the way of efficient deployment in clinical gene therapy. A totally different approach was suggested about 20 years ago when episomal non-integrative artificial chromosome-based vectors featuring large size inserts (even native gene loci) advanced to the stage. Since then numerous human artificial chromosome (HAC) vectors were developed by both de novo synthesis and top-down engineering technology. This approach so far is limited to ex vivo gene transfer and correction within highly proliferative or reversibly immortalized precursor stem cells or pluripotent stem cells. Recent breakthrough in generation of induced pluripotent stem cells and embryonic stem cell manipulation give the additional pivotal stimuli to integrate it with the HAC technology and to develop thereby novel approaches to replacement therapies of human genetic diseases. The HAC technology is complex and time consuming while nowadays it has significantly advanced and become notably closer to medical applications. In this review we discuss current advancements in the HAC technology, in particular, in terms of improvement of chromosome transfer method and achievements in developing mouse-based gene therapy tissue replacement models for several monogenic human diseases. The main progress has been done in elaboration of top-down type HAC technology in modeling and preclinical studies of gene therapy treatment for Duchenne muscular dystrophy (DMD) disease.

Genetic tool for fate mapping of Oct4 (Pou5f1)-expressing cells and their progeny past the pluripotency stage.

BACKGROUND: Methods based on site-specific recombinases are widely used in studying gene activities in vivo and in vitro. In these studies, constitutively active or inducible variants of these recombinases are expressed under the control of either lineage-specific or ubiquitous promoters. However, there is a need for more advanced schemes that combine these features with possibilities to choose a time point from which lineage tracing starts in an autonomous fashion. For example, the key mammalian germline gatekeeper gene Oct4 (Pou5f1) is expressed in the peri-implantation epiblast which gives rise to all cells within embryos. Thus the above techniques are hardly applicable to Oct4 tracing past the epiblast stage, and the establishment of genetic tools addressing such a limitation is a highly relevant pursuit. METHODS: The CRISPR/Cas9 tool was used to manipulate the genome of mouse embryonic stem cells (ESCs), and various cell culture technics-to maintain and differentiate ESCs to neural cell, lentivirus-based reprogramming technique-to generate induced pluripotent stem cells (iPSCs). RESULTS: In this paper, we have developed a two-component genetic system (referred to as O4S) that allows tracing Oct4 gene activity past the epiblast stage of development. The first component represents a knock-in of an ubiquitous promoter-driven inducible Cre, serving as a stop signal for downstream tdTomato. Upon activation of Cre activity with 4-hydroxytamoxifen (4-OHT) at any given time point, the recombinase excises a stop signal and poses the second component of the system-the FlpO recombinase, knocked into 3'UTR of Oct4, to be expressed upon activation of the latter gene. Oct4-driven expression of FlpO, in turn, triggers the tdTomato expression and thus, permanently marks Oct4+ cells and their progeny. We have validated the O4S system in cultured ESCs and shown that it is capable, for example, to timely capture an activation of Oct4 gene during the reprogramming of somatic cells into iPSCs. CONCLUSIONS: The developed O4S system can be used to detect Oct4 activation event, both permanent and transient, in somatic cell types outside the germline. The approach can be equally adjusted to other genes, provided the first component of the system is placed under transcriptional control of these genes, thus, making it a valuable tool for cell fate mapping in mice.

hnRNP-K Targets Open Chromatin in Mouse Embryonic Stem Cells in Concert with Multiple Regulators.

The transcription factor Oct4 plays a key regulatory role in the induction and maintenance of cellular pluripotency. In this article, we show that ubiquitous and multifunctional poly(C) DNA/RNA-binding protein hnRNP-K occupies Oct4 (Pou5f1) enhancers in embryonic stem cells (ESCs) but is dispensable for the initiation, maintenance, and downregulation of Oct4 gene expression. Nevertheless, hnRNP-K has an essential cell-autonomous function in ESCs to maintain their proliferation and viability. To better understand mechanisms of hnRNP-K action in ESCs, we have performed ChIP-seq analysis of genome-wide binding of hnRNP-K and identified several thousands of hnRNP-K target sites that are frequently co-occupied by pluripotency-related and common factors (Oct4, TATA-box binding protein, Sox2, Nanog, Otx2, etc.), as well as active histone marks. Furthermore, hnRNP-K localizes exclusively within open chromatin, implying its role in the onset and/or maintenance of this chromatin state. Stem Cells 2019;37:1018-1029.

Evidences against vesicle-dependent trafficking and involvement of extracellular proteasomes into cell-to-cell communications.

The ubiquitin proteasome system is involved in the regulation of most basic intracellular processes, and deregulation of this system can results in certain kinds of human diseases. Proteolytic core this system, the 20S proteasome, has been found in physiological fluids of both healthy humans and patients suffering from a variety of inflammatory, autoimmune, and neoplastic diseases. The concentration of these extracellular proteasomes has been found to correlate with the diseased state, being of a prognostic significance. The transport mechanisms and functions of these proteasomes, however, are largely unclear. Previous studies revealed that the transport of extracellular proteasomes may occur via microvesicles and exosomes, which led to the hypothesis that extracellular proteasomes are implicated in cell-to-cell communication process. Here we show that microvesicles and exosomes, two major known types of intercellular vehicles, contain no detectable proteasomes. Moreover, neither affinity purified nor naturally released into conditioned medium by donor cells 20S proteasomes could penetrate recipient HeLa cells. Taken together, these results suggest that extracellular proteasomes are unlikely to be involved in the cell-to-cell communication and that their release by cells serve other biological purposes.

Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications.

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.