Sumoylation and the oncogenic E17K mutation affect AKT1 subcellular distribution and impact on Nanog-binding dynamics to chromatin in embryonic stem cells.

AKT/PKB is a kinase involved in the regulation of a plethora of cell processes. Particularly, in embryonic stem cells (ESCs), AKT is crucial for the maintenance of pluripotency. Although the activation of this kinase relies on its recruitment to the cellular membrane and subsequent phosphorylation, multiple other post-translational modifications (PTMs), including SUMOylation, fine-tune its activity and target specificity. Since this PTM can also modify the localization and availability of different proteins, in this work we explored if SUMOylation impacts on the subcellular compartmentalization and distribution of AKT1 in ESCs. We found that this PTM does not affect AKT1 membrane recruitment, but it modifies the AKT1 nucleus/cytoplasm distribution, increasing its nuclear presence. Additionally, within this compartment, we found that AKT1 SUMOylation also impacts on the chromatin-binding dynamics of NANOG, a central pluripotency transcription factor. Remarkably, the oncogenic E17K AKT1 mutant produces major changes in all these parameters increasing the binding of NANOG to its targets, also in a SUMOylation dependent manner. These findings demonstrate that SUMOylation modulates AKT1 subcellular distribution, thus adding an extra layer of regulation of its function, possibly by affecting the specificity and interaction with its downstream targets.

Nucleus-cytoskeleton communication impacts on OCT4-chromatin interactions in embryonic stem cells.

BACKGROUND: The cytoskeleton is a key component of the system responsible for transmitting mechanical cues from the cellular environment to the nucleus, where they trigger downstream responses. This communication is particularly relevant in embryonic stem (ES) cells since forces can regulate cell fate and guide developmental processes. However, little is known regarding cytoskeleton organization in ES cells, and thus, relevant aspects of nuclear-cytoskeletal interactions remain elusive. RESULTS: We explored the three-dimensional distribution of the cytoskeleton in live ES cells and show that these filaments affect the shape of the nucleus. Next, we evaluated if cytoskeletal components indirectly modulate the binding of the pluripotency transcription factor OCT4 to chromatin targets. We show that actin depolymerization triggers OCT4 binding to chromatin sites whereas vimentin disruption produces the opposite effect. In contrast to actin, vimentin contributes to the preservation of OCT4-chromatin interactions and, consequently, may have a pro-stemness role. CONCLUSIONS: Our results suggest roles of components of the cytoskeleton in shaping the nucleus of ES cells, influencing the interactions of the transcription factor OCT4 with the chromatin and potentially affecting pluripotency and cell fate.

Pluripotency transcription factors at the focus: the phase separation paradigm in stem cells.

The transcription factors (TFs) OCT4, SOX2 and NANOG are key players of the gene regulatory network of pluripotent stem cells. Evidence accumulated in recent years shows that even small imbalances in the expression levels or relative concentrations of these TFs affect both, the maintenance of pluripotency and cell fate decisions. In addition, many components of the transcriptional machinery including RNA polymerases, cofactors and TFs such as those required for pluripotency, do not distribute homogeneously in the nucleus but concentrate in multiple foci influencing the delivery of these molecules to their DNA-targets. How cells control strict levels of available pluripotency TFs in this heterogeneous space and the biological role of these foci remain elusive. In recent years, a wealth of evidence led to propose that many of the nuclear compartments are formed through a liquid-liquid phase separation process. This new paradigm early penetrated the stem cells field since many key players of the pluripotency circuitry seem to phase-separate. Overall, the formation of liquid compartments may modulate the kinetics of biochemical reactions and consequently regulate many nuclear processes. Here, we review the state-of-the-art knowledge of compartmentalization in the cell nucleus and the relevance of this process for transcriptional regulation, particularly in pluripotent stem cells. We also highlight the recent advances and new ideas in the field showing how compartmentalization may affect pluripotency preservation and cell fate decisions.

The dynamical organization of the core pluripotency transcription factors responds to differentiation cues in early S-phase.

DNA replication in stem cells is a major challenge for pluripotency preservation and cell fate decisions. This process involves massive changes in the chromatin architecture and the reorganization of many transcription-related molecules in different spatial and temporal scales. Pluripotency is controlled by the master transcription factors (TFs) OCT4, SOX2 and NANOG that partition into condensates in the nucleus of embryonic stem cells. These condensates are proposed to play relevant roles in the regulation of gene expression and the maintenance of pluripotency. Here, we asked whether the dynamical distribution of the pluripotency TFs changes during the cell cycle, particularly during DNA replication. Since the S phase is considered to be a window of opportunity for cell fate decisions, we explored if differentiation cues in G1 phase trigger changes in the distribution of these TFs during the subsequent S phase. Our results show a spatial redistribution of TFs condensates during DNA replication which was not directly related to chromatin compaction. Additionally, fluorescence fluctuation spectroscopy revealed TF-specific, subtle changes in the landscape of TF-chromatin interactions, consistent with their particularities as key players of the pluripotency network. Moreover, we found that differentiation stimuli in the preceding G1 phase triggered a relatively fast and massive reorganization of pluripotency TFs in early-S phase. Particularly, OCT4 and SOX2 condensates dissolved whereas the lifetimes of TF-chromatin interactions increased suggesting that the reorganization of condensates is accompanied with a change in the landscape of TF-chromatin interactions. Notably, NANOG showed impaired interactions with chromatin in stimulated early-S cells in line with its role as naïve pluripotency TF. Together, these findings provide new insights into the regulation of the core pluripotency TFs during DNA replication of embryonic stem cells and highlight their different roles at early differentiation stages.

AKT1 induces Nanog promoter in a SUMOylation-dependent manner in different pluripotent contexts.

AKT/PKB is a kinase crucial for pluripotency maintenance in pluripotent stem cells. Multiple post-translational modifications modulate its activity. We have previously demonstrated that AKT1 induces the expression of the pluripotency transcription factor Nanog in a SUMOylation-dependent manner in mouse embryonic stem cells. Here, we studied different cellular contexts and main candidates that could mediate this induction. Our results strongly suggest the pluripotency transcription factors OCT4 and SOX2 are not essential mediators. Additionally, we concluded that this induction takes place in different pluripotent contexts but not in terminally differentiated cells. Finally, the cross-matching analysis of ESCs, iPSCs and MEFs transcriptomes and AKT1 phosphorylation targets provided new clues about possible factors that could be involved in the SUMOylation-dependent Nanog induction by AKT.

From the membrane to the nucleus: mechanical signals and transcription regulation.

Mechanical forces drive and modulate a wide variety of processes in eukaryotic cells including those occurring in the nucleus. Relevantly, forces are fundamental during development since they guide lineage specifications of embryonic stem cells. A sophisticated macromolecular machinery transduces mechanical stimuli received at the cell surface into a biochemical output; a key component in this mechanical communication is the cytoskeleton, a complex network of biofilaments in constant remodeling that links the cell membrane to the nuclear envelope. Recent evidence highlights that forces transmitted through the cytoskeleton directly affect the organization of chromatin and the accessibility of transcription-related molecules to their targets in the DNA. Consequently, mechanical forces can directly modulate transcription and change gene expression programs. Here, we will revise the biophysical toolbox involved in the mechanical communication with the cell nucleus and discuss how mechanical forces impact on the organization of this organelle and more specifically, on transcription. We will also discuss how live-cell fluorescence imaging is producing exquisite information to understand the mechanical response of cells and to quantify the landscape of interactions of transcription factors with chromatin in embryonic stem cells. These studies are building new biophysical insights that could be fundamental to achieve the goal of manipulating forces to guide cell differentiation in culture systems.

SOX2 Modulates the Nuclear Organization and Transcriptional Activity of the Glucocorticoid Receptor.

Steroid receptors (SRs) are ligand-dependent transcription factors (TFs) relevant to key cellular processes in both physiology and pathology, including some types of cancer. SOX2 is a master TF of pluripotency and self-renewal of embryonic stem cells, and its dysregulation is also associated with various types of human cancers. A potential crosstalk between these TFs could be relevant in malignant cells yet, to the best of our knowledge, no formal study has been performed thus far. Here we show, by quantitative live-cell imaging microscopy, that ectopic expression of SOX2 disrupts the formation of hormone-dependent intranuclear condensates of many steroid receptors (SRs), including those formed by the glucocorticoid receptor (GR). SOX2 also reduces GR's binding to specific DNA targets and modulates its transcriptional activity. SOX2-driven effects on GR condensates do not require the intrinsically disordered N-terminal domain of the receptor and, surprisingly, neither relies on GR/SOX2 interactions. SOX2 also alters the intranuclear dynamics and compartmentalization of the SR coactivator NCoA-2 and impairs GR/NCoA-2 interactions. These results suggest an indirect mechanism underlying SOX2-driven effects on SRs involving this coactivator. Together, these results highlight that the transcriptional program elicited by GR relies on its nuclear organization and is intimately linked to the distribution of other GR partners, such as the NCoA-2 coactivator. Abnormal expression of SOX2, commonly observed in many tumors, may alter the biological action of GR and, probably, other SRs as well. Understanding this crosstalk may help to improve steroid hormone-based therapies in cancers with elevated SOX2 expression.

SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells.

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-ß and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

Dynamical reorganization of the pluripotency transcription factors Oct4 and Sox2 during early differentiation of embryonic stem cells.

Pluripotency maintenance requires transcription factors (TFs) that induce genes necessary to preserve the undifferentiated state and repress others involved in differentiation. Recent observations support that the heterogeneous distribution of TFs in the nucleus impacts on gene expression. Thus, it is essential to explore how TFs dynamically organize to fully understand their role in transcription regulation. Here, we examine the distribution of pluripotency TFs Oct4 and Sox2 in the nucleus of embryonic stem (ES) cells and inquire whether their organization changes during early differentiation stages preceding their downregulation. Using ES cells expressing Oct4-YPet or Sox2-YPet, we show that Oct4 and Sox2 partition between nucleoplasm and a few chromatin-dense foci which restructure after inducing differentiation by 2i/LIF withdrawal. Fluorescence correlation spectroscopy showed distinct changes in Oct4 and Sox2 dynamics after differentiation induction. Specifically, we detected an impairment of Oct4-chromatin interactions whereas Sox2 only showed slight variations in its short-lived, and probably more unspecific, interactions with chromatin. Our results reveal that differentiation cues trigger early changes of Oct4 and Sox2 nuclear distributions that also include modifications in TF-chromatin interactions. This dynamical reorganization precedes Oct4 and Sox2 downregulation and may contribute to modulate their function at early differentiation stages.

Novel Interplay between p53 and HO-1 in Embryonic Stem Cells.

Stem cells genome safeguarding requires strict oxidative stress control. Heme oxygenase-1 (HO-1) and p53 are relevant components of the cellular defense system. p53 controls cellular response to multiple types of harmful stimulus, including oxidative stress. Otherwise, besides having a protective role, HO-1 is also involved in embryo development and in embryonic stem (ES) cells differentiation. Although both proteins have been extensively studied, little is known about their relationship in stem cells. The aim of this work is to explore HO-1-p53 interplay in ES cells. We studied HO-1 expression in p53 knockout (KO) ES cells and we found that they have higher HO-1 protein levels but similar HO-1 mRNA levels than the wild type (WT) ES cell line. Furthermore, cycloheximide treatment increased HO-1 abundance in p53 KO cells suggesting that p53 modulates HO-1 protein stability. Notably, H2O2 treatment did not induce HO-1 expression in p53 KO ES cells. Finally, SOD2 protein levels are also increased while Sod2 transcripts are not in KO cells, further suggesting that the p53 null phenotype is associated with a reinforcement of the antioxidant machinery. Our results demonstrate the existence of a connection between p53 and HO-1 in ES cells, highlighting the relationship between these stress defense pathways.

Kat6b Modulates Oct4 and Nanog Binding to Chromatin in Embryonic Stem Cells and Is Required for Efficient Neural Differentiation.

Chromatin remodeling is fundamental for the dynamical changes in transcriptional programs that occur during development and stem cell differentiation. The histone acetyltransferase Kat6b is relevant for neurogenesis in mouse embryos, and mutations of this gene cause intellectual disability in humans. However, the molecular mechanisms involved in Kat6b mutant phenotype and the role of this chromatin modifier in embryonic stem (ES) cells remain elusive. In this work, we show that Kat6b is expressed in ES cells and is repressed during differentiation. Moreover, we found that this gene is regulated by the pluripotency transcription factors Nanog and Oct4. To study the functional relevance of Kat6b in ES cells, we generated a Kat6b knockout ES cell line (K6b-/-) using CRISPR/Cas9. Fluorescence correlation spectroscopy analyses suggest a more compact chromatin organization in K6b-/- cells and impaired interactions of Oct4 and Nanog with chromatin. Remarkably, K6b-/- cells showed a reduced efficiency to differentiate to neural lineage. These results reveal a role of Kat6b as a modulator of chromatin plasticity, its impact on chromatin-transcription factors interactions and its influence on cell fate decisions during neural development.

Imaging transcription factors dynamics with advanced fluorescence microscopy methods.

Pluripotent stem cells (PSCs) are capable of self-renewing and producing all cell types derived from the three germ layers in response to developmental cues, constituting an important promise for regenerative medicine. Pluripotency depends on specific transcription factors (TFs) that induce genes required to preserve the undifferentiated state and repress other genes related to differentiation. The transcription machinery and regulatory components such as TFs are recruited dynamically on their target genes making it essential exploring their dynamics in living cells to understand the transcriptional output. Non-invasive and very sensitive fluorescence microscopy methods are making it possible visualizing the dynamics of TFs in living specimens, complementing the information extracted from studies in fixed specimens and bulk assays. In this work, we briefly describe the basis of these microscopy methods and review how they contributed to our knowledge of the function of TFs relevant to embryo development and cell differentiation in a variety of systems ranging from single cells to whole organisms.