The Role of ATP-Binding Cassette Proteins in Stem Cell Pluripotency.

Pluripotent stem cells (PSCs) are highly proliferative cells that can self-renew indefinitely in vitro. Upon receiving appropriate signals, PSCs undergo differentiation and can generate every cell type in the body. These unique properties of PSCs require specific gene expression patterns that define stem cell identity and dynamic regulation of intracellular metabolism to support cell growth and cell fate transitions. PSCs are prone to DNA damage due to elevated replicative and transcriptional stress. Therefore, mechanisms to prevent deleterious mutations in PSCs that compromise stem cell function or increase the risk of tumor formation from becoming amplified and propagated to progenitor cells are essential for embryonic development and for using PSCs including induced PSCs (iPSCs) as a cell source for regenerative medicine. In this review, we discuss the role of the ATP-binding cassette (ABC) superfamily in maintaining PSC homeostasis, and propose how their activities can influence cellular signaling and stem cell fate decisions. Finally, we highlight recent discoveries that not all ABC family members perform only canonical metabolite and peptide transport functions in PSCs; rather, they can participate in diverse cellular processes from genome surveillance to gene transcription and mRNA translation, which are likely to maintain the pristine state of PSCs.

Transcription factor SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating the neurogenic gene Hes5.

SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a postimplantation, epiblast stem cell state back to a preimplantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.

ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain.

OCT4 and SOX2 confer pluripotency by recruiting coactivators to activate stem cell­specific transcription. However, the composition of coactivator complexes and their roles in maintaining stem cell fidelity remain unclear. Here, we report the ATP-binding cassette subfamily F member 1 (ABCF1) as a coactivator for OCT4/SOX2 critical for stem cell self-renewal. The intrinsically disordered low-complexity domain (LCD) of ABCF1 contributes to phase separation in vitro and transcriptional activation of pluripotency genes by mediating multivalent interactions with SOX2 and co-dependent coactivators XPC and DKC1. These LCD-driven transcription factor­coactivator interactions critical for pluripotency gene expression are disrupted by DNA damage, likely due to LCD-dependent binding of ABCF1 to damage-generated intracellular DNA fragments instead of SOX2. This study identifies a transcriptional coactivator that uses its LCD to form selective multivalent interactions to regulate stem cell self-renewal and exit from pluripotency when genome integrity is compromised.

Low complexity domains, condensates, and stem cell pluripotency.

Biological reactions require self-assembly of factors in the complex cellular milieu. Recent evidence indicates that intrinsically disordered, low-complexity sequence domains (LCDs) found in regulatory factors mediate diverse cellular processes from gene expression to DNA repair to signal transduction, by enriching specific biomolecules in membraneless compartments or hubs that may undergo liquid-liquid phase separation (LLPS). In this review, we discuss how embryonic stem cells take advantage of LCD-driven interactions to promote cell-specific transcription, DNA damage response, and DNA repair. We propose that LCD-mediated interactions play key roles in stem cell maintenance and safeguarding genome integrity.

Small-Molecule PAPD5 Inhibitors Restore Telomerase Activity in Patient Stem Cells.

Genetic lesions that reduce telomerase activity inhibit stem cell replication and cause a range of incurable diseases, including dyskeratosis congenita (DC) and pulmonary fibrosis (PF). Modalities to restore telomerase in stem cells throughout the body remain unclear. Here, we describe small-molecule PAPD5 inhibitors that demonstrate telomere restoration in vitro, in stem cell models, and in vivo. PAPD5 is a non-canonical polymerase that oligoadenylates and destabilizes telomerase RNA component (TERC). We identified BCH001, a specific PAPD5 inhibitor that restored telomerase activity and telomere length in DC patient induced pluripotent stem cells. When human blood stem cells engineered to carry DC-causing PARN mutations were xenotransplanted into immunodeficient mice, oral treatment with a repurposed PAPD5 inhibitor, the dihydroquinolizinone RG7834, rescued TERC 3' end maturation and telomere length. These findings pave the way for developing systemic telomere therapeutics to counteract stem cell exhaustion in DC, PF, and possibly other aging-related diseases.

Multipotency of mouse trophoblast stem cells.

BACKGROUND: In a number of disease processes, the body is unable to repair injured tissue, promoting the need to develop strategies for tissue repair and regeneration, including the use of cellular therapeutics. Trophoblast stem cells (TSCs) are considered putative stem cells as they differentiate into other subtypes of trophoblast cells. To identify cells for future therapeutic strategies, we investigated whether TSCs have properties of stem/progenitor cells including self-renewal and the capacity to differentiate into parenchymal cells of fetal organs, in vitro and in vivo. METHODS: TSCs were isolated using anti-CD117 micro-beads, from embryonic day 18.5 placentas. In vitro, CD117+ TSCs were cultured, at a limiting dilution in growth medium for the development of multicellular clones and in specialized medium for differentiation into lung epithelial cells, cardiomyocytes, and retinal photoreceptor cells. CD117+ TSCs were also injected in utero into lung, heart, and the sub-retinal space of embryonic day 13.5 fetuses, and the organs were harvested for histological assessment after a natural delivery. RESULTS: We first identified CD117+ cells within the labyrinth zone and chorionic basal plate of murine placentas in late pregnancy, embryonic day 18.5. CD117+ TSCs formed multicellular clones that remained positive for CD117 in vitro, consistent with self-renewal properties. The clonal cells demonstrated multipotency, capable of differentiating into lung epithelial cells (endoderm), cardiomyocytes (mesoderm), and retinal photoreceptor cells (ectoderm). Finally, injection of CD117+ TSCs in utero into lungs, hearts, and the sub-retinal spaces of fetuses resulted in their engraftment on day 1 after birth, and the CD117+ TSCs differentiated into lung alveolar epithelial cells, heart cardiomyocytes, and retina photoreceptor cells, corresponding with the organs in which they were injected. CONCLUSIONS: Our findings demonstrate that CD117+ TSCs have the properties of stem cells including clonogenicity, self-renewal, and multipotency. In utero administration of CD117+ TSCs engraft and differentiate into resident cells of the lung, heart, and retina during mouse development.

Regulation of DNA demethylation by the XPC DNA repair complex in somatic and pluripotent stem cells.

Faithful resetting of the epigenetic memory of a somatic cell to a pluripotent state during cellular reprogramming requires DNA methylation to silence somatic gene expression and dynamic DNA demethylation to activate pluripotency gene transcription. The removal of methylated cytosines requires the base excision repair enzyme TDG, but the mechanism by which TDG-dependent DNA demethylation occurs in a rapid and site-specific manner remains unclear. Here we show that the XPC DNA repair complex is a potent accelerator of global and locus-specific DNA demethylation in somatic and pluripotent stem cells. XPC cooperates with TDG genome-wide to stimulate the turnover of essential intermediates by overcoming slow TDG-abasic product dissociation during active DNA demethylation. We further establish that DNA demethylation induced by XPC expression in somatic cells overcomes an early epigenetic barrier in cellular reprogramming and facilitates the generation of more robust induced pluripotent stem cells, characterized by enhanced pluripotency-associated gene expression and self-renewal capacity. Taken together with our previous studies establishing the XPC complex as a transcriptional coactivator, our findings underscore two distinct but complementary mechanisms by which XPC influences gene regulation by coordinating efficient TDG-mediated DNA demethylation along with active transcription during somatic cell reprogramming.

Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation.

Growth differentiation factor 11 (GDF11) and myostatin (or GDF8) are closely related members of the transforming growth factor ß superfamily and are often perceived to serve similar or overlapping roles. Yet, despite commonalities in protein sequence, receptor utilization and signaling, accumulating evidence suggests that these 2 ligands can have distinct functions in many situations. GDF11 is essential for mammalian development and has been suggested to regulate aging of multiple tissues, whereas myostatin is a well-described negative regulator of postnatal skeletal and cardiac muscle mass and modulates metabolic processes. In this review, we discuss the biochemical regulation of GDF11 and myostatin and their functions in the heart, skeletal muscle, and brain. We also highlight recent clinical findings with respect to a potential role for GDF11 and/or myostatin in humans with heart disease. Finally, we address key outstanding questions related to GDF11 and myostatin dynamics and signaling during development, growth, and aging.

Circulating Growth Differentiation Factor 11/8 Levels Decline With Age.

RATIONALE: Growth differentiation factor 11 (GDF11) and GDF8 are members of the transforming growth factor-ß superfamily sharing 89% protein sequence homology. We have previously shown that circulating GDF11 levels decrease with age in mice. However, a recent study by Egerman et al reported that GDF11/8 levels increase with age in mouse serum. OBJECTIVE: Here, we clarify the direction of change of circulating GDF11/8 levels with age and investigate the effects of GDF11 administration on the murine heart. METHODS AND RESULTS: We validated our previous finding that circulating levels of GDF11/8 decline with age in mice, rats, horses, and sheep. Furthermore, we showed by Western analysis that the apparent age-dependent increase in GDF11 levels, as reported by Egerman et al, is attributable to cross-reactivity of the anti-GDF11 antibody with immunoglobulin, which is known to increase with age. GDF11 administration in mice rapidly activated SMAD2 and SMAD3 signaling in myocardium in vivo and decreased cardiac mass in both young (2-month-old) and old (22-month-old) mice in a dose-dependent manner after only 9 days. CONCLUSIONS: Our study confirms an age-dependent decline in serum GDF11/8 levels in multiple mammalian species and that exogenous GDF11 rapidly activates SMAD signaling and reduces cardiomyocyte size. Unraveling the molecular basis for the age-dependent decline in GDF11/8 could yield insight into age-dependent cardiac pathologies.

Architecture of the human XPC DNA repair and stem cell coactivator complex.

The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.

Functional and mechanistic studies of XPC DNA-repair complex as transcriptional coactivator in embryonic stem cells.

The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex.

The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells.

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

The intertwined roles of transcription and repair proteins.

Transcription is apparently risky business. Its intrinsic mutagenic potential must be kept in check by networks of DNA repair factors that monitor the transcription process to repair DNA lesions that could otherwise compromise transcriptional fidelity and genome integrity. Intriguingly, recent studies point to an even more direct function of DNA repair complexes as coactivators of transcription and the unexpected role of "scheduled" DNA damage/repair at gene promoters. Paradoxically, spontaneous DNA double-strand breaks also induce ectopic transcription that is essential for repair. Thus, transcription, DNA damage, and repair may be more physically and functionally intertwined than previously appreciated.

Transcriptional regulation by coactivators in embryonic stem cells.

Embryonic stem (ES) cells, like all cell types, are defined by their unique transcriptional signatures. The ability of ES cells to self-renew or exit the pluripotent state and enter differentiation requires extensive changes in their transcriptome and epigenome. Remarkably, transcriptional programs governing each cell fate must remain sufficiently malleable so that expression of only a handful of transcriptional activators can override the pre-existing state by collaborating with an unexpectedly elaborate collection of coactivators to specify, restrict and stabilize the new state. Here, we discuss recent advances in our understanding of how the same coactivator can interpret multiple lines of information encoded by different activators and integrate signals from diverse regulators into stem cell-specific transcriptional outputs.

A DNA repair complex functions as an Oct4/Sox2 coactivator in embryonic stem cells.

The transcriptional activators Oct4, Sox2, and Nanog cooperate with a wide array of cofactors to orchestrate an embryonic stem (ES) cell-specific gene expression program that forms the molecular basis of pluripotency. Here, we report using an unbiased in vitro transcription-biochemical complementation assay to discover a multisubunit stem cell coactivator complex (SCC) that is selectively required for the synergistic activation of the Nanog gene by Oct4 and Sox2. Purification, identification, and reconstitution of SCC revealed this coactivator to be the trimeric XPC-nucleotide excision repair complex. SCC interacts directly with Oct4 and Sox2 and is recruited to the Nanog and Oct4 promoters as well as a majority of genomic regions that are occupied by Oct4 and Sox2. Depletion of SCC/XPC compromised both pluripotency in ES cells and somatic cell reprogramming of fibroblasts to induced pluripotent stem (iPS) cells. This study identifies a transcriptional coactivator with diversified functions in maintaining ES cell pluripotency and safeguarding genome integrity.