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Supplementary Figure 1 from N-Myc Regulates a Widespread Euchromatic Program in the Human Genome Partially Independent of Its Role as a Classical Transcription Factor

Myc proteins have long been modeled to operate strictly as classic gene-specific transcription factors; however, we find that N-Myc has a robust role in the human genome in regulating global cellular euchromatin, including that of intergenic regions. Strikingly, 90% to 95% of the total genomic euchromatic marks histone H3 acetylated at lysine 9 and methylated at lysine 4 is N-Myc–dependent. However, Myc regulation of transcription, even of genes it directly binds and at which it is required for the maintenance of active chromatin, is generally weak. Thus, Myc has a much more potent ability to regulate large domains of euchromatin than to influence the transcription of individual genes. Overall, Myc regulation of chromatin in the human genome includes both specific genes, but also expansive genomic domains that invoke functions independent of a classic transcription factor. These findings support a new dual model for Myc chromatin function with important implications for the role of Myc in cancer and stem cell biology, including that of induced pluripotent stem cells. [Cancer Res 2008;68(23):9654–62]

Histone variant H3.3 is encoded by two genes, H3f3a and H3f3b, which can be expressed differentially depending on tissue type. Previous work in our lab has shown that knockout of H3f3b causes some neonatal lethality and infertility in mice, and chromosomal defects in mouse embryonic fibroblasts (MEFs). Studies of H3f3a and H3f3b null mice by others have produced generally similar phenotypes to what we found in our H3f3b nulls, but the relative impacts of the loss of either H3f3a or H3f3b have varied depending on the approach and genetic background. Here we used a knockout-first approach to target the H3f3a gene for inactivation in C57BL6 mice. Homozygous H3f3a targeting produced a lethal phenotype at or before birth. E13.5 null embryos had some potential morphological differences from WT littermates including smaller size and reduced head size. An E18.5 null embryowas smaller than its control littermates with several potential defects including small head and brain size as well as small lungs, which would be consistent with a late gestation lethal phenotype. Despite a reduction in H3.3 and total H3 protein levels, the only histone H3 post-translational modification in the small panel assessed that was significantly altered was the unique H3.3 mark phospho-Serine31, which was consistently increased in null neurospheres. H3f3a null neurospheres also exhibited consistent gene expression changes including in protocadherins. Overall, our findings are consistent with the model that there are differential, cell-type-specific contributions of H3f3a and H3f3b to H3.3 functions in epigenetic and developmental processes.

Summary Many gene networks are shared between pluripotent stem cells and cancer; a concept exemplified by several DPPA factors such as DPPA2 and DPPA4, which are highly and selectively expressed in stem cells but also found to be reactivated in cancer. Despite their striking expression pattern, for many years the function of DPPA2 and DPPA4 remained a mystery; knockout of Dppa2 and Dppa4 did not affect pluripotency, but caused lung and skeletal defects late in development, long after Dppa2 and Dppa4 expression had been turned off. A number of recent papers have further clarified and defined the roles of these important factors, identifying roles in priming the chromatin and maintaining developmental competency through regulating both H3K4me3 and H3K27me3 at bivalent chromatin domains, and acting to remodel chromatin and facilitate reprogramming of somatic cells to induced pluripotency. These findings highlight an important regulatory role for DPPA2 and DPPA4 at the transitional boundary between pluripotency and differentiation and may have relevance to the functions of DPPA2 and 4 in the context of cancer cells as well., In this review, R. Klein and P. Knoepfler summarize the roles of DPPA family members in both pluripotency and oncogenesis, highlighting the connection between these two cellular states. They describe the functions of DPPA factors in priming the chromatin and acting at the boundary between pluripotency and differentiation.

Histone H3.3 mutations are a hallmark of pediatric gliomas, but their core oncogenic mechanisms are not well-defined. To identify major effectors, we used CRISPR-Cas9 to introduce H3.3K27M and G34R mutations into previously H3.3-wildtype brain cells, while in parallel reverting the mutations in glioma cells back to wildtype. ChIP-seq analysis broadly linked K27M to altered H3K27me3 activity including within super-enhancers, which exhibited perturbed transcriptional function. This was largely independent of H3.3 DNA binding. The K27M and G34R mutations induced several of the same pathways suggesting key shared oncogenic mechanisms including activation of neurogenesis and NOTCH pathway genes. H3.3 mutant gliomas are also particularly sensitive to NOTCH pathway gene knockdown and drug inhibition, reducing their viability in culture. Reciprocal editing of cells generally produced reciprocal effects on tumorgenicity in xenograft assays. Overall, our findings define common and distinct K27M and G34R oncogenic mechanisms, including potentially targetable pathways., Kuang-Yui Chen et al. show that histone H3.3 K27M and G34R mutations share key oncogenic mechanisms such as activation of neurogenesis and NOTCH pathway genes. They find that H3.3 mutant gliomas are sensitive to inhibition of the NOTCH pathway, suggesting a potentially targetable pathway in pediatric gliomas.

Summary In this issue of Cell Stem Cell, Funato et al. (2021) and Bressan et al. (2021) use stem cells as models to define functions of the histone H3.3 G34R mutation in childhood gliomas. Both studies find strong regional specificity to oncohistone activity and implicate specific elements of an aberrantly locked-in neural progenitor transcriptional circuitry.

It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence.

Aim: The industry of unproven stem cell clinics has rapidly mushroomed throughout the USA, posing risks to patients and the research field. In this study, the aim was to better define how this industry changes. Methods: I analyzed a large cohort of US stem cell clinic firms and their distinct clinic locations as defined in 2015–2016 for their status now in 2019. Results: About a quarter of the firms no longer marketed stem cells. Some lacked active websites, while others dropped stem cell services. Even so, the total number of clinics in this group increased because some firms greatly expanded their clinic numbers. Conclusion: Overall, the unproven clinic industry is a moving target requiring ongoing study and regulatory oversight.

Aim: There is a critical need for safe and effective treatments for COVID-19. One possible type of treatment is cellular medicine such as stem cell therapy, but its potential is unclear. Here, our aim was to assess the potential impact of the many cellular medicine trials for COVID-19. Materials & methods: We collected and analyzed data for defined criteria from trial registries. Results: Our data suggest that relatively few of these COVID-19 trials will produce high-level evidence, but that on average they may be somewhat more rigorous than typical cell therapy trials unrelated to COVID-19. Conclusion: Most COVID-19 cellular medicine trials have relatively low potential for rapid, concrete impact. We discuss the findings in the context of the cellular medicine field overall.

Ja, es stimmt – wir haben nichts als Drachen im Kopf. Wir sind besessen davon, einen Drachen zu erschaffen.

Seit vielen Tausend Jahren sind die Menschen von Drachen fasziniert. Wer wurde sich nicht wunschen, zumindest einen kurzen Blick auf einen zu erhaschen? Die noch kuhnere Idee, tatsachlich einen eigenen Drachen zu besitzen, hat die menschliche Fantasie noch starker gefangen genommen.

Bislang haben wir uns vorwiegend auf die Flugel unseres Drachens (damit er fliegen kann), seine typische Physiologie (damit er Feuer speien kann) und sein Gehirn (das das ganze System steuern soll) konzentriert. Aber ein Drache braucht weit mehr als das. Vom Kopf bis zur Schanzspitze unseres Drachens gibt s viele weitere Merkmale, die ernsthafte Uberlegungen verdienen.

Wir denken, dass wir, wenn wir einen Drachen bauen konnen, wohl auch andere interessante mythische Geschopfe schaffen konnen. Dazu konnten wir sogar einige derselben Techniken verwenden, zum Beispiel auf Stammzellen oder CRISPR basierende Methoden, wie wir sie im vorigen Kapitel diskutiert haben.

Mussen alle Wege zur Schaffung eines echten Drachens mit Sex beginnen oder von Sex abhangig sein? Nicht unbedingt, aber bei den Recherchen fur dieses Buch sind wir zu der Uberzeugung gelangt, dass andere mogliche wissenschaftliche Wege zum Bau eines Drachen alle in irgendeiner Weise auf Experimenten zur Reproduktions- oder Entwicklungsbiologie basieren mussen. Und diese Forschung wird sich uberwiegend auf sexuelle Fortpflanzung stutzen, ob spontan oder unterstutzt durch In-vitro-Fertilisation (IFV).

Hundreds of businesses in the US currently advertise a wide range of non-US FDA-approved stem cell interventions. Here we present a novel systematic temporal analysis of US companies engaged in direct-to-consumer marketing of putative stem cell treatments. Between 2009 and 2014, the number of new US stem cell businesses with websites grew rapidly, at least doubling on average every year. From 2014 to 2016, approximately 90–100 new stem cell business websites appeared per year. In contrast, from 2012 to the present, regulatory activity in the form of FDA warning letters has been limited. These data point to a problematic disconnect between a rapidly expanding US direct-to-consumer stem cell industry and limited FDA oversight of this marketplace. More consistent, timely and effective FDA actions are urgently needed.

Alterations in histone H3.3 are common driver mutations in high-grade pediatric gliomas, but the central oncogenic mechanisms remain an open question. To identify important mutant H3.3 effectors, we used CRISPR-Cas9 to precisely introduce H3.3 K27M and G34R mutations into previously H3.3-wildtype human astrocyte and glioma cells, while in parallel reverting mutations in glioma cells back to wildtype. K27M and G34R mutations invoked some strikingly similar epigenomic effects supporting a new model in which some major aspects of their oncogenic functions are shared. For instance, both K27M and G34R induced changes at many of the same genomic loci in specific histone marks, with the largest changes in H3K27me3 including in particular within super-enhancers, which also exhibited perturbed transcriptional function. K27M and G34R mutations induced some gene expression changes that were unique to each mutation, but both mutations changed similar functional ontological clusters and ASCL1 is a shared key putative effector. H3.3 mutant glioma cells are sensitive to ASCL1 knockdown or overexpression, resulting in cell viability that is reduced or increased, respectively. Comparison of our panel of glioma cells gene-edited with precise point mutations to edited glioma cells in other studies that performed gene knockout or overexpression reveals striking differences in the resulting phenotypes. We also determined that certain drugs exhibited specificity to H3.3 mutation-bearing cells including DAPT, JQ1, and ONC201. In vivo, we found that reversion of K27M to WT in glioma cells significantly reduced tumorigenicity in mouse xenograft assays and introduction of G34R mutations in previously WT glioma cells increased tumor growth. Overall, gene editing of gliomas and comparison of otherwise isogenic sets of cells defines both distinct and shared gliomagenesis mechanisms that can be targeted for development of oncohistone-based therapeutics.

Pediatric high-grade gliomas often contain mutations in the H3F3A gene encoding the histone variant H3.3 and more rarely in canonical histone H3 family genes, a feature distinguishing them from adult gliomas. To define specific functionally significant changes in epigenomic states driven by mutant H3.3, we have utilized CRISPR-Cas9 to introduce specific H3.3 mutations (K27M, G34R) into formerly H3.3 wildtype (WT) brain and glioma cells, while in parallel also precisely reverting the pre-existing K27M and G34R mutations in patient-derived glioma cells to WT. In each case, gene editing was conducted on endogenous H3F3A alleles. Analyses of this overall panel of H3.3 gene-edited cells indicate that CRISPR-introduced K27M or G34R mutations within formerly H3.3 WT cells leads to increased gliomatypic signatures: elevated expression of specific oncogenes as well as neurogenesis and Notch signaling pathway genes, and perturbation of specific histone post-translational marks. Conversely, gene editing-based reversion of histone mutations to WT in primary glioma cells partially reverses glioma-associated phenotypes. Gene editing of K27M also yields coherent phenotype changes in xenograft assays. K27M and G34R mutations appear to function via both shared and unique epigenomic mechanisms. Targeting the mutant H3.3 effector pathways identified by our analyses in our full panel of cells with specific inhibitory drugs plus or minus irradiation defines differential, largely opposite responses of the parental and gene-edited cells. These defined pathways may serve as entry points for development of novel therapies specific for H3.3-mutant pediatric glioma. Overall, this system of gene editing gain and loss of mutant H3.3 provides new insights into oncohistone mechanisms and therapeutic strategies.

The goal of editing the genomes of stem cells to generate model organisms and cell lines for genetic and biological studies has been pursued for decades. There is also exciting potential for future clinical impact in humans. While recent, rapid advances in targeted nuclease technologies have led to unprecedented accessibility and ease of gene editing, biology has benefited from past directed gene modification via homologous recombination, gene traps and other transgenic methodologies. Here we review the history of genome editing in stem cells (including via zinc finger nucleases, transcription activator-like effector nucleases and CRISPR–Cas9), discuss recent developments leading to the implementation of stem cell gene therapies in clinical trials and consider the prospects for future advances in this rapidly evolving field.

Pluripotency is maintained through a complex network of signaling pathways that interact with transcription factors including octamer binding protein 4, SRY- box 2 , and Nanog, chromatin modifying complexes, microRNAs, and the DNA structure itself to preserve the undifferentiated, self-renewing state. Whereas the signaling requirements for the maintenance of stem cells can vary by species of origin, state of the stem cells, and culturing conditions, the core transcriptional circuity activated and maintained by these pathways is highly conserved. The self-sustaining nature of the pluripotency network is evident in the finding that somatic cells can be reprogrammed to a pluripotent state through the transient introduction of a small group of factors, which serve to reestablish the stem cell signaling and transcriptional network. Growing understanding of the molecular circuitry of pluripotency has major implications for the field's view of developmental biology and disease pathogenesis and is important for the evolving field of regenerative medicine.

The stem cell and regenerative medicine arena has become increasingly complicated in recent years with thousands of people involved. There are as many as a dozen or more main groups of stakeholders, who together may be viewed as one ecosystem that is now rapidly evolving. The nature of the ecosystem and its evolution have major implications for not just those within it, but also for medicine and society at large. Here, I describe this ecosystem and its evolution, as well as the negative impacts within the ecosystem of a constellation of hundreds of unproven for-profit clinics and related businesses. Finally, I propose approaches for how to positively influence and drive the future of the global stem cell ecosystem.

Developmental pluripotency associated factor 4 (Dppa4) is a highly specific marker of pluripotent cells, and is also overexpressed in certain cancers, but its function in either of these contexts is poorly understood. In this study, we use ChIP-Seq to identify Dppa4 binding genome-wide in three distinct cell types: mouse embryonic stem cells (mESC), embryonal carcinoma cells, and 3T3 fibroblasts ectopically expressing Dppa4. We find a core set of Dppa4 binding sites shared across cell types, and also a substantial number of sites unique to each cell type. Across cell types Dppa4 shows a preference for binding to regions with active chromatin signatures, and can influence chromatin modifications at target genes. In 3T3 fibroblasts with enforced Dppa4 expression, Dppa4 represses the cell cycle inhibitor Cdkn2c and activates Ets family transcription factor Etv4, leading to alterations in the cell cycle that likely contribute to the oncogenic phenotype. Dppa4 also directly regulates Etv4 in mESC but represses it in this context, and binds with Oct4 to a set of shared targets that are largely independent of Sox2 and Nanog, indicating that Dppa4 functions independently of the core pluripotency network in stem cells. Together these data provide novel insights into Dppa4 function in both pluripotent and oncogenic contexts. Keywords: Pluripotent stem cells, Chromatin, Histone deacetylase, Cell cycle, Oncogene, Dppa4, Oct4

Regulators are now more often distinguishing between perceived good citizens and "bad actors" in stem cell and regenerative medicine clinical research, resulting in relatively more polar, carrot-and-stick oversight approaches. Here, I discuss why there may be too much carrot and not enough stick by regulators for effective enforcement.

The phrase "bench-to-bedside" is commonly used to describe the translation of basic discoveries such as those on stem cells to the clinic for therapeutic use in human patients. However, there is a key intermediate step in between the bench and the bedside involving governmental regulatory oversight such as by the Food and Drug Administration (FDA) in the United States (US). Thus, it might be more accurate in most cases to describe the stem cell biological drug development process in this way: from bench to FDA to bedside. The intermediate development and regulatory stage for stem cell-based biological drugs is a multifactorial, continually evolving part of the process of developing a biological drug such as a stem cell-based regenerative medicine product. In some situations, stem cell-related products may not be classified as biological drugs in which case the FDA plays a relatively minor role. However, this middle stage is generally a major element of the process and is often colloquially referred to in an ominous way as "The Valley of Death". This moniker seems appropriate because it is at this point, and in particular in the work that ensues after Phase 1, clinical trials that most drug product development is terminated, often due to lack of funding, diseases being refractory to treatment, or regulatory issues. Not surprisingly, workarounds to deal with or entirely avoid this difficult stage of the process are evolving both inside and outside the domains of official regulatory authorities. In some cases these efforts involve the FDA invoking new mechanisms of accelerating the bench to beside process, but in other cases these new pathways bypass the FDA in part or entirely. Together these rapidly changing stem cell product development and regulatory pathways raise many scientific, ethical, and medical questions. These emerging trends and their potential consequences are reviewed here.

Vor Kurzem ging eine Schockwelle um die Welt. Chinesische Wissenschaftler berichteten von der erstmaligen Erzeugung genetisch modifizierter menschlicher Embryos mittels einer neuen Gentechnologie und entfachten damit eine hitzige Kontroverse. Einige sind der Meinung, derlei Experimente mussten ein Ende haben, wahrend andere dafur pladieren, die humane Genmodifikation mit Volldampf voranzutreiben, um Erkrankungen vorzubeugen oder gar verbesserte Designerbabys zu erschaffen. Als Vollblutwissenschaftler fand ich mich zwischen den beiden Polen wieder. Uberzeugt davon, dass die Offentlichkeit sehr viel mehr uber diesen Prazedenzfall in der Wissenschafts- und Kulturgeschichte erfahren sollte, fasste ich den Entschluss, meine Kenntnisse mithilfe dieses Buches an Sie weiterzugeben.

Wie wurde man vorgehen, um einen GMO sapiens zu erschaffen? Ware es schwierig? Welche Schritte waren dafur erforderlich? Wie viele Vorkenntnisse musste man haben? Konnte man es als Do-it-yourself-Projekt betrachten, vielleicht sogar in der eigenen Garage, mit einer bei eBay erstandenen Ausrustung?

Im‌ Jahr 2014 beugte sich ein Wissenschaftler in Dr. Junjiu Huangs Labor an der Sun-Yat-sen-Universitat im chinesischen Guangdong uber ein Mikroskop und betrachtete einzellige menschliche Embryonen in einer Petrischale. Im Licht des Mikroskops sahen sie wie winzige durchscheinende Kugeln aus. Der Wissenschaftler stach mit einer sehr dunnen, scharfen Nadel in den ersten dieser Embryonen.

Der Philanthrop Charles M. Goethe war ein Naturliebhaber aus Sacramento, wo sich mein Forschungslabor befindet. Die Umweltschutzer von heute, insbesondere hier in Nordkalifornien, konnten von Goethes Vision zunachst einmal fasziniert sein. Er war ein Naturschutzer, der den Erdboden und Baume liebte und sich auch fur Padagogik interessierte. Er grundete sogar die Sacramento State University.

Ist die Erschaffung von GM-Menschen der Garant dafur, dass die Menschheit eine sich stets wandelnde, moglicherweise desastrose Zukunft uberlebt? Oder wurde die Genmodifikation beim Menschen vielmehr eine Katastrophe oder gar seine Vernichtung heraufbeschworen?

Muss alles, was technisch neu und leistungsstark ist, seinen Anfang in einer Garage nehmen? Manchmal hat es den Anschein.

Am 9. November 1977 durchbrachen zwei britische Wissenschaftler eine biologische Schallmauer und veranderten die Menschheit damit fur immer. Robert Edwards und Patrick Steptoe, mit denen wir weiter vorne im Buch schon Bekanntschaft geschlossen haben, war es gelungen, in einer Petrischale im Labor einen gesunden menschlichen Embryo zu erzeugen. Dieser Embryo wurde der Mutter erfolgreich eingepflanzt und entwickelte sich zu einem Kind.

Anno 1981 entwickelten Forscher in einer Garage in Davis, Kalifornien eine kuhne Idee. Es war einer dieser raren Geistesblitze mit dem Potenzial, die Welt zu verandern. Dieser Hightech-Heureka-Moment ereignete sich nicht weit entfernt von dem Ort, an dem ich das Buch, das Sie gerade in der Hand halten, geschrieben habe. Ich lebe in Davis, als Professor an der UC Davis School of Medicine.

Human pluripotent stem cells (hPSC) have great clinical potential through the use of their differentiated progeny, a population in which there is some concern over risks of tumorigenicity or other unwanted cellular behavior due to residual hPSC. Preclinical studies using human stem cells are most often performed within a xenotransplant context. In this study, we sought to measure how undifferentiated hPSC behave following xenotransplant. We directly transplanted undifferentiated human induced pluripotent stem cells (hIPSC) and human embryonic stem cells (hESC) into the adult mouse brain ventricle and analyzed their fates. No tumors or precancerous lesions were present at more than one year after transplantation. This result differed with the tumorigenic capacity we observed after allotransplantation of mouse ESC into the mouse brain. A substantial population of cellular derivatives of undifferentiated hESC and hIPSC engrafted, survived, and migrated within the mouse brain parenchyma. Within brain structures, transplanted cell distribution followed a very specific pattern, suggesting the existence of distinct microenvironments that offer different degrees of permissibility for engraftment. Most of the transplanted hESC and hIPSC that developed into brain cells were NeuN+ neuronal cells, and no astrocytes were detected. Substantial cell and nuclear fusion occurred between host and transplanted cells, a phenomenon influenced by microenvironment. Overall, hIPSC appear to be largely functionally equivalent to hESC in vivo. Altogether, these data bring new insights into the behavior of stem cells without prior differentiation following xenotransplantation into the adult brain.

The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics.

In order to identify novel pluripotency-related oncogenes, an expression screen for oncogenic foci-inducing genes within a retroviral human embryonic stem cell cDNA library was conducted. From this screen, we identified not only known oncogenes but also intriguingly the key pluripotency factor, DPPA4 (developmental pluripotency-associated four) that encodes a DNA binding SAP domain-containing protein. DPPA4 has not been previously identified as an oncogene but is highly expressed in embryonal carcinomas, pluripotent germ cell tumors, and other cancers. DPPA4 is also mutated in some cancers. In direct transformation assays, we validated that DPPA4 is an oncogene in both mouse 3T3 cells and immortalized human dermal fibroblasts. Overexpression of DPPA4 generates oncogenic foci (sarcoma cells) and causes anchorage-independent growth. The in vitro transformed cells also give rise to tumors in immunodeficient mice. Furthermore, functional analyses indicate that both the DNA-binding SAP domain and the histone-binding C-terminal domain are critical for the oncogenic transformation activity of DPPA4. Downregulation of DPPA4 in E14 mouse embryonic stem cells and P19 mouse embryonic carcinoma cells causes decreased cell proliferation in each case. In addition, DPPA4 overexpression induces cell proliferation through genes related to regulation of G1/S transition. Interestingly, we observed similar findings for family member DPPA2. Thus, we have identified a new family of pluripotency-related oncogenes consisting of DPPA2 and DPPA4. Our findings have important implications for stem cell biology and tumorigenesis.

A host of cancer types exhibit aberrant histone modifications. Recently, distinct and recurrent mutations in a specific histone variant, histone H3.3, have been implicated in a high proportion of malignant pediatric brain cancers. The presence of mutant H3.3 histone disrupts epigenetic post-translational modifications near genes involved in cancer processes and in brain function. Here, we propose several possible mechanisms by which mutant H3.3 histones may act to promote tumorigenesis. Furthermore, we discuss how perturbations in normal H3.3 chromatin-related and epigenetic functions may more broadly contribute to the formation of human cancers.

The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.

Direct-to-consumer marketing of unapproved stem cell interventions is a well-known phenomenon in countries with lax medical regulations. However, an examination of Internet-based marketing claims revealed widespread promotion of such interventions by businesses based in the United States. Such commercial activity suggests that regulatory agencies must better oversee this marketplace.

c-Myc is involved in the control of diverse cellular processes and implicated in the maintenance of different tissues including the neural crest. Here, we report that c-Myc is particularly important for pigment cell development and homeostasis. Targeting c-Myc specifically in the melanocyte lineage using the floxed allele of c-Myc and Tyr::Cre transgenic mice results in a congenital gray hair phenotype. The gray coat color is associated with a reduced number of functional melanocytes in the hair bulb and melanocyte stem cells in the hair bulge. Importantly, the gray phenotype does not progress with time, suggesting that maintenance of the melanocyte through the hair cycle does not involve c-Myc function. In embryos, at E13.5, c-Myc-deficient melanocyte precursors are affected in proliferation in concordance with a reduction in numbers, showing that c-Myc is required for the proper melanocyte development. Interestingly, melanocytes from c-Myc-deficient mice display elevated levels of the c-Myc paralog N-Myc. Double deletion of c-Myc and N-Myc results in nearly complete loss of the residual pigmentation, indicating that N-Myc is capable of compensating for c-Myc loss of function in melanocytes.

Induced pluripotent stem cells (iPSCs) hold great promise for autologous cell therapies, but significant roadblocks remain to translating iPSCs to the bedside. For example, concerns about the presumed autologous transplantation potential of iPSCs have been raised by a recent paper demonstrating that iPSC-derived teratomas were rejected by syngeneic hosts. Additionally, the reprogramming process can alter genomic and epigenomic states, so a key goal at this point is to determine the clinical relevance of these changes and minimize those that prove to be deleterious. Finally, thus far few studies have examined the efficacy and tumorigenicity of iPSCs in clinically relevant transplantation scenarios, an essential requirement for the FDA. We discuss potential solutions to these hurdles to provide a roadmap for iPSCs to "jump the dish" and become useful therapies.

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.

Basal progenitors (also called non-surface dividing or intermediate progenitors) have been proposed to regulate the number of neurons during neocortical development through expanding cells committed to a neuronal fate, although the signals that govern this population have remained largely unknown. Here, we show that N-myc mediates the functions of Wnt signaling in promoting neuronal fate commitment and proliferation of neural precursor cells in vitro. Wnt signaling and N-myc also contribute to the production of basal progenitors in vivo. Expression of a stabilized form of β-catenin, a component of the Wnt signaling pathway, or of N-myc increased the numbers of neocortical basal progenitors, whereas conditional deletion of the N-myc gene reduced these and, as a likely consequence, the number of neocortical neurons. These results reveal that Wnt signaling via N-myc is crucial for the control of neuron number in the developing neocortex.