Faculty Bibliography

Self-renewing naive mouse embryonic stem cells (mESCs) contain few mitochondria, which increase in number and volume at the onset of differentiation. KBP (encoded by Kif1bp) is an interactor of the mitochondrial-associated kinesin Kif1Balpha. We found that TDH, responsible for mitochondrial production of acetyl-CoA in mESCs, and the acetyltransferase GCN5L1 cooperate to acetylate Lys501 in KBP, allowing its recognition by and degradation via Fbxo15, an F-box protein transcriptionally controlled by the pluripotency core factors and repressed following differentiation. Defects in KBP degradation in mESCs result in an unscheduled increase in mitochondrial biogenesis, enhanced respiration and ROS production, and inhibition of cell proliferation. Silencing of Kif1Balpha reverts the aberrant increase in mitochondria induced by KBP stabilization. Notably, following differentiation, Kif1bp-/- mESCs display impaired expansion of the mitochondrial mass and form smaller embryoid bodies. Thus, KBP proteolysis limits the accumulation of mitochondria in mESCs to preserve their optimal fitness, whereas KBP accumulation promotes mitochondrial biogenesis in differentiating cells.

Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is typically an inefficient and asynchronous process. A variety of technological efforts have been made to accelerate and/or synchronize this process. To define a unified framework to study and compare the dynamics of reprogramming under different conditions, we developed an in silico analysis platform based on mathematical modeling. Our approach takes into account the variability in experimental results stemming from probabilistic growth and death of cells and potentially heterogeneous reprogramming rates. We suggest that reprogramming driven by the Yamanaka factors alone is a more heterogeneous process, possibly due to cell-specific reprogramming rates, which could be homogenized by the addition of additional factors. We validated our approach using publicly available reprogramming datasets, including data on early reprogramming dynamics as well as cell count data, and thus we demonstrated the general utility and predictive power of our methodology for investigating reprogramming and other cell fate change systems.

Genomic imprinting results in the monoallelic expression of genes that encode important regulators of growth and proliferation. Dysregulation of imprinted genes, such as those within the Dlk1-Dio3 locus, is associated with developmental syndromes and specific diseases. Our ability to interrogate causes of imprinting instability has been hindered by the absence of suitable model systems. Here, we describe a Dlk1 knockin reporter mouse that enables single-cell visualization of allele-specific expression and prospective isolation of cells, simultaneously. We show that this "imprinting reporter mouse" can be used to detect tissue-specific Dlk1 expression patterns in developing embryos. We also apply this system to pluripotent cell culture and demonstrate that it faithfully indicates DNA methylation changes induced upon cellular reprogramming. Finally, the reporter system reveals a role of elevated oxygen levels in eroding imprinted Dlk1 expression during prolonged culture and in vitro differentiation. The possibility to study allele-specific expression in different contexts makes our reporter system a useful tool to dissect the regulation of genomic imprinting in normal development and disease.

Pluripotent embryonic stem cells (ESCs) self-renew or differentiate into all tissues of the developing embryo and cell-specification factors are necessary to balance gene expression. Here we delineate the function of the PHD-finger protein 5a (Phf5a) in ESC self-renewal and ascribe its role in regulating pluripotency, cellular reprogramming and myoblast specification. We demonstrate that Phf5a is essential for maintaining pluripotency, since depleted ESCs exhibit hallmarks of differentiation. Mechanistically, we attribute Phf5a function to the stabilization of the Paf1 transcriptional complex and control of RNA polymerase II elongation on pluripotency loci. Apart from an ESC-specific factor, we demonstrate that Phf5a controls differentiation of adult myoblasts. Our findings suggest a potent mode of regulation by Phf5a in stem cells, which directs their transcriptional programme, ultimately regulating maintenance of pluripotency and cellular reprogramming.

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2, 3, 4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell- and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2-/- mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

A series of five related publications describe an alternative pluripotent state that is dependent on continuous high levels of exogenous reprogramming factor expression. A comprehensive effort to molecularly compare the acquisition of this state to induced pluripotency aims at providing new insights into the mechanisms underlying cellular reprogramming.

The differentiated state of somatic cells provides barriers for the derivation of induced pluripotent stem cells (iPSCs). To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. Surprisingly, inhibition of transforming growth factor beta (TGF-beta) together with activation of Wnt signaling in the presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after 1 week of reprogramming factor expression. In contrast, hepatic and blood progenitors predominantly required only TGF-beta inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner, and we demonstrate that expression of specific chromatin-modifying enzymes and reduced TGF-beta/mitogen-activated protein (MAP) kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Our observations define cell-type-specific requirements for the rapid and synchronous reprogramming of somatic cells.

For future application of induced pluripotent stem cell (iPSC) technology, the ability to assess the overall quality of iPSC clones will be an important issue. Here we show that the histone variant H2A.X is a functional marker that can distinguish the developmental potentials of mouse iPSC lines. We found that H2A.X is specifically targeted to and negatively regulates extraembryonic lineage gene expression in embryonic stem cells (ESCs) and prevents trophectoderm lineage differentiation. ESC-specific H2A.X deposition patterns are faithfully recapitulated in iPSCs that support the development of "all-iPS" animals via tetraploid complementation, the most stringent test available of iPSC quality. In contrast, iPSCs that fail to support all-iPS embryonic development show aberrant H2A.X deposition, upregulation of extraembryonic lineage genes, and a predisposition to extraembryonic differentiation. Thus, our work has highlighted an epigenetic mechanism for maintaining cell lineage commitment in ESCs and iPSCs that can be used to distinguish the quality of iPSC lines.

Chromatin structure determines DNA accessibility. We compare nucleosome occupancy in mouse and human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs) and differentiated cell types using MNase-seq. To address variability inherent in this technique, we developed a bioinformatic approach to identify regions of difference (RoD) in nucleosome occupancy between pluripotent and somatic cells. Surprisingly, most chromatin remains unchanged; a majority of rearrangements appear to affect a single nucleosome. RoDs are enriched at genes and regulatory elements, including enhancers associated with pluripotency and differentiation. RoDs co-localize with binding sites of key developmental regulators, including the reprogramming factors Klf4, Oct4/Sox2 and c-Myc. Nucleosomal landscapes in ESC enhancers are extensively altered, exhibiting lower nucleosome occupancy in pluripotent cells than in somatic cells. Most changes are reset during reprogramming. We conclude that changes in nucleosome occupancy are a hallmark of cell differentiation and reprogramming and likely identify regulatory regions essential for these processes.