Histone demethylase LSD1 regulates transcription by demethylating Lys 4 of histone H3. The crystal structure of the enzyme in complex with CoREST and a substrate-like peptide inhibitor highlights an intricate network of interactions and a folded conformation of the bound peptide. The core of the peptide structure is formed by Arg 2 , Gln 5 , and Ser 10 , which are engaged in specific intramolecular H-bonds. Several charged side chains on the surface of the substrate-binding pocket establish electrostatic interactions with the peptide. The three-dimensional structure predicts that methylated Lys 4 binds in a solvent inaccessible position in front of the flavin cofactor. This geometry is fully consistent with the demethylation reaction being catalyzed through a flavin-mediated oxidation of the substrate amino-methyl group. These features dictate the exquisite substrate specificity of LSD1 and provide a structural framework to explain the fine tuning of its catalytic activity and the active role of CoREST in substrate recognition.Lysine methylation is among the most well characterized histone modifications, and its existence has been known since the early days of chromatin research (1, 2). This type of epigenetic mark provides a huge potential for functional responses in that it can occur in different forms (mono-, di-, and tri-methylation) and on different histone sites, each having a specific physiological meaning. Histone methylation has been long thought to be a low turnover epigenetic mark, but the recent discovery of histone demethylases (3, 4) has challenged this view by demonstrating that histone lysine methylation can be actively and dynamically regulated. Two classes of histone demethylases have been uncovered; the enzymes of the JmjC family use iron as cofactor, whereas lysine-specific demethylase 1 (LSD1) 4 employs FAD as the prosthetic group (5).LSD1 catalyzes the oxidative demethylation of mono-and dimethyl Lys 4 of histone H3, generating hydrogen peroxide and formaldehyde (3, 4). The enzyme is implicated as a key component of distinct co-activator and co-repressor complexes in a surprisingly wide range of cellular processes where it participates in the dynamic transition of transcriptional programs (6). Its catalytic activity is finely tuned by the epigenetic marks present on the H3 N-terminal tail (7) and by other protein partners, such as CoREST, that form a stable complex with the enzyme (8, 9). The three-dimensional structure of LSD1 in its native state (10, 11) and in complex with the LSD1-binding domain of CoREST (12) have revealed that the catalytic center is located in the core of the enzyme main body. A protruding tower domain consisting of two remarkably long helices forms the docking site for the co-repressor protein (Fig. 1A).Here, we describe the structural analysis of LSD1-CoREST bound to a 21-amino acid H3 peptide in which pLys 4 ("p" is for peptide) is mutated to Met. The structural analysis illuminates the molecular properties that enable LSD1 to function as a key transcriptional reg...
We identify LSD1 (lysine-specific demethylase 1; also known as KDM1A and AOF2) as a key histone modifier that participates in the maintenance of pluripotency through the regulation of bivalent domains, a chromatin environment present at the regulatory regions of developmental genes that contains both H3K4 di/trimethylation and H3K27 trimethylation marks. LSD1 occupies the promoters of a subset of developmental genes that contain bivalent domains and are co-occupied by OCT4 and NANOG in human embryonic stem cells, where it controls the levels of H3K4 methylation through its demethylase activity. Thus, LSD1 has a role in maintaining the silencing of several developmental genes in human embryonic stem cells by regulating the critical balance between H3K4 and H3K27 methylation at their regulatory regions.
A variety of chromatin remodeling complexes are thought to orchestrate transcriptional programs that lead neuronal precursors from earliest commitment to terminal differentiation. Here we show that mammalian neurons have a specialized chromatin remodeling enzyme arising from a neurospecific splice variant of LSD1/KDM1, histone lysine specific demethylase 1, whose demethylase activity on Lys4 of histone H3 has been related to gene repression. We found that alternative splicing of LSD1 transcript generates four full-length isoforms from combinatorial retention of two identified exons: the 4 aa exon E8a is internal to the amine oxidase domain, and its inclusion is restricted to the nervous system. Remarkably, the expression of LSD1 splice variants is dynamically regulated throughout cortical development, particularly during perinatal stages, with a progressive increase of LSD1 neurospecific isoforms over the ubiquitous ones. Notably, the same LSD1 splice dynamics can be fairly recapitulated in cultured cortical neurons. Functionally, LSD1 isoforms display in vitro a comparable demethylase activity, yet the inclusion of the sole exon E8a reduces LSD1 repressor activity on a reporter gene. Additional distinction among isoforms is supported by the knockdown of neurospecific variants in cortical neurons resulting in the inhibition of neurite maturation, whereas overexpression of the same variants enhances it. Instead, perturbation of LSD1 isoforms that are devoid of the neurospecific exon elicits no morphogenic effect. Collectively, results demonstrate that the arousal of neuronal LSD1 isoforms pacemakes early neurite morphogenesis, conferring a neurospecific function to LSD1 epigenetic activity.
Cell reprogramming promises to make characterization of the impact of human genetic variation on health and disease experimentally tractable by enabling the bridging of genotypes to phenotypes in developmentally relevant human cell lineages. Here we apply this paradigm to two disorders caused by symmetrical copy number variations of 7q11.23, which display a striking combination of shared and symmetrically opposite phenotypes--Williams-Beuren syndrome and 7q-microduplication syndrome. Through analysis of transgene-free patient-derived induced pluripotent stem cells and their differentiated derivatives, we find that 7q11.23 dosage imbalance disrupts transcriptional circuits in disease-relevant pathways beginning in the pluripotent state. These alterations are then selectively amplified upon differentiation of the pluripotent cells into disease-relevant lineages. A considerable proportion of this transcriptional dysregulation is specifically caused by dosage imbalances in GTF2I, which encodes a key transcription factor at 7q11.23 that is associated with the LSD1 repressive chromatin complex and silences its dosage-sensitive targets.
The pig represents an ideal large-animal model, intermediate between rodents and humans, for the preclinical assessment of emerging cell therapies. As no validated pig embryonic stem (pES) cell lines have been derived so far, pig induced pluripotent stem cells (piPSCs) should offer an alternative source of undifferentiated cells to advance regenerative medicine research from bench to clinical trial. We report here for the first time the derivation of piPSCs from adult fibroblast with only three transcription factors: Sox2 (sex determining region Y-box 2), Klf4 (Krüppel-like factor 4), and c-Myc (avian myelocytomatosis viral oncogene homolog). We have been able to demonstrate that exogenous Pou5f1 (POU domain class 5 transcription factor 1; abbreviated as Octamer-4: Oct4) is dispensable to achieve and maintain pluripotency in the generation of piPSCs. To the best of our knowledge, this is also the first report of somatic reprogramming in any species without the overexpression, either directly or indirectly, of Oct4. Moreover, we were able to generate piPSCs without the use of feeder cells, approaching thus xeno-free conditions. Our work paves the way for the derivation of clinical grade piPSCs for regenerative medicine.
The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133 + cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression analysis, immunophenotyping, and electrophysiological analysis show that CBiNCs acquire a distinct neuronal phenotype characterized by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of future cell-replacement therapies.neurons | reprogramming | stem cells T he fate of adult somatic cells is not fixed rigidly and can be reprogrammed by experimental manipulation. The generation of induced pluripotent stem cells (iPSCs) represents the most dramatic evidence that the epigenome of somatic cells are remarkably plastic (1). Recently, it has been reported that fibroblasts can be converted by ectopic expression of defined transcription factors into postmitotic neurons (2-9), neural progenitors (10, 11), and self-renewing neural stem cells (NSCs) (12, 13). However, most reported methods rely on the use of multiple transcription factors and the use of fibroblasts as donor cells.Here we investigate if cord blood (CB) stem cells can be induced to acquire a neuronal phenotype by using only one transcription factor. It has been previously shown that stem cell populations are more amenable to reprogramming than other somatic cells, probably as a result of their preexisting epigenetic state (14,15). Moreover, because of their biological characteristics and availability, CB cells as a donor cell type could offer clear logistic advantages vs. other adult somatic cell types (16,17). These cells can be collected without any risk for the donor and are young cells carrying minimal somatic mutations.Strikingly, we show that forced expression of Sox2 is sufficient to convert CB CD133 + cells into induced neuronal progenitor (NP)-like cells, a conversion process that is augmented by the coexpression of c-Myc. Sox2 is highly expressed in adult NSCs, is one of the earliest functional markers of neuroectodermal specification in the embryo, and plays a key role in the neural lineage specification (18)(19)(20). We show that Sox2-transduced CB cells acquire a distinct neuronal morphology and express multiple neuronal markers in vitro, and that they can be expanded for as many as 25 passages when cultured in permissive condition culture. Moreover, we show that CB-induced neuronal-like cells (CB-iNCs) are able to differentiate in vitro and ...
RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods.
Environmental contextEnvironmental samples are best analysed in their native state, with minimal sample preparation, to fully understand the complex interactions and processes occurring in environmental systems. Nuclear magnetic resonance spectroscopy is a powerful tool used to study environmental samples but sample pre-treatment is often required to remove water and improve analysis. We introduce an experimental approach to remove water signals from environmental samples in their natural state, which opens the door to intact sample analysis and more environmentally relevant science. AbstractStudying environmental samples in their natural state is critical as drying, fractionating or extractions can alter the composition, structure, conformation and biological activity, as well as perturb essential interfaces and domains. Nuclear magnetic resonance (NMR) spectroscopy is a powerful and versatile tool that provides unprecedented levels of information regarding structure and interactions. Both high-resolution magic-angle-spinning and comprehensive-multiphase NMR probes facilitate the study of natural multiphase samples. 1H NMR spectroscopy is the most sensitive and provides unique information on swollen components and interfaces. However, samples such as plants, organisms and soil have a high aqueous content and a range of free, exchanging and bound water, leading to a broad and intense water signal that can span the entire 1H spectral region masking information from other components. In this manuscript, a water suppression approach termed Tailored Water suppression for Inhomogeneous Natural Samples (TWINS) is developed out of a practical need to study samples in their native state. TWINS builds upon the most effective approach to date (SPR-W5-WATERGATE) for natural samples with the addition of various elements to make the approach effective in the most challenging systems. TWINS was demonstrated on a range of environmental samples in both 1-D and 2-D NMR experiments. A lock capillary was developed to separate the lock solvent from the sample, further reducing sample alteration. In summary the more challenging the sample, the more TWINS outperformed conventional approaches. In turn this increases the range and diversity of samples that can be studied in their natural state critical for a wide variety of fields and applications.
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