Publications by authors named "Thomas Vierbuchen"

12 Publications

  • Page 1 of 1

Kathryn Anderson (1952-2020).

Cell 2021 03;184(5):1123-1126

Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065, USA. Electronic address:

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http://dx.doi.org/10.1016/j.cell.2021.01.054DOI Listing
March 2021

Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing.

Nat Neurosci 2018 12 19;21(12):1670-1679. Epub 2018 Nov 19.

Department of Neurobiology, Harvard Medical School, Boston, MA, USA.

In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single-nucleotide polymorphisms in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.
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http://dx.doi.org/10.1038/s41593-018-0270-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261686PMC
December 2018

AP-1 Transcription Factors and the BAF Complex Mediate Signal-Dependent Enhancer Selection.

Mol Cell 2017 12;68(6):1067-1082.e12

Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA.

Enhancer elements are genomic regulatory sequences that direct the selective expression of genes so that genetically identical cells can differentiate and acquire the highly specialized forms and functions required to build a functioning animal. To differentiate, cells must select from among the ∼10 enhancers encoded in the genome the thousands of enhancers that drive the gene programs that impart their distinct features. We used a genetic approach to identify transcription factors (TFs) required for enhancer selection in fibroblasts. This revealed that the broadly expressed, growth-factor-inducible TFs FOS/JUN (AP-1) play a central role in enhancer selection. FOS/JUN selects enhancers together with cell-type-specific TFs by collaboratively binding to nucleosomal enhancers and recruiting the SWI/SNF (BAF) chromatin remodeling complex to establish accessible chromatin. These experiments demonstrate how environmental signals acting via FOS/JUN and BAF coordinate with cell-type-specific TFs to select enhancer repertoires that enable differentiation during development.
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http://dx.doi.org/10.1016/j.molcel.2017.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744881PMC
December 2017

Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates.

Nature 2017 04 5;544(7649):245-249. Epub 2017 Apr 5.

Department of Pathology and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.

Normal differentiation and induced reprogramming require the activation of target cell programs and silencing of donor cell programs. In reprogramming, the same factors are often used to reprogram many different donor cell types. As most developmental repressors, such as RE1-silencing transcription factor (REST) and Groucho (also known as TLE), are considered lineage-specific repressors, it remains unclear how identical combinations of transcription factors can silence so many different donor programs. Distinct lineage repressors would have to be induced in different donor cell types. Here, by studying the reprogramming of mouse fibroblasts to neurons, we found that the pan neuron-specific transcription factor Myt1-like (Myt1l) exerts its pro-neuronal function by direct repression of many different somatic lineage programs except the neuronal program. The repressive function of Myt1l is mediated via recruitment of a complex containing Sin3b by binding to a previously uncharacterized N-terminal domain. In agreement with its repressive function, the genomic binding sites of Myt1l are similar in neurons and fibroblasts and are preferentially in an open chromatin configuration. The Notch signalling pathway is repressed by Myt1l through silencing of several members, including Hes1. Acute knockdown of Myt1l in the developing mouse brain mimicked a Notch gain-of-function phenotype, suggesting that Myt1l allows newborn neurons to escape Notch activation during normal development. Depletion of Myt1l in primary postmitotic neurons de-repressed non-neuronal programs and impaired neuronal gene expression and function, indicating that many somatic lineage programs are actively and persistently repressed by Myt1l to maintain neuronal identity. It is now tempting to speculate that similar 'many-but-one' lineage repressors exist for other cell fates; such repressors, in combination with lineage-specific activators, would be prime candidates for use in reprogramming additional cell types.
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http://dx.doi.org/10.1038/nature21722DOI Listing
April 2017

Genome-wide identification and characterization of functional neuronal activity-dependent enhancers.

Nat Neurosci 2014 Oct 7;17(10):1330-9. Epub 2014 Sep 7.

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA.

Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in mouse cortical neurons. We find that the subset of enhancers enriched for monomethylation of histone H3 Lys4 (H3K4me1) and binding of the transcriptional coactivator CREBBP (also called CBP) that shows increased acetylation of histone H3 Lys27 (H3K27ac) after membrane depolarization of cortical neurons functions to regulate activity-dependent transcription. A subset of these enhancers appears to require binding of FOS, which was previously thought to bind primarily to promoters. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function and provide a resource of functional cis-regulatory elements that may give insight into the genetic variants that contribute to brain development and disease.
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http://dx.doi.org/10.1038/nn.3808DOI Listing
October 2014

Hierarchical mechanisms for direct reprogramming of fibroblasts to neurons.

Cell 2013 Oct 24;155(3):621-35. Epub 2013 Oct 24.

Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University, Stanford, CA 94305, USA; Program in Cancer Biology, Stanford University, Stanford, CA 94305, USA.

Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.
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http://dx.doi.org/10.1016/j.cell.2013.09.028DOI Listing
October 2013

FOXO3 shares common targets with ASCL1 genome-wide and inhibits ASCL1-dependent neurogenesis.

Cell Rep 2013 Aug 25;4(3):477-91. Epub 2013 Jul 25.

Department of Genetics, Stanford University, Stanford, CA 94305, USA.

FOXO transcription factors are central regulators of longevity from worms to humans. FOXO3, the FOXO isoform associated with exceptional human longevity, preserves adult neural stem cell pools. Here, we identify FOXO3 direct targets genome-wide in primary cultures of adult neural progenitor cells (NPCs). Interestingly, FOXO3-bound sites are enriched for motifs for bHLH transcription factors, and FOXO3 shares common targets with the proneuronal bHLH transcription factor ASCL1/MASH1 in NPCs. Analysis of the chromatin landscape reveals that FOXO3 and ASCL1 are particularly enriched at the enhancers of genes involved in neurogenic pathways. Intriguingly, FOXO3 inhibits ASCL1-dependent neurogenesis in NPCs and direct neuronal conversion in fibroblasts. FOXO3 also restrains neurogenesis in vivo. Our study identifies a genome-wide interaction between the prolongevity transcription factor FOXO3 and the cell-fate determinant ASCL1 and raises the possibility that FOXO3's ability to restrain ASCL1-dependent neurogenesis may help preserve the neural stem cell pool.
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http://dx.doi.org/10.1016/j.celrep.2013.06.035DOI Listing
August 2013

Generation of oligodendroglial cells by direct lineage conversion.

Nat Biotechnol 2013 May 14;31(5):434-9. Epub 2013 Apr 14.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.

Transplantation of oligodendrocyte precursor cells (OPCs) is a promising potential therapeutic strategy for diseases affecting myelin. However, the derivation of engraftable OPCs from human pluripotent stem cells has proven difficult and primary OPCs are not readily available. Here we report the generation of induced OPCs (iOPCs) by direct lineage conversion. Forced expression of the three transcription factors Sox10, Olig2 and Zfp536 was sufficient to reprogram mouse and rat fibroblasts into iOPCs with morphologies and gene expression signatures resembling primary OPCs. More importantly, iOPCs gave rise to mature oligodendrocytes that could ensheath multiple host axons when co-cultured with primary dorsal root ganglion cells and formed myelin after transplantation into shiverer mice. We propose direct lineage reprogramming as a viable alternative approach for the generation of OPCs for use in disease modeling and regenerative medicine.
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http://dx.doi.org/10.1038/nbt.2564DOI Listing
May 2013

Molecular roadblocks for cellular reprogramming.

Mol Cell 2012 Sep;47(6):827-38

Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, and Cancer Biology Program, Stanford University School of Medicine, Stanford, CA 94305, USA.

During development, diverse cellular identities are established and maintained in the embryo. Although remarkably robust in vivo, cellular identities can be manipulated using experimental techniques. Lineage reprogramming is an emerging field at the intersection of developmental and stem cell biology in which a somatic cell is stably reprogrammed into a distinct cell type by forced expression of lineage-determining factors. Lineage reprogramming enables the direct conversion of readily available cells from patients (such as skin fibroblasts) into disease-relevant cell types (such as neurons and cardiomyocytes) or into induced pluripotent stem cells. Although remarkable progress has been made in developing novel reprogramming methods, the efficiency and fidelity of reprogramming need to be improved in order increase the experimental and translational utility of reprogrammed cells. Studying the mechanisms that prevent successful reprogramming should allow for improvements in reprogramming methods, which could have significant implications for regenerative medicine and the study of human disease. Furthermore, lineage reprogramming has the potential to become a powerful system for dissecting the mechanisms that underlie cell fate establishment and terminal differentiation processes. In this review, we will discuss how transcription factors interface with the genome and induce changes in cellular identity in the context of development and reprogramming.
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http://dx.doi.org/10.1016/j.molcel.2012.09.008DOI Listing
September 2012

Direct lineage conversions: unnatural but useful?

Nat Biotechnol 2011 Oct;29(10):892-907

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Classic experiments such as somatic cell nuclear transfer into oocytes and cell fusion demonstrated that differentiated cells are not irreversibly committed to their fate. More recent work has built on these conclusions and discovered defined factors that directly induce one specific cell type from another, which may be as distantly related as cells from different germ layers. This suggests the possibility that any specific cell type may be directly converted into any other if the appropriate reprogramming factors are known. Direct lineage conversion could provide important new sources of human cells for modeling disease processes or for cellular-replacement therapies. For future applications, it will be critical to carefully determine the fidelity of reprogramming and to develop methods for robustly and efficiently generating human cell types of interest.
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http://dx.doi.org/10.1038/nbt.1946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3222779PMC
October 2011

Induction of human neuronal cells by defined transcription factors.

Nature 2011 May 26;476(7359):220-3. Epub 2011 May 26.

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 265 Campus Drive, Stanford, California 94305, USA.

Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.
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http://dx.doi.org/10.1038/nature10202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159048PMC
May 2011

Direct conversion of fibroblasts to functional neurons by defined factors.

Nature 2010 Feb 27;463(7284):1035-41. Epub 2010 Jan 27.

Institute for Stem Cell Biology and Regenerative Medicine, Department of Pathology, Stanford University School of Medicine, 1050 Arastradero Road, Palo Alto, California 94304, USA.

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.
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http://dx.doi.org/10.1038/nature08797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829121PMC
February 2010
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