Singh et al. reply | Nature

Singh et al. reply | Nature

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ABSTRACT Replying to: H. F. Jørgensen, Z.-F. Chen, M. Merkenschlager & A. G. Fisher _Nature_ 457, 10.1038/nature07783; N. J. Buckley, R. Johnson, Y.-M. Sun & L. W. Stanton _Nature_ 457, 10.1038/nature07784 (2009) In contrast to the comments made by Jørgensen _et al._1 and Buckley _et al._2, our experiments showed that REST maintains the self-renewal and pluripotency of mouse embryonic stem cells (mESCs)3. Two recent papers support our work: ref. 4 indicated that REST is indeed in the network that regulates ESC self-renewal and pluripotency and ref. 5 showed that mESCs with lower REST levels derived from a mouse model of Down’s syndrome have decreased levels of self-renewal markers and a higher propensity towards differentiation, even when cultured in the presence of LIF. We note that Buckley and Stanton also recently concluded that REST is part of the Oct4–Sox2–Nanog regulatory network and has “a key role in the maintenance of the ESC phenotype”6. We proposed that REST represses a set of microRNAs that potentially target self-renewal genes. At least one of them, miR-21, represses self-renewal, probably by destabilizing the messenger RNAs of Sox2 and/or Nanog (not Tbx3 or c-Myc as suggested by Buckley _et al._2). In this model, changes in the cellular environment that counter this function of REST or stimulate the mRNA levels of Sox2 or Nanog could minimize the effect of REST. For this reason, in our study we used mESCs with a low passage number, and we cultured them without feeder cells to avoid possible contributions of the feeder cells or an adaptive response to high passage. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Subscribe to this journal Receive 51 print issues and online access $199.00 per year only $3.90 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS PARALLEL GENOME-SCALE CRISPR-CAS9 SCREENS UNCOUPLE HUMAN PLURIPOTENT STEM CELL IDENTITY VERSUS FITNESS Article Open access 17 October 2024 DUSP6 IS A MEMORY RETENTION FEEDBACK REGULATOR OF ERK SIGNALING FOR CELLULAR RESILIENCE OF HUMAN PLURIPOTENT STEM CELLS IN RESPONSE TO DISSOCIATION Article Open access 07 April 2023 A RETINOID ANALOGUE, TTNPB, PROMOTES CLONAL EXPANSION OF HUMAN PLURIPOTENT STEM CELLS BY UPREGULATING _CLDN2_ AND _HOXA1_ Article Open access 16 February 2024 REFERENCES * Jørgensen, H. F., Chen, Z.-F., Merkenschlager, M. & Fisher, A. G. Is REST required for ESC pluripotency? _Nature_ 457 10.1038/nature07783 (2009) * Buckley, N. J., Johnson, R., Sun, Y.-M. & Stanton, L. W. Is REST a regulator of pluripotency? _Nature_ 457 10.1038/nature07784 (2009) * Singh, S. K., Kagalwala, M. N., Parker-Thornburg, J., Adams, H. & Majumder, S. REST maintains self-renewal and pluripotency of embryonic stem cells. _Nature_ 453, 223–227 (2008) Article  ADS  CAS  Google Scholar  * Kim, J., Chu, J., Shen, X., Wang, J. & Orkin, S. H. An extended transcriptional network for pluripotency of embryonic stem cells. _Cell_ 132, 1049–1061 (2008) Article  CAS  Google Scholar  * Canzonetta, C. et al. DYRK1A-dosage imbalance perturbs NRSF/REST levels, deregulating pluripotency and embryonic stem cell fate in Down syndrome. _Am. J. Hum. Genet._ 83, 388–400 (2008) Article  CAS  Google Scholar  * Johnson, R. et al. REST regulates distinct transcriptional networks in embryonic and neural stem cells. _PLoS Biol._ 6, e256 (2008) Article  Google Scholar  * Ying, Q. L. & Smith, A. G. Defined conditions for neural commitment and differentiation. _Methods Enzymol._ 365, 327–341 (2003) Article  CAS  Google Scholar  * Ballas, N., Grunseich, C., Lu, D. D., Speh, J. C. & Mandel, G. REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis. _Cell_ 121, 645–657 (2005) Article  CAS  Google Scholar  * Westbrook, T. F. et al. SCFbeta-TRCP controls oncogenic transformation and neural differentiation through REST degradation. _Nature_ 452, 370–374 (2008) Article  ADS  CAS  Google Scholar  * Houbaviy, H. B., Murray, M. F. & Sharp, P. A. Embryonic stem cell-specific microRNAs. _Dev. Cell_ 5, 351–358 (2003) Article  CAS  Google Scholar  * Loh, Y. H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. _Nature Genet._ 38, 431–440 (2006) Article  CAS  Google Scholar  * Chen, Z. F., Paquette, A. J. & Anderson, D. J. NRSF/REST is required _in vivo_ for repression of multiple neuronal target genes during embryogenesis. _Nature Genet._ 20, 136–142 (1998) Article  CAS  Google Scholar  * Davis, A. C. et al. A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice. _Genes Dev._ 7, 671–682 (1993) Article  CAS  Google Scholar  * Nichols, J., Chambers, I., Taga, T. & Smith, A. Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines. _Development_ 128, 2333–2339 (2001) CAS  Google Scholar  Download references AUTHOR INFORMATION Author notes * Present address: Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California 92037, USA AUTHORS AND AFFILIATIONS * Department of Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, Texas, USA Sanjay K. Singh, Mohamedi N. Kagalwala, Henry Adams & Sadhan Majumder * Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, Texas, USA Jan Parker-Thornburg * Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, Texas, USA Sadhan Majumder * The Brain Tumor Center, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, Texas, USA Sadhan Majumder * Center for Stem Cell and Developmental Biology, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, Texas, USA Sanjay K. Singh, Mohamedi N. Kagalwala & Sadhan Majumder * Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, 77030, Texas, USA Sadhan Majumder Authors * Sanjay K. Singh View author publications You can also search for this author inPubMed Google Scholar * Mohamedi N. Kagalwala View author publications You can also search for this author inPubMed Google Scholar * Jan Parker-Thornburg View author publications You can also search for this author inPubMed Google Scholar * Henry Adams View author publications You can also search for this author inPubMed Google Scholar * Sadhan Majumder View author publications You can also search for this author inPubMed Google Scholar RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Singh, S., Kagalwala, M., Parker-Thornburg, J. _et al._ _Singh_ et al. _reply_. _Nature_ 457, E7 (2009). https://doi.org/10.1038/nature07785 Download citation * Issue Date: 26 February 2009 * DOI: https://doi.org/10.1038/nature07785 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative

ABSTRACT Replying to: H. F. Jørgensen, Z.-F. Chen, M. Merkenschlager & A. G. Fisher _Nature_ 457, 10.1038/nature07783; N. J. Buckley, R. Johnson, Y.-M. Sun & L. W. Stanton _Nature_


457, 10.1038/nature07784 (2009) In contrast to the comments made by Jørgensen _et al._1 and Buckley _et al._2, our experiments showed that REST maintains the self-renewal and pluripotency of


mouse embryonic stem cells (mESCs)3. Two recent papers support our work: ref. 4 indicated that REST is indeed in the network that regulates ESC self-renewal and pluripotency and ref. 5


showed that mESCs with lower REST levels derived from a mouse model of Down’s syndrome have decreased levels of self-renewal markers and a higher propensity towards differentiation, even


when cultured in the presence of LIF. We note that Buckley and Stanton also recently concluded that REST is part of the Oct4–Sox2–Nanog regulatory network and has “a key role in the


maintenance of the ESC phenotype”6. We proposed that REST represses a set of microRNAs that potentially target self-renewal genes. At least one of them, miR-21, represses self-renewal,


probably by destabilizing the messenger RNAs of Sox2 and/or Nanog (not Tbx3 or c-Myc as suggested by Buckley _et al._2). In this model, changes in the cellular environment that counter this


function of REST or stimulate the mRNA levels of Sox2 or Nanog could minimize the effect of REST. For this reason, in our study we used mESCs with a low passage number, and we cultured them


without feeder cells to avoid possible contributions of the feeder cells or an adaptive response to high passage. Access through your institution Buy or subscribe This is a preview of


subscription content, access via your institution ACCESS OPTIONS Access through your institution Subscribe to this journal Receive 51 print issues and online access $199.00 per year only


$3.90 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout


ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS PARALLEL GENOME-SCALE


CRISPR-CAS9 SCREENS UNCOUPLE HUMAN PLURIPOTENT STEM CELL IDENTITY VERSUS FITNESS Article Open access 17 October 2024 DUSP6 IS A MEMORY RETENTION FEEDBACK REGULATOR OF ERK SIGNALING FOR


CELLULAR RESILIENCE OF HUMAN PLURIPOTENT STEM CELLS IN RESPONSE TO DISSOCIATION Article Open access 07 April 2023 A RETINOID ANALOGUE, TTNPB, PROMOTES CLONAL EXPANSION OF HUMAN PLURIPOTENT


STEM CELLS BY UPREGULATING _CLDN2_ AND _HOXA1_ Article Open access 16 February 2024 REFERENCES * Jørgensen, H. F., Chen, Z.-F., Merkenschlager, M. & Fisher, A. G. Is REST required for


ESC pluripotency? _Nature_ 457 10.1038/nature07783 (2009) * Buckley, N. J., Johnson, R., Sun, Y.-M. & Stanton, L. W. Is REST a regulator of pluripotency? _Nature_ 457 10.1038/nature07784


(2009) * Singh, S. K., Kagalwala, M. N., Parker-Thornburg, J., Adams, H. & Majumder, S. REST maintains self-renewal and pluripotency of embryonic stem cells. _Nature_ 453, 223–227


(2008) Article  ADS  CAS  Google Scholar  * Kim, J., Chu, J., Shen, X., Wang, J. & Orkin, S. H. An extended transcriptional network for pluripotency of embryonic stem cells. _Cell_ 132,


1049–1061 (2008) Article  CAS  Google Scholar  * Canzonetta, C. et al. DYRK1A-dosage imbalance perturbs NRSF/REST levels, deregulating pluripotency and embryonic stem cell fate in Down


syndrome. _Am. J. Hum. Genet._ 83, 388–400 (2008) Article  CAS  Google Scholar  * Johnson, R. et al. REST regulates distinct transcriptional networks in embryonic and neural stem cells.


_PLoS Biol._ 6, e256 (2008) Article  Google Scholar  * Ying, Q. L. & Smith, A. G. Defined conditions for neural commitment and differentiation. _Methods Enzymol._ 365, 327–341 (2003)


Article  CAS  Google Scholar  * Ballas, N., Grunseich, C., Lu, D. D., Speh, J. C. & Mandel, G. REST and its corepressors mediate plasticity of neuronal gene chromatin throughout


neurogenesis. _Cell_ 121, 645–657 (2005) Article  CAS  Google Scholar  * Westbrook, T. F. et al. SCFbeta-TRCP controls oncogenic transformation and neural differentiation through REST


degradation. _Nature_ 452, 370–374 (2008) Article  ADS  CAS  Google Scholar  * Houbaviy, H. B., Murray, M. F. & Sharp, P. A. Embryonic stem cell-specific microRNAs. _Dev. Cell_ 5,


351–358 (2003) Article  CAS  Google Scholar  * Loh, Y. H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. _Nature Genet._ 38, 431–440


(2006) Article  CAS  Google Scholar  * Chen, Z. F., Paquette, A. J. & Anderson, D. J. NRSF/REST is required _in vivo_ for repression of multiple neuronal target genes during


embryogenesis. _Nature Genet._ 20, 136–142 (1998) Article  CAS  Google Scholar  * Davis, A. C. et al. A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and


reduced fertility in heterozygous female mice. _Genes Dev._ 7, 671–682 (1993) Article  CAS  Google Scholar  * Nichols, J., Chambers, I., Taga, T. & Smith, A. Physiological rationale for


responsiveness of mouse embryonic stem cells to gp130 cytokines. _Development_ 128, 2333–2339 (2001) CAS  Google Scholar  Download references AUTHOR INFORMATION Author notes * Present


address: Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, California 92037, USA AUTHORS AND AFFILIATIONS * Department of Genetics, The University of Texas M. D.


Anderson Cancer Center, Houston, 77030, Texas, USA Sanjay K. Singh, Mohamedi N. Kagalwala, Henry Adams & Sadhan Majumder * Department of Biochemistry and Molecular Biology, The


University of Texas M. D. Anderson Cancer Center, Houston, 77030, Texas, USA Jan Parker-Thornburg * Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center,


Houston, 77030, Texas, USA Sadhan Majumder * The Brain Tumor Center, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, Texas, USA Sadhan Majumder * Center for Stem Cell


and Developmental Biology, The University of Texas M. D. Anderson Cancer Center, Houston, 77030, Texas, USA Sanjay K. Singh, Mohamedi N. Kagalwala & Sadhan Majumder * Program in Genes


and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, 77030, Texas, USA Sadhan Majumder Authors * Sanjay K. Singh View author publications You


can also search for this author inPubMed Google Scholar * Mohamedi N. Kagalwala View author publications You can also search for this author inPubMed Google Scholar * Jan Parker-Thornburg


View author publications You can also search for this author inPubMed Google Scholar * Henry Adams View author publications You can also search for this author inPubMed Google Scholar *


Sadhan Majumder View author publications You can also search for this author inPubMed Google Scholar RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE


Singh, S., Kagalwala, M., Parker-Thornburg, J. _et al._ _Singh_ et al. _reply_. _Nature_ 457, E7 (2009). https://doi.org/10.1038/nature07785 Download citation * Issue Date: 26 February 2009


* DOI: https://doi.org/10.1038/nature07785 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not


currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative