Limits...
Variability of Gene Expression Identifies Transcriptional Regulators of Early Human Embryonic Development.

Hasegawa Y, Taylor D, Ovchinnikov DA, Wolvetang EJ, de Torrenté L, Mar JC - PLoS Genet. (2015)

Bottom Line: Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression.The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes.Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America; Division of Life Science, Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.

ABSTRACT
An analysis of gene expression variability can provide an insightful window into how regulatory control is distributed across the transcriptome. In a single cell analysis, the inter-cellular variability of gene expression measures the consistency of transcript copy numbers observed between cells in the same population. Application of these ideas to the study of early human embryonic development may reveal important insights into the transcriptional programs controlling this process, based on which components are most tightly regulated. Using a published single cell RNA-seq data set of human embryos collected at four-cell, eight-cell, morula and blastocyst stages, we identified genes with the most stable, invariant expression across all four developmental stages. Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression. The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes. Genes with low expression variability at early stages of development are involved in regulation of DNA methylation, responses to hypoxia and telomerase activity, whereas by the blastocyst stage, low-variability genes are enriched for metabolic processes as well as telomerase signaling. Based on changes in expression variability, we identified a putative set of gene expression markers of morulae and blastocyst stages. Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells. Collectively our analyses identified new regulators involved in human embryonic development that would have otherwise been missed using methods that focus on assessment of the average expression levels; in doing so, we highlight the value of studying expression variability for single cell RNA-seq data.

No MeSH data available.


Related in: MedlinePlus

Activation of the endogenous HDDC2 locus using an inducible Cas9-VP64 system attenuates neural differentiation of human pluripotent stem cells.(A) Upregulation of the HDDC2 mRNA levels after 2 days of induction of activation in pluripotent hES cells. (B-D) Effect of Cas9-VP64-driven HDDC2 up-regulation on gene expression during the early stages (day 3) of neural differentiation. We observed that artificially-maintained levels of HDDC2 expression (B) resulted in more sustained NANOG expression (C) and lower induction of PAX6(D), a definitive neuroectodermal marker.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4546122&req=5

pgen.1005428.g007: Activation of the endogenous HDDC2 locus using an inducible Cas9-VP64 system attenuates neural differentiation of human pluripotent stem cells.(A) Upregulation of the HDDC2 mRNA levels after 2 days of induction of activation in pluripotent hES cells. (B-D) Effect of Cas9-VP64-driven HDDC2 up-regulation on gene expression during the early stages (day 3) of neural differentiation. We observed that artificially-maintained levels of HDDC2 expression (B) resulted in more sustained NANOG expression (C) and lower induction of PAX6(D), a definitive neuroectodermal marker.

Mentions: To demonstrate the functional impact of the stage-specific variability markers, we validated one of the blastocyst variability markers, HDDC2, by shRNA-mediated knockdown in a human iPSC line. qPCR experiments confirmed that the knockdown of HDDC2 mRNA caused a significant decrease in the expression of key pluripotency markers DNMT3B and NANOG, two genes that are critical for embryonic development and maintenance of pluripotency in iPSCs (see Fig 6). We also tested the impact of the up-regulation of the HDDC2 locus on hESC differentiation using a hESC line with a stably-integrated inducible CRISPRa/Cas9-VP64 artificial transcriptional activator system. Over-expression of HDDC2 attenuates the drop in expression of the pluripotency marker NANOG and induction of the neuroepithelial marker PAX6 during the early stages of neural differentiation (see Fig 7). While these experiments cannot provide evidence for the effects of HDDC2 on cell viability or embryonic development, the results from the HDDC2 knockdown suggest that this gene plays a role in the maintenance of pluripotency. From the transcriptional effects caused by HDDC2 over-expression on early neural differentiation, we can infer that HDDC2 is able to either reinforce the persistence of the pluripotent phenotype, or is involved with specific interference of the neural differentiation process, or both.


Variability of Gene Expression Identifies Transcriptional Regulators of Early Human Embryonic Development.

Hasegawa Y, Taylor D, Ovchinnikov DA, Wolvetang EJ, de Torrenté L, Mar JC - PLoS Genet. (2015)

Activation of the endogenous HDDC2 locus using an inducible Cas9-VP64 system attenuates neural differentiation of human pluripotent stem cells.(A) Upregulation of the HDDC2 mRNA levels after 2 days of induction of activation in pluripotent hES cells. (B-D) Effect of Cas9-VP64-driven HDDC2 up-regulation on gene expression during the early stages (day 3) of neural differentiation. We observed that artificially-maintained levels of HDDC2 expression (B) resulted in more sustained NANOG expression (C) and lower induction of PAX6(D), a definitive neuroectodermal marker.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4546122&req=5

pgen.1005428.g007: Activation of the endogenous HDDC2 locus using an inducible Cas9-VP64 system attenuates neural differentiation of human pluripotent stem cells.(A) Upregulation of the HDDC2 mRNA levels after 2 days of induction of activation in pluripotent hES cells. (B-D) Effect of Cas9-VP64-driven HDDC2 up-regulation on gene expression during the early stages (day 3) of neural differentiation. We observed that artificially-maintained levels of HDDC2 expression (B) resulted in more sustained NANOG expression (C) and lower induction of PAX6(D), a definitive neuroectodermal marker.
Mentions: To demonstrate the functional impact of the stage-specific variability markers, we validated one of the blastocyst variability markers, HDDC2, by shRNA-mediated knockdown in a human iPSC line. qPCR experiments confirmed that the knockdown of HDDC2 mRNA caused a significant decrease in the expression of key pluripotency markers DNMT3B and NANOG, two genes that are critical for embryonic development and maintenance of pluripotency in iPSCs (see Fig 6). We also tested the impact of the up-regulation of the HDDC2 locus on hESC differentiation using a hESC line with a stably-integrated inducible CRISPRa/Cas9-VP64 artificial transcriptional activator system. Over-expression of HDDC2 attenuates the drop in expression of the pluripotency marker NANOG and induction of the neuroepithelial marker PAX6 during the early stages of neural differentiation (see Fig 7). While these experiments cannot provide evidence for the effects of HDDC2 on cell viability or embryonic development, the results from the HDDC2 knockdown suggest that this gene plays a role in the maintenance of pluripotency. From the transcriptional effects caused by HDDC2 over-expression on early neural differentiation, we can infer that HDDC2 is able to either reinforce the persistence of the pluripotent phenotype, or is involved with specific interference of the neural differentiation process, or both.

Bottom Line: Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression.The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes.Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America; Division of Life Science, Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.

ABSTRACT
An analysis of gene expression variability can provide an insightful window into how regulatory control is distributed across the transcriptome. In a single cell analysis, the inter-cellular variability of gene expression measures the consistency of transcript copy numbers observed between cells in the same population. Application of these ideas to the study of early human embryonic development may reveal important insights into the transcriptional programs controlling this process, based on which components are most tightly regulated. Using a published single cell RNA-seq data set of human embryos collected at four-cell, eight-cell, morula and blastocyst stages, we identified genes with the most stable, invariant expression across all four developmental stages. Stably-expressed genes were found to be enriched for those sharing indispensable features, including essentiality, haploinsufficiency, and ubiquitous expression. The stable genes were less likely to be associated with loss-of-function variant genes or human recessive disease genes affected by a DNA copy number variant deletion, suggesting that stable genes have a functional impact on the regulation of some of the basic cellular processes. Genes with low expression variability at early stages of development are involved in regulation of DNA methylation, responses to hypoxia and telomerase activity, whereas by the blastocyst stage, low-variability genes are enriched for metabolic processes as well as telomerase signaling. Based on changes in expression variability, we identified a putative set of gene expression markers of morulae and blastocyst stages. Experimental validation of a blastocyst-expressed variability marker demonstrated that HDDC2 plays a role in the maintenance of pluripotency in human ES and iPS cells. Collectively our analyses identified new regulators involved in human embryonic development that would have otherwise been missed using methods that focus on assessment of the average expression levels; in doing so, we highlight the value of studying expression variability for single cell RNA-seq data.

No MeSH data available.


Related in: MedlinePlus