Limits...
LIF-independent JAK signalling to chromatin in embryonic stem cells uncovered from an adult stem cell disease.

Griffiths DS, Li J, Dawson MA, Trotter MW, Cheng YH, Smith AM, Mansfield W, Liu P, Kouzarides T, Nichols J, Bannister AJ, Green AR, Göttgens B - Nat. Cell Biol. (2010)

Bottom Line: Leukaemia inhibitory factor (LIF) signalling through the JAK-signal transducer and activator of transcription (STAT) pathway enables self-renewal of embryonic stem (ES) cells.Furthermore, Nanog was required for factor independence of JAK2V617F ES cells.Taken together, these results uncover a previously unrecognized role for direct signalling to chromatin by JAK2 as an important mediator of ES cell self-renewal.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematology and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK. dsg29@cam.ac.uk

ABSTRACT
Activating mutations in the tyrosine kinase Janus kinase 2 (JAK2) cause myeloproliferative neoplasms, clonal blood stem cell disorders with a propensity for leukaemic transformation. Leukaemia inhibitory factor (LIF) signalling through the JAK-signal transducer and activator of transcription (STAT) pathway enables self-renewal of embryonic stem (ES) cells. Here we show that mouse ES cells carrying the human JAK2V617F mutation were able to self-renew in chemically defined conditions without cytokines or small-molecule inhibitors, independently of JAK signalling through the STAT3 or phosphatidylinositol-3-OH kinase pathways. Phosphorylation of histone H3 tyrosine 41 (H3Y41) by JAK2 was recently shown to interfere with binding of heterochromatin protein 1α (HP1α). Levels of chromatin-bound HP1α were lower in JAK2V617F ES cells but increased following inhibition of JAK2, coincident with a global reduction in histone H3Y41 phosphorylation. JAK2 inhibition reduced levels of the pluripotency regulator Nanog, with a reduction in H3Y41 phosphorylation and concomitant increase in HP1α levels at the Nanog promoter. Furthermore, Nanog was required for factor independence of JAK2V617F ES cells. Taken together, these results uncover a previously unrecognized role for direct signalling to chromatin by JAK2 as an important mediator of ES cell self-renewal.

Show MeSH

Related in: MedlinePlus

JAK2V617F sustains ES cells in a self-renewing state without any additional factorsa. Targeting strategy to insert patient cDNA containing JAK2V617F mutation into the jak2 allele by homologous recombination.b. JAK2V617F ES cells are made factor-independent by transferring ES cells growing in N2B27 plus LIF and BMP4 into N2B27 only. The ES cells then undergo a crisis; detaching and forming spheres, these spheres can then be reattached by transferring into fresh N2B27 on freshly gelatinised flasks.c. ES cells were plated at 1 × 103 cells per well of a 12 well plate, 6 days later the cells were fixed and stained for alkaline phosphatase to identify ES cell colonies. Only factor-independent JAK2V617F ES cells (JAK2V617F) can form colonies in N2B27 only, this ability is lost following the addition of AG490.d. Parental ES cells grown in N2B27 plus LIF and BMP4 have a similar pattern of staining for the key ES cell transcription factors Nanog and Oct4 to factor-independent JAK2V617F ES cells in N2B27 alone. Of note, factor-independent JAK2V617F cells have the characteristic variable levels of Nanog seen with wild type ES cells. Scale bar 20μm.e. Microarray analysis of the three ES cell lines demonstrates that the majority of expressed genes are common to all three ES cell lines. Correlation coefficients were calculated using the mean of each two-way comparisons and show a strong correlation coefficient between all three datasets (red values).f. Genes known to be critical for ES cell self-renewal are expressed at similar levels in all datasets, and there is no up-regulation of genes expressed in more differentiated cell types. Values are mean of three biological replicates, error bars represent S.E.M.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3008749&req=5

Figure 1: JAK2V617F sustains ES cells in a self-renewing state without any additional factorsa. Targeting strategy to insert patient cDNA containing JAK2V617F mutation into the jak2 allele by homologous recombination.b. JAK2V617F ES cells are made factor-independent by transferring ES cells growing in N2B27 plus LIF and BMP4 into N2B27 only. The ES cells then undergo a crisis; detaching and forming spheres, these spheres can then be reattached by transferring into fresh N2B27 on freshly gelatinised flasks.c. ES cells were plated at 1 × 103 cells per well of a 12 well plate, 6 days later the cells were fixed and stained for alkaline phosphatase to identify ES cell colonies. Only factor-independent JAK2V617F ES cells (JAK2V617F) can form colonies in N2B27 only, this ability is lost following the addition of AG490.d. Parental ES cells grown in N2B27 plus LIF and BMP4 have a similar pattern of staining for the key ES cell transcription factors Nanog and Oct4 to factor-independent JAK2V617F ES cells in N2B27 alone. Of note, factor-independent JAK2V617F cells have the characteristic variable levels of Nanog seen with wild type ES cells. Scale bar 20μm.e. Microarray analysis of the three ES cell lines demonstrates that the majority of expressed genes are common to all three ES cell lines. Correlation coefficients were calculated using the mean of each two-way comparisons and show a strong correlation coefficient between all three datasets (red values).f. Genes known to be critical for ES cell self-renewal are expressed at similar levels in all datasets, and there is no up-regulation of genes expressed in more differentiated cell types. Values are mean of three biological replicates, error bars represent S.E.M.

Mentions: To gain new insights into the molecular consequences of the JAK2V617F mutation, a human JAK2 cDNA containing the V617F mutation was introduced by homologous recombination into the jak2 locus of murine embryonic stem (ES) cells (fig 1a). The mutant cDNA was under the normal regulatory control of endogenous jak2 and the JAK2V617F allele was expressed at an equal level to the wild type allele15. ES cells can be maintained in chemically defined media with two small molecule inhibitors of ERK and GSK3 signalling; known as 2i13. JAK signalling in this context was thought to be unimportant because 2i obviates the requirement for STAT3 phosphorylation by JAK kinases13. However, when JAK2V617F ES cells were grown in 2i conditions at clonal density, there was a substantial increase in the number of ES cell colonies compared to wild type ES cells. This observation lead us to hypothesise that there may be a previously unknown requirement for Janus kinase signalling in ES cells.


LIF-independent JAK signalling to chromatin in embryonic stem cells uncovered from an adult stem cell disease.

Griffiths DS, Li J, Dawson MA, Trotter MW, Cheng YH, Smith AM, Mansfield W, Liu P, Kouzarides T, Nichols J, Bannister AJ, Green AR, Göttgens B - Nat. Cell Biol. (2010)

JAK2V617F sustains ES cells in a self-renewing state without any additional factorsa. Targeting strategy to insert patient cDNA containing JAK2V617F mutation into the jak2 allele by homologous recombination.b. JAK2V617F ES cells are made factor-independent by transferring ES cells growing in N2B27 plus LIF and BMP4 into N2B27 only. The ES cells then undergo a crisis; detaching and forming spheres, these spheres can then be reattached by transferring into fresh N2B27 on freshly gelatinised flasks.c. ES cells were plated at 1 × 103 cells per well of a 12 well plate, 6 days later the cells were fixed and stained for alkaline phosphatase to identify ES cell colonies. Only factor-independent JAK2V617F ES cells (JAK2V617F) can form colonies in N2B27 only, this ability is lost following the addition of AG490.d. Parental ES cells grown in N2B27 plus LIF and BMP4 have a similar pattern of staining for the key ES cell transcription factors Nanog and Oct4 to factor-independent JAK2V617F ES cells in N2B27 alone. Of note, factor-independent JAK2V617F cells have the characteristic variable levels of Nanog seen with wild type ES cells. Scale bar 20μm.e. Microarray analysis of the three ES cell lines demonstrates that the majority of expressed genes are common to all three ES cell lines. Correlation coefficients were calculated using the mean of each two-way comparisons and show a strong correlation coefficient between all three datasets (red values).f. Genes known to be critical for ES cell self-renewal are expressed at similar levels in all datasets, and there is no up-regulation of genes expressed in more differentiated cell types. Values are mean of three biological replicates, error bars represent S.E.M.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008749&req=5

Figure 1: JAK2V617F sustains ES cells in a self-renewing state without any additional factorsa. Targeting strategy to insert patient cDNA containing JAK2V617F mutation into the jak2 allele by homologous recombination.b. JAK2V617F ES cells are made factor-independent by transferring ES cells growing in N2B27 plus LIF and BMP4 into N2B27 only. The ES cells then undergo a crisis; detaching and forming spheres, these spheres can then be reattached by transferring into fresh N2B27 on freshly gelatinised flasks.c. ES cells were plated at 1 × 103 cells per well of a 12 well plate, 6 days later the cells were fixed and stained for alkaline phosphatase to identify ES cell colonies. Only factor-independent JAK2V617F ES cells (JAK2V617F) can form colonies in N2B27 only, this ability is lost following the addition of AG490.d. Parental ES cells grown in N2B27 plus LIF and BMP4 have a similar pattern of staining for the key ES cell transcription factors Nanog and Oct4 to factor-independent JAK2V617F ES cells in N2B27 alone. Of note, factor-independent JAK2V617F cells have the characteristic variable levels of Nanog seen with wild type ES cells. Scale bar 20μm.e. Microarray analysis of the three ES cell lines demonstrates that the majority of expressed genes are common to all three ES cell lines. Correlation coefficients were calculated using the mean of each two-way comparisons and show a strong correlation coefficient between all three datasets (red values).f. Genes known to be critical for ES cell self-renewal are expressed at similar levels in all datasets, and there is no up-regulation of genes expressed in more differentiated cell types. Values are mean of three biological replicates, error bars represent S.E.M.
Mentions: To gain new insights into the molecular consequences of the JAK2V617F mutation, a human JAK2 cDNA containing the V617F mutation was introduced by homologous recombination into the jak2 locus of murine embryonic stem (ES) cells (fig 1a). The mutant cDNA was under the normal regulatory control of endogenous jak2 and the JAK2V617F allele was expressed at an equal level to the wild type allele15. ES cells can be maintained in chemically defined media with two small molecule inhibitors of ERK and GSK3 signalling; known as 2i13. JAK signalling in this context was thought to be unimportant because 2i obviates the requirement for STAT3 phosphorylation by JAK kinases13. However, when JAK2V617F ES cells were grown in 2i conditions at clonal density, there was a substantial increase in the number of ES cell colonies compared to wild type ES cells. This observation lead us to hypothesise that there may be a previously unknown requirement for Janus kinase signalling in ES cells.

Bottom Line: Leukaemia inhibitory factor (LIF) signalling through the JAK-signal transducer and activator of transcription (STAT) pathway enables self-renewal of embryonic stem (ES) cells.Furthermore, Nanog was required for factor independence of JAK2V617F ES cells.Taken together, these results uncover a previously unrecognized role for direct signalling to chromatin by JAK2 as an important mediator of ES cell self-renewal.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematology and Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK. dsg29@cam.ac.uk

ABSTRACT
Activating mutations in the tyrosine kinase Janus kinase 2 (JAK2) cause myeloproliferative neoplasms, clonal blood stem cell disorders with a propensity for leukaemic transformation. Leukaemia inhibitory factor (LIF) signalling through the JAK-signal transducer and activator of transcription (STAT) pathway enables self-renewal of embryonic stem (ES) cells. Here we show that mouse ES cells carrying the human JAK2V617F mutation were able to self-renew in chemically defined conditions without cytokines or small-molecule inhibitors, independently of JAK signalling through the STAT3 or phosphatidylinositol-3-OH kinase pathways. Phosphorylation of histone H3 tyrosine 41 (H3Y41) by JAK2 was recently shown to interfere with binding of heterochromatin protein 1α (HP1α). Levels of chromatin-bound HP1α were lower in JAK2V617F ES cells but increased following inhibition of JAK2, coincident with a global reduction in histone H3Y41 phosphorylation. JAK2 inhibition reduced levels of the pluripotency regulator Nanog, with a reduction in H3Y41 phosphorylation and concomitant increase in HP1α levels at the Nanog promoter. Furthermore, Nanog was required for factor independence of JAK2V617F ES cells. Taken together, these results uncover a previously unrecognized role for direct signalling to chromatin by JAK2 as an important mediator of ES cell self-renewal.

Show MeSH
Related in: MedlinePlus