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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.

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JAK2 is present in the nucleus of ES cells and JAK2 dynamically regulates HP1α access to chromatin by phosphorylating H3Y41.a. Immunohistochemistry for phosphorylated JAK2 in wild type ES cells growing in 2i. Orthogonal view confirms the presence of phosphorylated JAK2 in the nucleus. Scale bar 20μm.b. Immunohistochemistry confirms that HP1α was present at lower levels in JAK2V617F ES cells compared to parental cells when they are maintained in multiple ES cell conditions, but Oct4 remains unchanged. Scale bar 20μm.c. Immunohistochemistry for HP1α and Nanog in steady state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 hours. There was a significant increase in the level of HP1α and decrease in Nanog following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number. Scale bar 20μm.d. Immunohistochemistry for H3Y41ph and Nanog in steady state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 hours. There was a significant decrease in the levels of H3Y41ph and Nanog following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number. Scale bar 20μm.
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Figure 4: JAK2 is present in the nucleus of ES cells and JAK2 dynamically regulates HP1α access to chromatin by phosphorylating H3Y41.a. Immunohistochemistry for phosphorylated JAK2 in wild type ES cells growing in 2i. Orthogonal view confirms the presence of phosphorylated JAK2 in the nucleus. Scale bar 20μm.b. Immunohistochemistry confirms that HP1α was present at lower levels in JAK2V617F ES cells compared to parental cells when they are maintained in multiple ES cell conditions, but Oct4 remains unchanged. Scale bar 20μm.c. Immunohistochemistry for HP1α and Nanog in steady state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 hours. There was a significant increase in the level of HP1α and decrease in Nanog following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number. Scale bar 20μm.d. Immunohistochemistry for H3Y41ph and Nanog in steady state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 hours. There was a significant decrease in the levels of H3Y41ph and Nanog following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number. Scale bar 20μm.

Mentions: Studies in Drosophila have shown that JAK signalling globally counteracts heterochromatic gene silencing by antagonising the function of heterochromatin protein 1 (HP1)31,32. Moreover, we have recently identified a novel role for JAK2 in the nucleus of haematopoietic cells where it can phosphorylate tyrosine 41 of histone H3 (H3Y41) which interferes with HP1α binding14 and thus provides a molecular explanation for the JAK2 activity uncovered from Drosophila genetics. Phosphorylated JAK2 was present in the nucleus of ES cells (fig 4a) and we therefore investigated whether JAK2V617F alters the distribution of HP1α. Chromatin bound HP1α, but not Oct4, was lower in JAK2 mutant compared to wild type ES cells, under multiple growth conditions (fig 4b).


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)

JAK2 is present in the nucleus of ES cells and JAK2 dynamically regulates HP1α access to chromatin by phosphorylating H3Y41.a. Immunohistochemistry for phosphorylated JAK2 in wild type ES cells growing in 2i. Orthogonal view confirms the presence of phosphorylated JAK2 in the nucleus. Scale bar 20μm.b. Immunohistochemistry confirms that HP1α was present at lower levels in JAK2V617F ES cells compared to parental cells when they are maintained in multiple ES cell conditions, but Oct4 remains unchanged. Scale bar 20μm.c. Immunohistochemistry for HP1α and Nanog in steady state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 hours. There was a significant increase in the level of HP1α and decrease in Nanog following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number. Scale bar 20μm.d. Immunohistochemistry for H3Y41ph and Nanog in steady state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 hours. There was a significant decrease in the levels of H3Y41ph and Nanog following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number. Scale bar 20μm.
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Related In: Results  -  Collection

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Figure 4: JAK2 is present in the nucleus of ES cells and JAK2 dynamically regulates HP1α access to chromatin by phosphorylating H3Y41.a. Immunohistochemistry for phosphorylated JAK2 in wild type ES cells growing in 2i. Orthogonal view confirms the presence of phosphorylated JAK2 in the nucleus. Scale bar 20μm.b. Immunohistochemistry confirms that HP1α was present at lower levels in JAK2V617F ES cells compared to parental cells when they are maintained in multiple ES cell conditions, but Oct4 remains unchanged. Scale bar 20μm.c. Immunohistochemistry for HP1α and Nanog in steady state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 hours. There was a significant increase in the level of HP1α and decrease in Nanog following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number. Scale bar 20μm.d. Immunohistochemistry for H3Y41ph and Nanog in steady state factor-independent JAK2V617F ES cells and following treatment with TG101209 for 2 hours. There was a significant decrease in the levels of H3Y41ph and Nanog following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number. Scale bar 20μm.
Mentions: Studies in Drosophila have shown that JAK signalling globally counteracts heterochromatic gene silencing by antagonising the function of heterochromatin protein 1 (HP1)31,32. Moreover, we have recently identified a novel role for JAK2 in the nucleus of haematopoietic cells where it can phosphorylate tyrosine 41 of histone H3 (H3Y41) which interferes with HP1α binding14 and thus provides a molecular explanation for the JAK2 activity uncovered from Drosophila genetics. Phosphorylated JAK2 was present in the nucleus of ES cells (fig 4a) and we therefore investigated whether JAK2V617F alters the distribution of HP1α. Chromatin bound HP1α, but not Oct4, was lower in JAK2 mutant compared to wild type ES cells, under multiple growth conditions (fig 4b).

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