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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 not the only JAK which can phosphorylate H3Y41a. Immunohistochemistry of JAK2 Null ES cells growing in 2i media demonstrated that H3Y41 is phosphorylated in the absence of JAK2, which in turn was dynamically regulated by JAK inhibition treatment with AG490 for 16 hours. There was a significant decrease in the levels of H3Y41ph and Oct4 following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number.b. Orthogonal view of immunohistochemistry for phosphorylated JAK1 in wild type ES cells maintained in N2B27 plus 2i.
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Figure 6: JAK2 is not the only JAK which can phosphorylate H3Y41a. Immunohistochemistry of JAK2 Null ES cells growing in 2i media demonstrated that H3Y41 is phosphorylated in the absence of JAK2, which in turn was dynamically regulated by JAK inhibition treatment with AG490 for 16 hours. There was a significant decrease in the levels of H3Y41ph and Oct4 following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number.b. Orthogonal view of immunohistochemistry for phosphorylated JAK1 in wild type ES cells maintained in N2B27 plus 2i.

Mentions: Knock-outs of single Janus kinase family genes can survive beyond the epiblast stage of development 37-39, which in light of our JAK inhibitor data suggest functional redundancy. We therefore wanted to ascertain if this new regulatory role for JAK2 signalling in ES cell self-renewal can also be conferred by other Janus kinase family members. We derived JAK2 ES cells (Supplementary figure 3), in order to identify whether H3Y41 was phosphorylated independently from JAK2. H3Y41ph was present in JAK2 ES cells, and was dynamically regulated by inhibiting JAK signalling. Moreover, there was a significant reduction in the global level of H3Y41ph (fig 6a) following treatment with AG490, which was accompanied by differentiation of these inhibitor-treated JAK2 ES cells, as seen by loss of Oct4 expression (figure 6a). JAK1 has long been implicated in ES cell self-renewal 40. We now show that JAK1 was also present in the nucleus of wild type ES cells grown in 2i (fig 6b) and that JAK1 can directly phosphorylate H3Y41 (Supplementary figure 6). Moreover, ES cells express all Janus kinase family members (Supplementary figure 2), and JAKs other than JAK1 and JAK2 may also be involved in phosphorylating H3Y41. Our observation that H3Y41 can be phosphorylated by JAKs other than Jak2 coupled with our analysis of Jak2 ES-cells suggests that while this pathway was discovered using JAK2, it likely involves multiple members of the Janus kinase family.


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 not the only JAK which can phosphorylate H3Y41a. Immunohistochemistry of JAK2 Null ES cells growing in 2i media demonstrated that H3Y41 is phosphorylated in the absence of JAK2, which in turn was dynamically regulated by JAK inhibition treatment with AG490 for 16 hours. There was a significant decrease in the levels of H3Y41ph and Oct4 following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number.b. Orthogonal view of immunohistochemistry for phosphorylated JAK1 in wild type ES cells maintained in N2B27 plus 2i.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: JAK2 is not the only JAK which can phosphorylate H3Y41a. Immunohistochemistry of JAK2 Null ES cells growing in 2i media demonstrated that H3Y41 is phosphorylated in the absence of JAK2, which in turn was dynamically regulated by JAK inhibition treatment with AG490 for 16 hours. There was a significant decrease in the levels of H3Y41ph and Oct4 following inhibitor treatment, two independent experiments combined in box and whisker plot, difference determined by Students T-Test. N is cell number.b. Orthogonal view of immunohistochemistry for phosphorylated JAK1 in wild type ES cells maintained in N2B27 plus 2i.
Mentions: Knock-outs of single Janus kinase family genes can survive beyond the epiblast stage of development 37-39, which in light of our JAK inhibitor data suggest functional redundancy. We therefore wanted to ascertain if this new regulatory role for JAK2 signalling in ES cell self-renewal can also be conferred by other Janus kinase family members. We derived JAK2 ES cells (Supplementary figure 3), in order to identify whether H3Y41 was phosphorylated independently from JAK2. H3Y41ph was present in JAK2 ES cells, and was dynamically regulated by inhibiting JAK signalling. Moreover, there was a significant reduction in the global level of H3Y41ph (fig 6a) following treatment with AG490, which was accompanied by differentiation of these inhibitor-treated JAK2 ES cells, as seen by loss of Oct4 expression (figure 6a). JAK1 has long been implicated in ES cell self-renewal 40. We now show that JAK1 was also present in the nucleus of wild type ES cells grown in 2i (fig 6b) and that JAK1 can directly phosphorylate H3Y41 (Supplementary figure 6). Moreover, ES cells express all Janus kinase family members (Supplementary figure 2), and JAKs other than JAK1 and JAK2 may also be involved in phosphorylating H3Y41. Our observation that H3Y41 can be phosphorylated by JAKs other than Jak2 coupled with our analysis of Jak2 ES-cells suggests that while this pathway was discovered using JAK2, it likely involves multiple members of the Janus kinase family.

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