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ESCs JNK their way to Neurogenesis – A chromatin-modifying function of JNK during stem cell differentiation

From Nature Genetics
By Stuart P. Atkinson

c-Jun N-terminal kinases (JNKs) are a subgroup of mitogen-activated protein kinases (MAPKs), and have been suggested to mediate transcriptional changes through their downstream effectors (Bogoyevitch and Kobe and Pearson et al), such as AP-1 (a heterodimeric transcription factor composed of proteins belonging to the c-Fos, c-Jun, ATF and JDP families). Additionally, some signal-activated kinases have been suggested to bind to chromatin at certain genes. Mouse embryonic stem cells (mESCs) lacking JNK1 (Mapk8) show dysregulation of lineage-commitment genes (Amura et al) and fail to undergo neuronal differentiation, suggesting a vital role for JNK1 in the lineage specific differentiation of mESCs. Researchers from the group of Christian Beisel from the Eidgenössische Technische Hochschule (ETH) Zürich, Basel, Switzerland have now undertaken a detailed analysis of the JNK proteins in mESCs during self renewal and differentiation, finding that gene specific JNK-mediated chromatin modification is critical for neuronal differentiation (Tiwari et al).

JNK function was studied during the process of the differentiation of mESCs to committed neuronal progenitors (NPs) and further to terminally differentiated pyramidal glutamatergic neurons (TNs). At the mRNA and protein levels, JNK2 (Mapk9) remained constant during differentiation while levels of JNK1 and JNK3 (Mapk10) rose in TNs, where JNK phosphorylation was uniquely detected. Further, the JNK proteins were localised to the nucleus in TNs suggesting that they may bind to chromatin and mediate gene expression changes during differentiation. Subsequent chromatin immunoprecipitation (ChIP) analysis found genome-wide JNK enrichment in cells throughout the differentiation process, mainly at promoter regions and near transcription start sites; a finding also observed when similar ChIP assays were undertaken in whole brains of 3.5-week-old adult mice. Detailed analysis found that JNK1/3 target genes became specifically enriched for JNK binding during differentiation, concomitant with increases in JNK1/3 expression and phosphorylation and, therefore, activation of JNKs. Further analysis of JNK target genes found a significant enrichment for genes involved in developmental processes, and said genes were also found to be characteristic of canonical JNK signaling biology (Weston and Davis). JNK target genes also demonstrated several characteristics of active genes, such as having high RNA polymerase II (RNAPII) occupancy and an elevated occurrence of the permissive H3K4me2 chromatin mark and a low level of the repressive H3K27me3 chromatin mark.

Surprisingly, analysis of binding site motifs surrounding JNK enriched areas did not find any AP-1 binding sites even though many genes responsive to JNK signaling harbour AP-1 consensus sequences in their promoter regions and are known targets of transcription factors, such as the Jun and Fos dimers that function in a JNK-dependent fashion (Karin and Gallagher and Shaulian). However, sequence analysis found two enriched motifs; a consensus site for NF-Y and an SP-1-like motif, and analysis of published NF-Y binding data revealed a close correspondence to the JNK ChIP data. Detailed analysis of NF-YA, the DNA-binding subunit of the trimeric NF-Y complex, found overlapping JNK binding throughout differentiation and increased signal strength in TNs. Further proof of the role of NF-YA in recruiting JNK1/3 was demonstrated by the overexpression of a dominant-negative repressor of NF-YA in HEK293 cells from human embryonic kidney, which led to a significant reduction in JNK binding at target gene promoters.

Specific inhibition of JNK signalling in mESC did not affect pluripotency, in accordance with its inactivity in mESCs, but when JNK signalling was inhibited during differentiation, neurogenesis was severely abrogated and massive cell death was observed suggesting that JNK kinase activity is crucial for terminal neuronal differentiation.   This suggests that phosphorylation by JNK1/3 when recruited to chromatin is essential for neurogenesis, and through histone substrate screens, Serine 10 on Histone H3 (H3S10) was found to be a major target, with subsequent ChIP analysis confirming an increase in H3S10 phosphorylation at JNK target genes.   JNK inhibition in TNs led to a significant decrease in JNK phosphorylation, although without cell death, and this was found to correspond with preferential down-regulation of JNK target genes, although this only affected genes expressed at low levels, and not highly expressed genes.

Overall, this study suggests a new pathway in mammalian cells, whereby MAPKs directly regulate gene expression at the chromatin level in a similar mechanism to that observed for other signalling kinases; MSK1 (Gehani et al), AMPK2 (Bungard et al) and JAK2 (Dawson et al), and has created an important link between signalling kinases, chromatin mediated gene regulation and lineage specification in ESCs.

 

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