MK3 controls Polycomb target gene expression via negative feedback on ERK

Abstract

BackgroundGene-environment interactions are mediated by epigenetic mechanisms. Polycomb Group proteins constitute part of an epigenetic cellular transcriptional memory system that is subject to dynamic modulation during differentiation. Molecular insight in processes that control dynamic chromatin association and dissociation of Polycomb repressive complexes during and beyond development is limited. We recently showed that MK3 interacts with Polycomb repressive complex 1 (PRC1). The functional relevance of this interaction, however, remained poorly understood. MK3 is activated downstream of mitogen- and stress-activated protein kinases (M/SAPKs), all of which fulfill crucial roles during development. We here use activation of the immediate-early response gene ATF3, a bona fide PRC1 target gene, as a model to study how MK3 and its effector kinases MAPK/ERK and SAPK/P38 are involved in regulation of PRC1-dependent ATF3 transcription.ResultsOur current data show that mitogenic signaling through ERK, P38 and MK3 regulates ATF3 expression by PRC1/chromatin dissociation and epigenetic modulation. Mitogenic stimulation results in transient P38-dependent H3S28 phosphorylation and ERK-driven PRC1/chromatin dissociation at PRC1 targets. H3S28 phosphorylation by itself appears not sufficient to induce PRC1/chromatin dissociation, nor ATF3 transcription, as inhibition of MEK/ERK signaling blocks BMI1/chromatin dissociation and ATF3 expression, despite induced H3S28 phosphorylation. In addition, we establish that concomitant loss of local H3K27me3 promoter marking is not required for ATF3 activation. We identify pERK as a novel signaling-induced binding partner of PRC1, and provide evidence that MK3 controls ATF3 expression in cultured cells via negative regulatory feedback on M/SAPKs. Dramatically increased ectopic wing vein formation in the absence of Drosophila MK in a Drosophila ERK gain-of-function wing vein patterning model, supports the existence of MK-mediated negative feedback regulation on pERK.ConclusionWe here identify and characterize important actors in a PRC1-dependent epigenetic signal/response mechanism, some of which appear to be nonspecific global responses, whereas others provide modular specificity. Our findings provide novel insight into a Polycomb-mediated epigenetic mechanism that dynamically controls gene transcription and support a direct link between PRC1 and cellular responses to changes in the microenvironment.

Hypoxia increases genome-wide bivalent epigenetic marking by specific gain of H3K27me3

“…However, PRC proteins are known to localize at active RNA polymerase 2 (POLR2) promoters in Drosophila and to interact with basal transcription factors (TF) [59]. In keeping with this notion, we recently showed that transcriptional activation at a defined PRC1-target locus did not require loss of H3K27me3 marking, but PCR1/chromatin dissociation [60]. In addition, PRC associates with splicing factors, revealing an as of yet poorly understood additional role in regulation of gene expression [61].…”

PRC1 Prevents Replication Stress during Chondrogenic Transit Amplification

“…We have recently shown that transcriptional activity persists during differentiation or physiological stress, despite increased genic H3K27me3-enrichment [33,[55][56][57]. Conversely, PRC1 was suggested to be required for gene expression in specific settings [58][59][60][61][62], illustrating the complexity of the functional interaction between PRC1 and H3K27me3 in the context of transcriptional regulation.…”

“…Of relevance, PRC1-proteins were shown to occupy promoters of actively transcribed genes and are required for POLR2A complex disassembly [41,75]. We recently showed transcriptional activation of the immediate early gene ATF3 in response to mitogenic signaling, involves release of chromatin-bound PRC1-complex members without the need for local change in promoter H3K27me3-marking [55]. PRC1-mediated transcriptional repression, instead, appears to be dependent on ubiquitylation of H2AK119, which is also involved in DNA repair [76][77][78].…”

“…Quantitative analysis was carried out using Chemi Luminescent Image capture software (Chemidoc XRS, Biorad, München, Germany). Cell fractionation assays were carried out as described before: quantitative comparison between different fractions was enabled by loading volume equivalents [55].…”

“…Equal chromatin-input in IPs was assessed by DNA concentration measurement. For TOP2A/chromatin-association, no fixation was applied; a mild lysis was used (250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 50 mM HEPES, pH 7.0; supplemented with inhibitor cocktail) to separate total soluble from chromatin-bound fractions as described before [55]. Soluble fractions were collected after centrifugation; chromatin pellets were washed; all samples were sonicated before analysis (see: Western analysis).…”