Abstract
Heterochromatin protein 1 γ (HP1γ) is a well-known chromatin protein, which regulates gene silencing during the execution of processes associated with embryogenesis, organ maturation, and cell differentiation. We find that, in vivo, the levels of HP1γ are downregulated during nervous system development. Similar results are recapitulated in vitro during nerve growth factor (NGF)-induced neuronal cell differentiation in PC12 cells. Mechanistically, our experiments demonstrate that in differentiating PC12 cells, NGF treatment decreases the association of HP1γ to silent heterochromatin, leads to phosphorylation of this protein at S83 via protein kinase A (PKA), and ultimately results in its degradation. Genome-wide experiments, using gain-of-function (overexpression) and loss-of-function (RNAi) paradigms, demonstrate that changing the level of HP1γ impacts on PC12 differentiation, at least in part, through gene networks involved in this process. Hence, inactivation of HP1γ by different post-translational mechanisms, including reduced heterochromatin association, phosphorylation, and degradation, is necessary for neuronal cell differentiation to occur. Indeed, we show that the increase of HP1γ levels has the reverse effect, namely antagonizing neuronal cell differentiation, supporting that this protein acts as a barrier for this process. Thus, these results describe the regulation and participation of HP1γ in a novel membrane-to-nucleus pathway, through NGF-PKA signaling, which is involved in NGF-induced neuronal cell differentiation.
Highlights
We have demonstrated that HP1 can form complexes with sequence-specific transcription factors (e.g. KLF11), which are involved in the regulation of neuron specific genes, such as the Dopamine receptor
We found that Heterochromatin protein 1 γ (HP1γ) levels significantly decreased during maturation of whole embryonic brain until the time of birth (Fig. 1C; E18; 38.8 ± 8.2%, p < 0.05; P1; 35.4 ± 5.5%, p < 0.01 compared to E13; and Supplementary Fig. 1B)
The current study provides direct evidence supporting a role for HP1γ as a barrier in models of neuronal cell differentiation
Summary
We have demonstrated that HP1 can form complexes with sequence-specific transcription factors (e.g. KLF11), which are involved in the regulation of neuron specific genes, such as the Dopamine receptor. Other groups have implicated a role of HP1γ in neuronal differentiation[11,12]. The mechanism by which HP1γ regulates neuronal cell differentiation and cell functions remains to be established. We used well-established cell models for studying NGF-mediated neuronal differentiation in combination with the transduction of genetic mutants, signaling experiments, and genome-wide transcriptional profiling. Combined, these approaches allow us to provide evidence supporting the notion that the timely regulation of the intracellular concentration and post-translational modification of HP1γ is necessary for proper differentiation that occurs through an NGF-PKA-HP1γ pathway
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