Abstract
The X-linked Mecp2 is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell in vitro differentiation. Therefore, we modified murine Mecp2 deficient (Mecp2 −/y) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in Mecp2 deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of Mecp2 wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found Mecp2 deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of Mecp2 deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state.
Highlights
Heterochromatin is defined as chromatin that stays densely packed during interphase
Mecp22/y tEG and Mecp2wt tEG stem cells were differentiated (Figure 1; Figure S2– S3) using a single step neural in vitro differentiation protocol [59]. This approach combined the advantage of a feeder free stem cell culture with neuronal specific EGFP reporter expression driven by the tau promoter and allowed us to focus directly on developing Mecp2 wild type or Mecp2 deficient neural cells
In the presence of differentiation medium and upon leukemia inhibitory factor (LIF) withdrawal, Mecp2 wild type embryonic stem (ES) cells start to differentiate as described by Ying et al [62] and give rise to a variety of cell types including astroglia positive for glial fibrillary acidic protein (GFAP+) and neurons positive for EGFP (EGFP+) (Figure 1B)
Summary
Heterochromatin is defined as chromatin that stays densely packed during interphase. Cytologically, heterochromatin can be further subdivided into constitutive and facultative heterochromatin [1,2], with facultative heterochromatin usually differing between cell types [3,4,5,6]. We compared Mecp2wt tEG to Mecp22/y tEG stem cells and evaluated chromocenter numbers at selected time points of differentiation (undifferentiated cells, days 7, 13, and 21 or 23 after LIF withdrawal and plating in differentiation medium).
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