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Rett syndrome is a human intellectual disability disorder that is associated with mutations in the X-linked MECP2 gene. The epigenetic reader MeCP2 binds to methylated cytosines on the DNA and regulates chromatin organization. We have shown previously that MECP2 Rett syndrome missense mutations are impaired in chromatin binding and heterochromatin reorganization. Here, we performed a proteomics analysis of post-translational modifications of MeCP2 isolated from adult mouse brain. We show that MeCP2 carries various post-translational modifications, among them phosphorylation on S80 and S421, which lead to minor changes in either heterochromatin binding kinetics or clustering. We found that MeCP2 is (di)methylated on several arginines and that this modification alters heterochromatin organization. Interestingly, we identified the Rett syndrome mutation site R106 as a dimethylation site. In addition, co-expression of protein arginine methyltransferases (PRMT)1 and PRMT6 lead to a decrease of heterochromatin clustering. Altogether, we identified and validated novel modifications of MeCP2 in the brain and show that these can modulate its ability to bind as well as reorganize heterochromatin, which may play a role in the pathology of Rett syndrome.

Corneal endothelial cells (CECs) are essential for maintaining corneal stromal hydration and ensuring its transparency, which is necessary for normal vision. Dysfunction of CECs leads to stromal decompensation, loss of transparency and corneal blindness. Corneal endothelium has low proliferative potential compared to surface epithelial cells leading to poor regeneration of CEC following injury. Additionally, the tissue exhibits age related decline in endothelial cell density with re-organisation of the cell layer, but no regeneration. The mechanisms which control proliferation and differentiation of neural crest derived CEC progenitors are yet to be clearly elucidated. Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifiers are important for driving differentiation of a variety of cellular types. Many Prdm proteins are expressed in specific precursor cell populations and are necessary for their progression to a fully differentiated phenotype. In the present work, we sought to identify members of the Prdm gene family which are specifically expressed in human (h) CECs with a view to begin addressing their potential roles in CEC biology, focussing especially on Prdm 4 and 5 genes. By performing semi-quantitative reverse transcription coupled to PCR amplification we found that in addition to Prdm4 and Prdm5, Prdm2 and Prdm10 genes are expressed in hCECs. We further found that cultured primary hCECs or immortalised HCEC-12 cells express all of the Prdm genes found in CECs, but also express additional Prdm transcripts. This difference is most pronounced between Prdm gene expression patterns of CECs isolated from healthy human corneas and immortalised HCEC-12 cells. We further investigated Prdm 4 and Prdm 5 protein expression in cultured primary hCECs and HCEC-12 cells as well as in a human cadaveric whole cornea. Both Prdm 4 and Prdm 5 are expressed in human corneal endothelium, primary hCECs and in HCECs-12 cells, characterised by expression of the Na+/K+-ATPase. We observed that both proteins exhibit cytosolic (intracellular, but non-nuclear and distinct from extracellular fluid) as well as nuclear localisation within the endothelial layer, with Prdm 5 being more concentrated in the nuclei of the endothelial cells than Prdm 4. Thus, our work identifies novel Prdm genes specifically expressed in corneal endothelial cells which may be important in the control of CEC differentiation and proliferation., Highlights • The analysis of Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifying genes in corneal endothelial cells (CECs) was undertaken. • Prdm 2, 4, 5 and 10 genes were found to be expressed in human CECs. • Prdm 4 and 5 protein expression was characterised in CECs, ex vivo cultured corneal endothelial cells, and the intact cornea.

During neuronal development, the neuroepithelial stem cells (NSCs) initially undergo proliferative divisions, later switching to neurogenic ones whereby one NSC and a post-mitotic neuron are generated. We recently showed that a member of the PRDM family of transcriptional regulators, PRDM4/SC1, recruits a type II protein arginine methyltransferase, PRMT5, to maintain the “stem-like” cellular state of the embryonic mouse cortical NSCs. However, little is known about the regulation of activity of this complex under proliferation- or differentiation-inducing growth conditions.In the present work I investigate the regulation of SC1/PRMT5-mediated methylation activity in PC12 cells treated with EGF or NGF. I present evidence that NGF down-regulates SC1/PRMT5 methyltransferase (MTase) activity and that the reduction in SC1/PRMT5 MTase activity occurs mainly in the nucleus. I suggest that high levels of SC1/PRMT5 activity are associated with the proliferative state of the cells.

An unresolved question is how HIV-1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle-associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/MEK/ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1-like phase macrophages at the single-cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle-associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV-1. We observe both embryo-derived and monocyte-derived tissue-resident macrophages in a G1-like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV-1 replication in vivo Finally, we reveal a SAMHD1-dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host-directed therapeutic approaches aimed at limiting HIV-1 burden in macrophages that may contribute to curative interventions.