Analysis of the interplay between MeCP2 and histone H1 during in vitro differentiation of human ReNCell neural progenitor cells.

An immortalized neural cell line derived from the human ventral mesencephalon, called ReNCell, and its MeCP2 knock out were used. With it, we characterized the chromatin compositional transitions undergone during differentiation, with special emphasis on linker histones. While the WT cells displayed the development of dendrites and axons the KO cells did not, despite undergoing differentiation as monitored by NeuN. ReNCell expressed minimal amounts of histone H1.0 and their linker histone complement consisted mainly of histone H1.2, H1.4 and H1.5. The overall level of histone H1 exhibited a trend to increase during the differentiation of MeCP2 KO cells. The phosphorylation levels of histone H1 proteins decreased dramatically during ReNCell's cell differentiation independently of the presence of MeCP2. Immunofluorescence analysis showed that MeCP2 exhibits an extensive co-localization with linker histones. Interestingly, the average size of the nucleus decreased during differentiation but in the MeCP2 KO cells, the smaller size of the nuclei at the start of differentiation increased by almost 40% after differentiation by 8 days (8 DIV). In summary, our data provide a compelling perspective on the dynamic changes of H1 histones during neural differentiation, coupled with the intricate interplay between H1 variants and MeCP2.Abbreviations: ACN, acetonitrile; A230, absorbance at 230 nm; bFGF, basic fibroblast growth factor; CM, chicken erythrocyte histone marker; CNS, central nervous system; CRISPR, clustered regulated interspaced short palindromic repeatsDAPI, 4,'6-diaminidino-2-phenylindole; DIV, days in vitro (days after differentiation is induced); DMEM, Dulbecco's modified Eagle medium; EGF, epidermal growth factor; ESC, embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic proteinHPLC, high-performance liquid chromatography; IF, immunofluorescence; iPSCs, induced pluripotent stem cells; MAP2, microtubule-associated protein 2; MBD, methyl-binding domain; MeCP2, methyl-CpG binding protein 2; MS, mass spectrometry; NCP, nucleosome core particle; NeuN, neuron nuclear antigen; NPC, neural progenitor cellPAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PFA, paraformaldehyde; PTM, posttranslational modification; RP-HPLC, reversed phase HPLC; ReNCells, ReNCells VM; RPLP0, ribosomal protein lateral stalk subunit P0; RT-qPCR, reverse transcription quantitative polymerase-chain reaction; RTT, Rett Syndrome; SDS, sodium dodecyl sulphate; TAD, topologically associating domain; Triple KO, triple knockout.

Phosphorylation Promotes DELLA Activity by Enhancing Its Binding to Histone H2A at Target Chromatin in Arabidopsis.

DELLA proteins are conserved master growth regulators that play a central role in controlling plant development in response to internal and environmental cues. DELLAs function as transcription regulators, which are recruited to target promoters by binding to transcription factors (TFs) and histone H2A via its GRAS domain. Recent studies showed that DELLA stability is regulated post-translationally via two mechanisms, phytohormone gibberellin-induced polyubiquitination for its rapid degradation, and Small Ubiquitin-like Modifier (SUMO)- conjugation to alter its accumulation. Moreover, DELLA activity is dynamically modulated by two distinct glycosylations: DELLA-TF interactions are enhanced by O -fucosylation, but inhibited by O -linked N -acetylglucosamine ( O -GlcNAc) modification. However, the role of DELLA phosphorylation remains unclear. Here, we identified phosphorylation sites in REPRESSOR OF ga1-3 (RGA, an AtDELLA) purified from Arabidopsis by tandem mass spectrometry analysis, and showed that phosphorylation of the RGA LKS-peptide in the poly- S/T region enhances RGA-H2A interaction and RGA association with target promoters. Interestingly, phosphorylation does not affect RGA-TF interactions. Our study has uncovered that phosphorylation is a new regulatory mechanism of DELLA activity.