X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells

Abstract

We provide a systematic characterization of the nanosecond groundstate lactam-lactim tautomerization of pyridone derivatives in aqueous solution under ambient conditions using temperature-jump transient 2D IR spectroscopy. Although electronic excited-state tautomerization has been widely studied, experimental work on the ground electronic state, most relevant to chemistry and biology, is lacking. Using 2D IR spectroscopy, lactam and lactim tautomers of 6-chloro-2-pyridone and 2-chloro-4-pyridone are unambiguously identified by their unique cross-peak patterns. Monitoring the correlated exponential relaxation of these signals in response to a laser temperature jump provides a direct measurement of the nanosecond tautomerization kinetics. By studying the temperature, concentration, solvent, and pH dependence, we extract a thermodynamic and kinetic characterization and conclude that the tautomerization proceeds through a two-state concerted mechanism. We find that the intramolecular proton transfer is mediated by bridging water molecules and the reaction barrier is dictated by the release of a proton from pyridone, as would be expected for an efficient Grothusstype proton transfer mechanism.keto-enol tautomerism | multidimensional | time-resolved spectroscopy | ultrafast T automerism of aromatic heterocycles has been extensively studied owing to its importance in biochemical processes such as enzyme catalysis (1), ligand binding (2), and spontaneous mutagenesis (3). To characterize the tautomeric equilibria, researchers have used techniques such as UV absorption (4), circular dichroism (5), X-ray crystallography (3), NMR (6), Raman (7), and IR absorption spectroscopy (8). However, these methods face several challenges to characterizing thermodynamic and kinetic data for tautomeric equilibria and exchange processes. For example, electronic spectra are broad and featureless, which complicates spectral interpretation, particularly for systems with multiple tautomers. Although NMR provides excellent structural resolution, it can only directly monitor chemical exchange in real time on millisecond and longer time scales, rather than the picosecond to nanosecond time scales expected for proton transfer under physiological conditions. From the theoretical point of view, quantum mechanical calculations have been performed extensively to characterize the relative stability of tautomeric systems, their activation barriers, and the reaction mechanisms (9, 10); nevertheless, experimental validation of these predictions is scarce. To provide a characterization under ambient aqueous conditions, we need a technique with both the structural sensitivity to unambiguously identify various tautomers and the time resolution to probe the rapid proton transfer dynamics. To this end, we demonstrate the capability of 2D IR spectroscopy coupled with a nanosecond temperature-jump (Tjump) laser to reveal the nonequilibrium lactam-lactim tautomerization kinetics of pyridone derivatives.Time-resolved studies of tautomerism have a long and storie...

Xist Deficiency and Disorders of X-Inactivation in Rabbit Embryonic Stem Cells Can Be Rescued by Transcription-Factor-Mediated Conversion

“…Since the deficient expression of Xist in hESCs and hiPSCs can greatly affect the differentiation potential [16,17,27], particularly in vivo [16], we further examined the differentiation ability of these three female rbESC lines. In vitro differentiation was firstly performed using EB formation assay.…”

“…The XaXa state in mice appears to be a specific hallmark of the naive state. However, the X chromosome status in both hESCs and hiPSCs is unstable even in early passages [15][16][17]. Both female hESCs and hiPSCs have been grouped into three classes based on their X chromosome status: class I lines initially carry two active X chromosomes (XaXa) and undergo XCI upon differentiation, similar to mESCs; class II lines possess one inactive X coated by XIST RNA (Xi XIST Xa), similar to mEpiSCs; class III lines have a silent Xi but lack of XIST expression (Xi w/oXIST Xa).…”

“…The class III cells with Xi w/oXIST Xa or XeXa will retain XIST deficiency, and neither XIST expression nor XIST RNA coating can be detected in the differentiated cells. Loss of XIST in hESCs and hiPSCs is strongly correlated with the upregulation of oncogenes and differentiation deficiencies in vivo [16,17]. Therefore, X chromosome instability should be taken seriously in both basic research and clinical applications.…”

“…The dispersed cells were placed onto glass slides. RNA FISH was performed as previously described [17,24].…”

Inactivation du chromosome X chez l’humain

“…Dans la majeure partie des cellules, un des deux X est inactivé, mais il se réactive fréquemment en culture [5,6]. L'inactivation semble donc incomplète et pas totalement verrouillée (Figure 1).…”