Nuclear positioning and pairing of X-chromosome inactivation centers are not primary determinants during initiation of random X-inactivation
Authors: Tim Pollex
Edith Heard
Journal: Nature Genetics (2019)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: X-chromosome inactivation; X-inactivation centers; Xist; nuclear envelope; Xic pairing
Affiliations: Mammalian Developmental Epigenetics Group, Genetics and Developmental Biology Unit, Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, France., European Molecular Biology Laboratory, Heidelberg, Germany., Mammalian Developmental Epigenetics Group, Genetics and Developmental Biology Unit, Institut Curie, PSL Research University, CNRS UMR3215, INSERM U934, Paris, France. .

Abstract:  During X-chromosome inactivation (XCI), one of the two X-inactivation centers (Xics) upregulates the noncoding RNA Xist to initiate chromosomal silencing in cis. How one Xic is chosen to upregulate Xist remains unclear. Models proposed include localization of one Xic at the nuclear envelope or transient homologous Xic pairing followed by asymmetric transcription factor distribution at Xist's antisense Xite/Tsix locus. Here, we use a TetO/TetR system that can inducibly relocate one or both Xics to the nuclear lamina in differentiating mouse embryonic stem cells. We find that neither nuclear lamina localization nor reduction of Xic homologous pairing influences monoallelic Xist upregulation or choice-making. We also show that transient pairing is associated with biallelic expression, not only at Xist/Tsix but also at other X-linked loci that can escape XCI. Finally, we show that Xic pairing occurs in wavelike patterns, coinciding with genome dynamics and the onset of global regulatory programs during early differentiation. 

Retracted: No

PubPeer comments:
 See paper by Aeby et al, Nature Cell Biology 2020 Sep;22(9):1116-1129, about technical problem with the cell line used in paper by Pollex and Heard, impacting their negative conclusions.  Tethering the pairing centre to the nuclear lamina to constrain Xu2013X pairing did not affect XCI63. In this system, a Te t O array was integrated into both Xic (XTe t OXTe t O) and doxycycline (Dox)-repressible TetRu2013laminB1 fusion was used to tether both Xic copies to the nuclear lamina. However, because it is known that Xu2013X pairing takes place at the nuclear periphery19,25and X-telomeres (which often localize at the periphery) direct pair-ing in 3D space25, Xicu2013lamin tethering might not disrupt pairing. Furthermore, Xicu2013lamin association occurred in only approximately 40% of cells at day 0, and no frequency was reported for the pairingtime window63. We therefore revisited the XTe t OXTe t O and TetRu2013eGFPu2013laminB1 system under reported experimental conditions.Under adherent growth conditions63, pairing occurred in the untethered (+Dox) condition between days 1 and 4 (Extended Data Fig. 7b,c), as expected. When Dox was withdrawn to induce teth-ering, pairing still occurred and Xist was upregulated (Extended Data Fig. 7bu2013e). When ES cells were cultured as embryoid bodies in suspension culture14,24,25, we similarly observed pairing and Xist upregulation (Extended Data Fig. 7fu2013i). Thus, contrasting with the previous report63, pairing still took place in XTe t OXTe t O TetRu2013eGFPu2013laminB1 cells. Pairing did appear in a u2018wave-likeu2019 pattern in the XTe t OXTe t O TetRu2013eGFPu2013laminB1 cells, as reported63. Because this was not seen in other female ES cells14,24,25 (Figs. 2, 4 and 5), the pat-tern may be an idiosyncrasy of the XTe t OXTe t O TetRu2013eGFPu2013laminB1 cell line. Most critically, Xicu2013lamin tethering did not occur dur-ing the pairing time window (days 2u20134). Under +Dox conditions (u2018untetheredu2019), 17.7% (day 0), 27.6% (day 1), 27.0% (day 2), 28.6% (day 3) and 24.6% (day 4) of nuclei showed Xicu2013laminB1 associa-tion. In u2212Dox conditions (u2018tetheredu2019), 47% (day 0), 44.1% (day 1), 34.4% (day 2), 29.7% (day 3) and 25.7% (day 4) showed Xicu2013lam-inB1 association. Thus, tethering in XTe t OXTe t O TetRu2013eGFPu2013laminB1 cells was not robust. We conclude that the system cannot be used to address the role of pairing during XCI. , 
 #1 The Aeby paper in NCB says that Pollex and Heard did not check if their cell line can tether XIC to the nuclear envelope. It is hard to believe that a paper published in Nature Genetics would miss this important control, which is required to interpret the rest of the paper. If Aeby is right that there is no tethering, how did the authors hinder XIC pairing? What were they looking at instead? Can Pollex and Heard shed light on this? , 
 After reading the papers, I agree that there are fundamental problems with the T Pollex, E Heard paper. If there are problems with the cell lines and they are not working as authors claimed, conclusions derived from them must be called into question. From Aeby et al, it looks like pairing is a real phenomenon and controlling X inactivation. I am not in this field directly but I am interested in pairing as a mechanism. Can someone with more knowledge comment on this? ,