Mechanistic dissection of SPEN functionduring X chromosome inactivation

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Submitted on 28 May 2021

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Mechanistic dissection of SPEN function during X

chromosome inactivation

François Dossin

To cite this version:

François Dossin. Mechanistic dissection of SPEN function during X chromosome inactivation. Genet-ics. Université Paris sciences et lettres, 2021. English. �NNT : 2021UPSLS042�. �tel-03240606�

Préparée à l’Institut Curie

Mechanistic dissection of SPEN function

during X chromosome inactivation

Fonctions et mécanismes de SPEN pendant l’inactivation du chromosome X

Soutenue par

François Dossin

Le 15 Mars 2021

École doctorale no₅₇₇

Structure et Dynamique des Systèmes Vivants

Spécialités

Sciences de la vie et de la santé

Composition du jury :

Pierre CAPY Président

Professeur, Université Paris-Saclay (Orsay)

Claire ROUGEULLE Rapportrice

Directrice de recherche, Epigenetics and Cell Fate Institute (CNRS, Paris)

Reiner VEITIA Rapporteur

Professeur, Université de Paris (Paris); Institut F. Jacob (Fontenay-aux-Roses)

Geneviève ALMOUZNI Examinatrice

Directrice de recherche, Institut Curie (CNRS, Paris)

Dirk SCHÜBELER Examinateur

Professeur, Friedrich Miescher Institute (Basel)

Edith HEARD Directrice de thèse

Professeure, Collège de France (Paris); European Molecular Biology Laboratory (EMBL, Heidelberg)

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■■ ▼❛t❡r✐❛❧ ❛♥❞ ▼❡t❤♦❞s

✸✼

✸ ❊①♣❡r✐♠❡♥t❛❧ ✇♦r❦ ✸✽ ✸✳✶ P❧❛s♠✐❞ ❝♦♥str✉❝t✐♦♥ ❢♦r ❡♥❞♦❣❡♥♦✉s t❛❣❣✐♥❣ ♦❢ ❙♣❡♥ ✫ ❍❞❛❝✸ ✐♥ ♠❊❙❈s ✳ ✳ ✸✽ ✸✳✷ ❈❡❧❧ ❝✉❧t✉r❡ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✸✾ ✸✳✸ ❈❡❧❧ tr❛♥s❢❡❝t✐♦♥ ❛♥❞ ❝❧♦♥❡ ✐s♦❧❛t✐♦♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✸✾ ✸✳✹ ❈❡❧❧ tr❡❛t♠❡♥ts ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✸✾ ✸✳✺ Pr♦t❡✐♥ ❡①tr❛❝t✐♦♥ ❛♥❞ ✇❡st❡r♥ ❜❧♦tt✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✵ ✸✳✻ ❘◆❆ ❡①tr❛❝t✐♦♥✱ ❘❚✱ P②r♦s❡q✉❡♥❝✐♥❣ ❛♥❞ ❘◆❆s❡q ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✵ ✸✳✼ ❘◆❆ ❋■❙❍ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✵ ✸✳✽ ❍❛❧♦❚❛❣ ❧❛❜❡❧✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✶ ✸✳✾ ❍❛❧♦❚❛❣ ❧❛❜❡❧✐♥❣ ❢♦❧❧♦✇❡❞ ❜② ❘◆❆ ❋■❙❍ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✶ ✸✳✶✵ ■♠♠✉♥♦✢✉♦r❡s❝❡♥❝❡ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✷ ✸✳✶✶ ❍✐✲❈ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✷ ✸✳✶✷ ▼♦✉s❡ ❜r❡❡❞✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✷ ✸✳✶✸ ❊♠❜r②♦ ❝♦❧❧❡❝t✐♦♥ ❛♥❞ s✐♥❣❧❡✲❡♠❜r②♦ ❘◆❆s❡q ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✸ ✸✳✶✹ ❚❛r❣❡t✐♥❣ ❇❣❧ st❡♠ ❧♦♦♣s ❛t t❤❡ ❳✐st ❧♦❝✉s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✸ ✸✳✶✺ ▲✐✈❡✲❝❡❧❧ ✐♠❛❣✐♥❣ ❛♥❞ ♠❛❝❤✐♥❡ ❧❡❛r♥✐♥❣ ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✹ ✸✳✶✻ ❈r♦ss✲❧✐♥❦❡❞ ❈❯❚✫❘❯◆ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✹ ✸✳✶✼ ❙P❊◆ ✫ ❇❣❧●✲❙P❖❈ ❝♦♠♣❧❡♠❡♥t❛t✐♦♥ ❝♦♥str✉❝ts ❞❡s✐❣♥ ❛♥❞ ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✹✻ ✸✳✶✽ ❙P❖❈✲●❋P ✐♠♠✉♥♦♣r❡❝✐♣✐t❛t✐♦♥ ✭●❋P✲❚r❛♣✮ ♠❛ss✲s♣❡❝tr♦♠❡tr② ✳ ✳ ✳ ✳ ✳ ✳ ✹✻ ✸✳✶✾ ❈❧♦♥✐♥❣ ♦❢ ❙P❖❈ ❝♦♥str✉❝ts ❢♦r ❜❛❝t❡r✐❛❧ ❡①♣r❡ss✐♦♥ ✐♥ ❘♦s❡tt❛✷ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✼ ✸✳✷✵ ❇❛❝t❡r✐❛❧ ❡①♣r❡ss✐♦♥ ❛♥❞ ♣✉r✐✜❝❛t✐♦♥ ♦❢ ❙P❖❈ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✽ ✸✳✷✶ ❈r②st❛❧❧✐③❛t✐♦♥ ♦❢ ❙P❖❈ ❛❧♦♥❡ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✽ ✸✳✷✷ Pr❡♣❛r❛t✐♦♥ ♦❢ ❘◆❆P■■✲❈❚❉ ♣❤♦s♣❤♦♣❡♣t✐❞❡s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✾ ✸✳✷✸ ■s♦t❤❡r♠❛❧ t✐tr❛t✐♦♥ ❝❛❧♦r✐♠❡tr② ✭■❚❈✮ ❛ss❛②s ♦❢ ❜✐♥❞✐♥❣ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ ❘◆❆P■■✲❈❚❉ ♣❡♣t✐❞❡s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✹✾ ✸✳✷✹ ❈♦✲❝r②st❛❧❧✐③❛t✐♦♥ ♦❢ ❙P❖❈ ❛♥❞ ❘◆❆P■■✲❈❚❉ ❙❡r✺P ♣❡♣t✐❞❡s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✵ ✹ ❇✐♦✐♥❢♦r♠❛t✐❝s ❛♥❛❧②s❡s ✺✶ ✹✳✶ ●❡♥♦♠✐❝s ❛♥❛❧②s❡s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✶ ✹✳✶✳✶ ❘◆❆s❡q ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✶ ✹✳✶✳✶✳✶ ❙P❊◆✲❞❡❣r♦♥ ♠❊❙❈s ❘◆❆s❡q ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✷ ✹✳✶✳✶✳✷ ❙♣❡♥ ❑❖ ❊✸✳✺ ❡♠❜r②♦ ❘◆❆s❡q ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✷ ✹✳✶✳✶✳✸ ❙P❊◆✲❞❡❣r♦♥ ◆P❈ ❘◆❆s❡q ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✷ ✹✳✶✳✷ ❈❯❚✫❘❯◆ ❜✐♦✐♥❢♦r♠❛t✐❝s ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✸ ✹✳✶✳✷✳✶ P❡❛❦ ✜❧t❡r✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✸

✹✳✶✳✷✳✷ ❚♦t❛❧ ❙♣❡♥ ❡♥r✐❝❤♠❡♥t ✐♥ ♣r♦♠♦t❡r ✇✐♥❞♦✇s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✸ ✹✳✶✳✷✳✸ ●❡♥♦♠✐❝ ❢❡❛t✉r❡s ❛♥❞ ✐♥t❡❣r❛t✐♦♥ ✇✐t❤ ❘◆❆s❡q ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✸ ✹✳✶✳✷✳✹ ■♥t❡❣r❛t✐♦♥ ✇✐t❤ ♣✉❜❧✐❝❛❧❧② ❛✈❛✐❧❛❜❧❡ ❈❤■Ps❡q ❞❛t❛ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✹ ✹✳✶✳✸ ❍✐✲❈ ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✹ ✹✳✷ Pr♦t❡♦♠✐❝s ❞❛t❛ ❛♥❛❧②s✐s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✹ ✹✳✸ Pr♦t❡✐♥ str✉❝t✉r❡ ❞❡t❡r♠✐♥❛t✐♦♥ ❛♥❞ r❡✜♥❡♠❡♥t ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✺ ✹✳✹ ❉❛t❛ ❛✈❛✐❧❛❜✐❧✐t② ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✺

■■■ ❘❡s✉❧ts

✺✻

✺ ❈❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳❈■ ✭❩②❧✐❝③ ❡t ❛❧✳✱ ❈❡❧❧✳ ✷✵✶✾✮ ✺✼ ✺✳✶ ●❡♥❡r❛❧ ♠❡t❤♦❞♦❧♦❣② ❜❡❤✐♥❞ ❩②❧✐❝③ ❡t ❛❧✳✱ ✷✵✶✾ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✼ ✺✳✶✳✶ ❚❤❡ ❚❳✶✵✼✷ ❝❡❧❧✉❧❛r s②st❡♠ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✼ ✺✳✶✳✷ ❊①♣❡r✐♠❡♥t❛❧ ❛♣♣r♦❛❝❤ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✺✾ ✺✳✷ ❑❡② r❡s✉❧ts ✐♥❞❡♣❡♥❞❡♥t ♦❢ ♠② ❝♦♥tr✐❜✉t✐♦♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✻✵ ✺✳✷✳✶ ❙♣r❡❛❞✐♥❣ ♦❢ P♦❧②❝♦♠❜ ♠❛r❦s ❞✉r✐♥❣ ❳❈■ r❡q✉✐r❡s ❣❡♥❡ s✐❧❡♥❝✐♥❣ ✳ ✳ ✳ ✳ ✳ ✻✵ ✺✳✷✳✷ P❘❈✶ r❡❝r✉✐t♠❡♥t ♣r❡❝❡❞❡s P❘❈✷ ❞✉r✐♥❣ ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✻✵ ✺✳✷✳✸ ❍❉❆❈✸ ✐s ✐♠♣♦rt❛♥t ❢♦r ❳✐st✲❞❡♣❡♥❞❡♥t ❣❡♥❡ s✐❧❡♥❝✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✻✵ ✺✳✸ ❑❡② r❡s✉❧ts t♦ ✇❤✐❝❤ ■ ❝♦♥tr✐❜✉t❡❞ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✻✶ ✺✳✸✳✶ ●❡♥❡r❛t✐♦♥ ❛♥❞ ❝❤❛r❛❝t❡r✐③❛t✐♦♥ ♦❢ ❡♥❞♦❣❡♥♦✉s ❋▲❆●✲t❛❣❣❡❞ ❍❉❆❈✸ ♠❊❙❈s ✻✶ ✺✳✸✳✷ ❍❉❆❈✸ ✐s ♣r❡✲❜♦✉♥❞ t♦ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡ ❛♥❞ ❢✉♥❝t✐♦♥s str✐❝t❧② t❤r♦✉❣❤ ◆❈♦❘✴❙▼❘❚ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✻✷ ✺✳✹ ❆rt✐❝❧❡ ✶✿ ❚❤❡ ✐♠♣❧✐❝❛t✐♦♥ ♦❢ ❡❛r❧② ❝❤r♦♠❛t✐♥ ❝❤❛♥❣❡s ✐♥ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝✲ t✐✈❛t✐♦♥ ✭❈❡❧❧✳ ✷✵✷✵✮ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✻✸ ✻ ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ♠❡❝❤❛♥✐s♠s ✐♥ ❳❈■ ✭❉♦ss✐♥ ❡t ❛❧✳✱ ◆❛t✉r❡✳ ✷✵✷✵✮ ✶✵✹ ✻✳✶ ❘❛t✐♦♥❛❧❡ ❢♦r ✐♥✈❡st✐❣❛t✐♥❣ ❙P❊◆ ❛♥❞ ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✵✹ ✻✳✷ ●♦❛❧ ✶✿ ❆❞❞r❡ss✐♥❣ ❙P❊◆ ❢✉♥❝t✐♦♥ ❛♥❞ ✇✐♥❞♦✇ ♦❢ ♦♣♣♦rt✉♥✐t② ❞✉r✐♥❣ ❳❈■ ✳ ✶✵✻ ✻✳✷✳✶ ❉✐ss❡❝t✐♥❣ ❙P❊◆ ❢✉♥❝t✐♦♥ ✐♥ ✐♥✐t✐❛t✐♥❣ ❳❈■ ✐♥ ✈✐tr♦ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✵✻ ✻✳✷✳✶✳✶ ●❡♥❡r❛t✐♦♥ ♦❢ ❙P❊◆ ❛✉①✐♥✲✐♥❞✉❝✐❜❧❡ ❞❡❣r♦♥ ♠❊❙❈s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✵✼ ✻✳✷✳✶✳✷ ❙P❊◆ ✐s ❡ss❡♥t✐❛❧ ❢♦r ✐♥✐t✐❛t✐♥❣ ❳❈■ ✐♥ ✈✐tr♦ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✵✽ ✻✳✷✳✷ ❉✐ss❡❝t✐♥❣ ❙P❊◆ ❢✉♥❝t✐♦♥ ✐♥ ✐♥✐t✐❛t✐♥❣ ✐♠♣r✐♥t❡❞ ❳❈■ ✐♥ ✈✐✈♦ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✵✽ ✻✳✷✳✷✳✶ ▼♦✉s❡ ❝r♦ss✐♥❣ str❛t❡❣② ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✵✽ ✻✳✷✳✷✳✷ ❙P❊◆ ✐s ❡ss❡♥t✐❛❧ ❢♦r ✐♠♣r✐♥t❡❞ ❳❈■ ✐♥ ✈✐✈♦ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✵✾ ✻✳✷✳✸ ❉✐ss❡❝t✐♥❣ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ♠❛✐♥t❡♥❛♥❝❡ ♦❢ ❳❈■ ✐♥ s♦♠❛t✐❝ ❝❡❧❧s ✳ ✳ ✳ ✶✵✾ ✻✳✷✳✸✳✶ ❙P❊◆ ✐s ❞✐s♣❡♥s❛❜❧❡ ❢♦r ❦❡❡♣✐♥❣ ❣❡♥❡s s✐❧❡♥❝❡❞✱ ❜✉t ❜✉✛❡rs tr❛♥s❝r✐♣t✐♦♥ ♦❢ ❡s❝❛♣❡❡s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✶✵ ✻✳✷✳✸✳✷ ❚❤❡ ♣❡❝✉❧✐❛r ♦r❣❛♥✐③❛t✐♦♥ ♦❢ t❤❡ ❳✐ ✐s ♠❛✐♥t❛✐♥❡❞ ✐♥❞❡♣❡♥❞❡♥t❧② ♦❢ ❙P❊◆ ✶✶✵ ✻✳✷✳✹ ■♠❛❣✐♥❣ ♦❢ ❙P❊◆ ❛♥❞ ❳✐st ❘◆❆ ❞②♥❛♠✐❝s ❞✉r✐♥❣ ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✶✶

✻✳✷✳✹✳✶ ❋✐①❡❞✲❝❡❧❧ ✐♠❛❣✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✶✶ ✻✳✷✳✹✳✷ ▲✐✈❡✲❝❡❧❧ ✐♠❛❣✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✶✶ ✻✳✸ ●♦❛❧ ✷✿ ●❛✐♥✐♥❣ ♠❡❝❤❛♥✐st✐❝ ✐♥s✐❣❤ts ✐♥t♦ ❙P❊◆ ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ✳ ✳ ✳ ✳ ✶✶✸ ✻✳✸✳✶ Pr♦✜❧✐♥❣ ❙P❊◆ ❝❤r♦♠❛t✐♥ ❜✐♥❞✐♥❣ ❞✉r✐♥❣ ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✶✸ ✻✳✸✳✶✳✶ ❆❞❛♣t✐♥❣ ❈❯❚✫❘❯◆ ❢♦r ❙P❊◆ ❛♥❞ ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✶✸ ✻✳✸✳✶✳✷ ❙P❊◆ ❈❯❚✫❘❯◆ s❤❡❞s ❧✐❣❤t ♦♥ ✐ts ♠❡❝❤❛♥✐s♠ ♦❢ ❛❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ✳ ✳ ✶✶✺ ✻✳✸✳✷ ■❞❡♥t✐❢②✐♥❣ t❤❡ ❞♦♠❛✐♥s ♦❢ ❙P❊◆ r❡q✉✐r❡❞ ❢♦r ✐ts ❢✉♥❝t✐♦♥ ❞✉r✐♥❣ ❳❈■ ✳ ✳ ✳ ✶✶✽ ✻✳✸✳✷✳✶ ❙P❊◆ ❢✉♥❝t✐♦♥❛❧ ❝♦♠♣❧❡♠❡♥t❛t✐♦♥ ♠❡t❤♦❞♦❧♦❣② ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✶✽ ✻✳✸✳✷✳✷ ❙P❊◆✬s ❘❘▼✷✲✹ ❛♥❞ ❙P❖❈ ❞♦♠❛✐♥s ❛♣♣❡❛r ✈✐t❛❧ ❢♦r ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✷✵ ✻✳✸✳✷✳✸ ❊♥❞♦❣❡♥♦✉s ❙P❖❈✲❞❡❧❡t✐♦♥ ❝♦♥✜r♠s ✐ts ❡ss❡♥t✐❛❧✐t② ✐♥ ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✷✵ ✻✳✸✳✷✳✹ ❙P❖❈ t❡t❤❡r✐♥❣ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐s s✉✣❝✐❡♥t t♦ ♠❡❞✐❛t❡ ❣❡♥❡ s✐❧❡♥❝✐♥❣✶✷✶ ✻✳✸✳✸ ■❞❡♥t✐❢②✐♥❣ t❤❡ ♣r♦t❡✐♥ ♣❛rt♥❡rs ♦❢ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✷✶ ✻✳✸✳✸✳✶ ❘❛t✐♦♥❛❧❡ ❢♦r ❝❤❛r❛❝t❡r✐③✐♥❣ t❤❡ ❙P❖❈✲✐♥t❡r❛❝t♦♠❡ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✷✷ ✻✳✸✳✸✳✷ ❙P❖❈ ♣r♦t❡♦♠✐❝s ♠❡t❤♦❞♦❧♦❣② ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✷✸ ✻✳✸✳✸✳✸ ❙P❖❈ ✐♥t❡r❛❝ts ✇✐t❤ ❛ ✈❛r✐❡t② ♦❢ ❝♦♠♣❧❡①❡s ✐♥✈♦❧✈❡❞ ✐♥ ❞✐r❡❝t tr❛♥s❝r✐♣✲ t✐♦♥ r❡❣✉❧❛t✐♦♥✱ ❘◆❆ ♠❡t❛❜♦❧✐s♠✱ ❝❤r♦♠❛t✐♥ ♠♦❞✐✜❝❛t✐♦♥ ❛♥❞ ♥✉❝❧❡♦s♦♠❡ r❡♠♦❞❡❧✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✷✸ ✻✳✹ ❆rt✐❝❧❡ ✷✿ ❙P❊◆ ✐♥t❡❣r❛t❡s tr❛♥s❝r✐♣t✐♦♥❛❧ ❛♥❞ ❡♣✐❣❡♥❡t✐❝ ❝♦♥tr♦❧ ♦❢ ❳✲✐♥❛❝✲ t✐✈❛t✐♦♥ ✭◆❛t✉r❡✳ ✷✵✷✵✮ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✷✻ ✼ ❙tr✉❝t✉r❛❧✴❜✐♦♣❤②s✐❝❛❧ ❛♥❛❧②s✐s ♦❢ t❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ✐♥t❡r❛❝t✐♦♥ ✶✺✵ ✼✳✶ ❘❛t✐♦♥❛❧❡ ❢♦r ✐♥✈❡st✐❣❛t✐♥❣ ❛ ♣♦t❡♥t✐❛❧ ♣❤♦s♣❤♦❞❡♣❡♥❞❡♥t ✐♥t❡r❛❝t✐♦♥ ❜❡t✇❡❡♥ ❙P❖❈ ❛♥❞ t❤❡ ❈❚❉ ♦❢ ❘◆❆P■■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✵ ✼✳✶✳✶ ❚❤❡ ❈❚❉ ♦❢ ❘◆❆P■■ ❛♥❞ ❢✉♥❝t✐♦♥ ♦❢ t❤❡ ❈❚❉ ❝♦❞❡ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✶ ✼✳✶✳✶✳✶ ❋✉♥❝t✐♦♥ ♦❢ ❙❡r✺ ♣❤♦s♣❤♦r②❧❛t✐♦♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✷ ✼✳✶✳✶✳✷ ❋✉♥❝t✐♦♥ ♦❢ ❙❡r✷ ♣❤♦s♣❤♦r②❧❛t✐♦♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✷ ✼✳✶✳✶✳✸ ❋✉♥❝t✐♦♥ ♦❢ ❙❡r✼ ♣❤♦s♣❤♦r②❧❛t✐♦♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✷ ✼✳✶✳✷ ❙P❖❈ ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ ◆❈♦❘✴❙▼❘❚ ✐s ♣❤♦s♣❤♦✲❞❡♣❡♥❞❡♥t ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✸ ✼✳✷ ❇❛❝t❡r✐❛❧ ❡①♣r❡ss✐♦♥ ❛♥❞ ♣✉r✐✜❝❛t✐♦♥ ♦❢ t❤❡ ❙P❖❈ ❞♦♠❛✐♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✹ ✼✳✸ ❙tr✉❝t✉r❡ ♦❢ ♠♦✉s❡ ❙P❖❈ ❛t ✶✳✷➴ r❡s♦❧✉t✐♦♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✻ ✼✳✹ ❙P❖❈ ✐♥t❡r❛❝ts ✇✐t❤ t❤❡ ❈❚❉ ♦❢ ❘◆❆P■■ s♣❡❝✐✜❝❛❧❧② ✇❤❡♥ ❙❡r✺ ✐s ♣❤♦s♣❤♦✲ r②❧❛t❡❞ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✺✽ ✼✳✺ ❙❡r✷✴✼ ♣❤♦s♣❤♦r②❧❛t✐♦♥s ❞♦ ♥♦t ❛❧t❡r ❙P❖❈ ❜✐♥❞✐♥❣ t♦ t❤❡ ❙❡r✺P✲❈❚❉ ♦❢ ❘◆❆P■■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✻✵ ✼✳✻ ❚❤❡ ❙P❖❈✴❙❡r✺P✲❈❚❉ ❝♦♠♣❧❡① ❛t ✶✳✽➴ r❡s♦❧✉t✐♦♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✻✷ ✼✳✼ ❚❤❡ ❜❛s✐❝ ♣❛t❝❤ ♦❢ ❙P❖❈ ♠❡❞✐❛t❡s ❞✐r❡❝t ✐♥t❡r❛❝t✐♦♥ ✇✐t❤ t❤❡ ❙❡r✺P✲❈❚❉ ♦❢ ❘◆❆P■■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✻✹

■❱ ❉✐s❝✉ss✐♦♥

✶✻✼

✽ ❙P❊◆ ✐s t❤❡ ♠❛st❡r ❡✛❡❝t♦r ♦❢ ❣❡♥❡ s✐❧❡♥❝✐♥❣ ❞✉r✐♥❣ ❳❈■ ✶✻✽ ✽✳✶ ▼♦st ❳✲❧✐♥❦❡❞ ❣❡♥❡s ❛r❡ s✐❧❡♥❝❡❞ ❡♥t✐r❡❧② t❤r♦✉❣❤ ❙P❊◆✲❞❡♣❡♥❞❡♥t ♠❡❝❤❛♥✐s♠s✳✶✻✽ ✽✳✷ ❆ ❢❡✇ ❳✲❧✐♥❦❡❞ ❣❡♥❡s ❛r❡ s✐❧❡♥❝❡❞ t❤r♦✉❣❤ ❙P❊◆✲✐♥❞❡♣❡♥❞❡♥t ♠❡❝❤❛♥✐s♠s✳ ✳ ✶✻✾ ✽✳✸ ❙P❊◆ ✐s ❝r✐t✐❝❛❧ ❢♦r s✐❧❡♥❝✐♥❣ ❤✐❣❤❧② ❡①♣r❡ss❡❞ ❣❡♥❡s✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✵ ✽✳✹ ❙P❊◆ ✐s ❡ss❡♥t✐❛❧ ❢♦r ✐♠♣r✐♥t❡❞ ❳❈■ ✐♥ ✈✐✈♦ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✵ ✽✳✺ ❙P❊◆ ✐s ❞✐s♣❡♥s❛❜❧❡ t♦ ♠❛✐♥t❛✐♥ ❣❡♥❡ s✐❧❡♥❝✐♥❣ ❜✉t ❜✉✛❡rs ❡①♣r❡ss✐♦♥ ♦❢ ❡s✲ ❝❛♣❡❡s ✐♥ s♦♠❛t✐❝ ❝❡❧❧s ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✶ ✽✳✻ ❳✐st r❡❝r✉✐ts ❙P❊◆ t♦ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✷ ✽✳✻✳✶ ❙P❊◆ ❛❝ts ❞♦✇♥str❡❛♠ ♦❢ ❳✐st ❝♦❛t✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✷ ✽✳✻✳✷ ❙P❊◆ ❧♦ss ❛✛❡❝ts ❳✐st ❡①♣r❡ss✐♦♥ ❞✐✛❡r❡♥t❧② ✐♥ ✈✐tr♦ ❛♥❞ ✐♥ ✈✐✈♦ ✳ ✳ ✳ ✳ ✳ ✶✼✷ ✽✳✻✳✸ ❳✐st ✉♣r❡❣✉❧❛t✐♦♥ ✐♠♠❡❞✐❛t❡❧② tr✐❣❣❡rs ❙P❊◆ r❡❝r✉✐t♠❡♥t ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✸ ✽✳✻✳✹ ❙P❊◆ r❡❝r✉✐t♠❡♥t ✐s r❡q✉✐r❡❞ ❢♦r ❣❡♥❡ s✐❧❡♥❝✐♥❣ ❛♥❞ ❞❡♣❡♥❞s ♦♥ ❘❘▼✷✲✹ ✳ ✶✼✹ ✾ ▼❡❝❤❛♥✐s♠s ♦❢ ❙P❊◆✲♠❡❞✐❛t❡❞ tr❛♥s❝r✐♣t✐♦♥❛❧ s✐❧❡♥❝✐♥❣ ✶✼✺ ✾✳✶ ❙P❊◆ ✐♥t❡r❛❝ts ✇✐t❤ ❝❤r♦♠❛t✐♥ t♦ ♣r♦♠♦t❡ tr❛♥s❝r✐♣t✐♦♥❛❧ s✐❧❡♥❝✐♥❣ ✳ ✳ ✳ ✳ ✳ ✶✼✺ ✾✳✶✳✶ ❙P❊◆ t❛r❣❡ts ♣r♦♠♦t❡rs ❛♥❞ ❡♥❤❛♥❝❡rs ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✺ ✾✳✶✳✷ ❆❝t✐✈❡ tr❛♥s❝r✐♣t✐♦♥ ✐s r❡q✉✐r❡❞ ❢♦r ❙P❊◆ ❜✐♥❞✐♥❣ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✻ ✾✳✷ ❙P❖❈✲❝❡♥t❡r❡❞ ❣❡♥❡ s✐❧❡♥❝✐♥❣ ♠❡❝❤❛♥✐s♠s ❞✉r✐♥❣ ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✼ ✾✳✷✳✶ ❙P❊◆✬s ❙P❖❈ ❞♦♠❛✐♥ ✐s ♥❡❝❡ss❛r② ❛♥❞ s✉✣❝✐❡♥t ❢♦r ❣❡♥❡ s✐❧❡♥❝✐♥❣ ✳ ✳ ✳ ✳ ✶✼✼ ✾✳✷✳✷ ❙P❖❈ ✐s ❛ ♠♦❧❡❝✉❧❛r ✐♥t❡❣r❛t♦r ♦❢ ❡♣✐❣❡♥❡t✐❝ ❛♥❞ tr❛♥s❝r✐♣t✐♦♥❛❧ ❝♦♥tr♦❧ ✳ ✶✼✽ ✾✳✷✳✷✳✶ ❘❡✲❡✈❛❧✉❛t✐♥❣ t❤❡ ❡ss❡♥t✐❛❧✐t② ♦❢ t❤❡ ❙P❊◆✲◆❈♦❘✴❙▼❘❚✲❍❉❆❈✸ ❛①✐s ✳ ✶✼✽ ✾✳✷✳✷✳✷ ❆ ♣♦ss✐❜❧❡ r♦❧❡ ❢♦r ♥✉❝❧❡♦s♦♠❡ r❡♠♦❞❡❧✐♥❣ ❞✉r✐♥❣ ❳❈■ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✾ ✾✳✷✳✷✳✸ ❙P❖❈ ❛s ❛ ❞✐r❡❝t r❡♣r❡ss♦r ♦❢ ❘◆❆P■■ ❢✉♥❝t✐♦♥ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✼✾ ✾✳✷✳✷✳✹ ❙P❖❈ ✐s ❛ ♥♦✈❡❧ ✐♥t❡r❛❝t✐♦♥ ❞♦♠❛✐♥ ♦❢ t❤❡ ♠✻_{❆ ✇r✐t✐♥❣ ♠❛❝❤✐♥❡r② ✳ ✳ ✳ ✶✽✷} ✾✳✸ ❙P❊◆✲❢❛♠✐❧② ♣r♦t❡✐♥s ❛s ❘◆❆✲❣✉✐❞❡❞ tr❛♥s❝r✐♣t✐♦♥❛❧ r❡♣r❡ss♦rs ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✶✽✸

❱ ❘❡❢❡r❡♥❝❡s

✶✽✻

❱■ ❆♣♣❡♥❞✐①

✷✶✺

❆ ❆❞❞✐t✐♦♥❛❧ ♣✉❜❧✐❝❛t✐♦♥s ✷✶✻ ❆✳✶ ❆rt✐❝❧❡ ✸✿ ♠✻_{❆ ❘◆❆ ♠❡t❤②❧❛t✐♦♥ r❡❣✉❧❛t❡s t❤❡ ❢❛t❡ ♦❢ ❡♥❞♦❣❡♥♦✉s r❡tr♦✈✐r✉s❡s} ✭◆❛t✉r❡✳ ✷✵✷✶✮ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✷✶✻ ❆✳✷ ❆rt✐❝❧❡ ✹✿ ❙P❊◆ ✐s r❡q✉✐r❡❞ ❢♦r ❳✐st ✉♣r❡❣✉❧❛t✐♦♥ ❞✉r✐♥❣ ✐♥✐t✐❛t✐♦♥ ♦❢ ❳ ❝❤r♦✲ ♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ✭❇✐♦❘①✐✈✳ ✷✵✷✵✮ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✳ ✷✹✾ ❇ ▲♦♥❣ rés✉♠é ❡♥ ❢r❛♥ç❛✐s ✷✾✶

P❛rt ■

■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥

❈❤❛♣t❡r ✶

❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥

❉✐s❝❧❛✐♠❡r✿ ❚❤❡ ❝♦r❡ ♦❢ ♠② P❤❉ ❡①♣❡r✐♠❡♥t❛❧ ✇♦r❦ ❤❛s ❜❡❡♥ t♦ ❝❤❛r❛❝t❡r✐③❡ t❤❡ ❢✉♥❝t✐♦♥ ♦❢ ❙P❊◆ ✐♥ t❤❡ ♣r♦❝❡ss ♦❢ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ❚❤❡ ❢♦❧❧♦✇✐♥❣ ❝❤❛♣t❡r ❞♦❡s ♥♦t ❛✐♠ t♦ ♣r♦✈✐❞❡ ❛♥ ❡①❤❛✉st✐✈❡ r❡✈✐❡✇ ♦♥ t❤❡ t♦♣✐❝ ♦❢ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ■♥st❡❛❞✱ ✐t ❛✐♠s t♦ ❛❞❞r❡ss ❝❡rt❛✐♥ ❛s♣❡❝ts ♦❢ t❤✐s ♣r♦❝❡ss ✇❤✐❝❤ ❛r❡ ❡ss❡♥t✐❛❧ t♦ ✉♥❞❡rst❛♥❞ t❤❡ q✉❡st✐♦♥s ■ ✐♥✈❡st✐❣❛t❡❞ ❛♥❞ ❣r❛s♣ t❤❡ r❡❧❡✈❛♥❝❡ ♦❢ t❤❡ r❡s✉❧ts ■ ♦❜t❛✐♥❡❞✳

✶✳✶ ❙❡① ❝❤r♦♠♦s♦♠❡ ❡✈♦❧✉t✐♦♥ ✐♥ ♠❛♠♠❛❧s

■♥ t❤❡r✐❛♥ ♠❛♠♠❛❧s✱ s❡① ✐s ❣❡♥❡t✐❝❛❧❧② ❞❡t❡r♠✐♥❡❞ ❜② ❛ ♣❛✐r ♦❢ s❡① ❝❤r♦♠♦s♦♠❡s ❝❛❧❧❡❞ ❳ ❛♥❞ ❨ ✿ ❢❡♠❛❧❡s ❤❛r❜♦r t✇♦ ❳ ❝❤r♦♠♦s♦♠❡s ✇✐t❤✐♥ t❤❡✐r ❣❡♥♦♠❡✱ ✇❤✐❧❡ ♠❛❧❡s ♦♥❧② ❤❛r❜♦r ♦♥❡ ❳ ❝❤r♦♠♦s♦♠❡ ❛s ✇❡❧❧ ❛s ❛♥ ❛❞❞✐t✐♦♥❛❧ ❨ ❝❤r♦♠♦s♦♠❡ ❬▼❛rs❤❛❧❧ ●r❛✈❡s ❛♥❞ ❙❤❡tt②✱ ✷✵✵✶❪✳ ❆❧t❤♦✉❣❤ t❤❡ ❳ ❛♥❞ ❨ ❝❤r♦♠♦s♦♠❡s ❜♦t❤ ❡✈♦❧✈❡❞ ❢r♦♠ t❤❡ s❛♠❡ ♣❛✐r ♦❢ ❛✉t♦s♦♠❡s ✭♣r♦t♦✲s❡① ❝❤r♦♠♦s♦♠❡s✱ ❋✐❣✉r❡ ✶✳✶❆✮✱ t❤❡② ❞✐✛❡r ❞r❛st✐❝❛❧❧② ❢r♦♠ ♦♥❡ ❛♥♦t❤❡r ❛s ❛ ❝♦♥s❡q✉❡♥❝❡ ♦❢ ♠♦r❡ t❤❛♥ ✸✵✵ ♠✐❧❧✐♦♥ ②❡❛rs ♦❢ ❡✈♦❧✉t✐♦♥ ✭❋✐❣✉r❡ ✶✳✶✮ ❬●r❛✈❡s✱ ✷✵✵✻❪✳ ❚♦❞❛②✱ ✇❤✐❧❡ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐s ✶✻✺▼❜ ❧♦♥❣ ✐♥ ❤✉♠❛♥s✱ ❡♥❝♦❞✐♥❣ ♦✈❡r ✽✵✵ ♣r♦t❡✐♥✲❝♦❞✐♥❣ ❣❡♥❡s✱ t❤❡ ❨ ❝❤r♦♠♦s♦♠❡ ✐s ♦♥❧② ✻✵▼❜ ❧♦♥❣ ❛♥❞ ❡♥❝♦❞❡s ♥♦ ♠♦r❡ t❤❛♥ ✺✵ ♣r♦t❡✐♥✲❝♦❞✐♥❣ ❣❡♥❡s ❬●r❛✈❡s ❡t ❛❧✳✱ ✷✵✵✻❪✳ ❆♣♣r♦①✐♠❛t❡❧② ✸✵✵ ♠✐❧❧✐♦♥ ②❡❛rs ❛❣♦✱ ❛ ♠✉t❛t✐♦♥ ♦❢ t❤❡ ❙❖❳✸ ❣❡♥❡ ❧❡❞ t♦ t❤❡ ❣❡♥✲ ❡r❛t✐♦♥ ♦❢ ❛ ♥❡✇ ✈❛r✐❛♥t✱ ❝❛❧❧❡❞ ❙❘❨✱ ♦♥ ♦♥❡ ♦❢ t❤❡ ♣r♦t♦✲s❡① ❝❤r♦♠♦s♦♠❡s✿ t❤❡ ♣r♦t♦✲❨ ✭❋✐❣✉r❡ ✶✳✶❇✮ ❬●r❛✈❡s✱ ✷✵✵✻❪✳ ❚❤✐s ✈❛r✐❛♥t ❝♦♥❢❡rr❡❞ t❤❡ ❛❜✐❧✐t② t♦ ✐ts ❝❛rr✐❡rs t♦ ❜❡ ♠❛❧❡✱ ✇❤✐❧❡ ✐♥❞✐✈✐❞✉❛❧s ❝❛rr②✐♥❣ t❤❡ ✉♥♠♦❞✐✜❡❞ ♣r♦t♦✲❳ r❡♠❛✐♥❡❞ ❢❡♠❛❧❡✳ ❲✐t❤ ❡✈♦✲ ❧✉t✐♦♥❛r② t✐♠❡✱ ❣❡♥❡s t❤❛t ✇❡r❡ ❜❡♥❡✜❝✐❛❧ t♦ ♠❛❧❡s ❜❡❝❛♠❡ ❡♥r✐❝❤❡❞ s♣❡❝✐✜❝❛❧❧② ♦♥ t❤❡ ♣r♦t♦✲❨ ✭❋✐❣✉r❡ ✶✳✶❈✮ ❬❇❛❝❤tr♦❣✱ ✷✵✶✸❪✳ ❈♦♥s❡q✉❡♥t❧②✱ ♠❡✐♦t✐❝ r❡❝♦♠❜✐♥❛t✐♦♥ ❜❡t✇❡❡♥ t❤❡ ♣r♦t♦ ❳ ❛♥❞ ❨ ✇♦✉❧❞ ❧❡❛❞ t♦ ✉♥❞❡s✐r❡❞ ✏✐♥t❡rs❡①✑ st❛t❡s✱ ✇❤❡r❡✐♥ ♠❛❧❡s ✇♦✉❧❞ ❧♦s❡ ✼

■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ s♦♠❡ ✏❜❡♥❡✜❝✐❛❧✑ ♠❛❧❡✲s♣❡❝✐✜❝ ❣❡♥❡s✱ ✇❤✐❧❡ ❢❡♠❛❧❡s ✇♦✉❧❞ ✐♥❤❡r✐t ✏❤❛r♠❢✉❧✑ ❣❡♥❡s ♣r❡✈✐✲ ♦✉s❧② ❢♦✉♥❞ ♦♥❧② ♦♥ t❤❡ ♣r♦t♦✲❨ ✭❋✐❣✉r❡ ✶✳✶❉✮✳ ❚❤❡r❡❢♦r❡✱ ♠❡✐♦t✐❝ r❡❝♦♠❜✐♥❛t✐♦♥ ❛r♦✉♥❞ t❤❡ ♠❛❧❡✲s♣❡❝✐✜❝ ❣❡♥❡s ✇❛s s✉♣♣r❡ss❡❞ s♦ ❛s t♦ ♣r❡s❡r✈❡ ❛ str✐❝t ❞✐st✐♥❝t✐♦♥ ❜❡t✇❡❡♥ t❤❡ ♣r♦t♦✲❳ ❛♥❞ t❤❡ ♣r♦t♦✲❨ ✭❋✐❣✉r❡ ✶✳✶❊✮✳ ❯♥❛❜❧❡ t♦ r❡❝♦♠❜✐♥❡ ✇✐t❤ t❤❡ ♣r♦t♦✲❳✱ t❤❡ ♣r♦t♦✲❨ ❛❝❝✉♠✉❧❛t❡❞ ❞❡❧❡t❡r✐♦✉s ♠✉t❛t✐♦♥s ✇❤✐❝❤ ❝♦✉❧❞ ♥❡✈❡r ❜❡ ❜❛❧❛♥❝❡❞ ♦✉t ❞✉r✐♥❣ ♠❡✐♦s✐s ✭❋✐❣✉r❡ ✶✳✶❊✮ ❬▼✉❧❧❡r✱ ✶✾✶✽❀ ❘✐❝❡✱ ✶✾✽✼✱ ✶✾✾✻❪✳ ❚❤✐s ♠✉t❛t✐♦♥❛❧ ❜✉r❞❡♥ ❧❡❞ t♦ t❤❡ ❞❡❣❡♥❡r❛t✐♦♥ ♦❢ ♠♦st ♦❢ t❤❡ ❨ ❝❤r♦♠♦s♦♠❡ ❛♥❞ ✐ts ❡♥❝♦❞✐♥❣ ❣❡♥❡s✱ t❤❡r❡❜② ♣r♦✈✐❞✐♥❣ ❛♥ ❡①♣❧❛♥❛t✐♦♥ ❢♦r t❤❡ ❞✐✛❡r❡♥❝❡ ❜❡t✇❡❡♥ t❤❡ ♠♦❞❡r♥ ❳ ❛♥❞ ❨ ❝❤r♦♠♦s♦♠❡s ✐♥ ♠❛♠✲ ♠❛❧s ✭❋✐❣✉r❡ ✶✳✶❊✮ ❬❈❤❛r❧❡s✇♦rt❤✱ ✶✾✾✻❀ ❈❤❛r❧❡s✇♦rt❤ ❡t ❛❧✳✱ ✷✵✵✺❀ ●r❛✈❡s✱ ✷✵✵✻❀ ●r❛✈❡s ❡t ❛❧✳✱ ✷✵✵✻❀ ❱✐❝♦s♦ ❛♥❞ ❈❤❛r❧❡s✇♦rt❤✱ ✷✵✵✻❪✳ A Autosome pair SOX3 proto-X proto-Y SRY

proto-X proto-Y proto-X proto-Y

XCI male-specific genes meiotic crossovers deleterious mutations B E F C D proto-X proto-Y x2 proto-X proto-Y Y x2 x2 X x2 x2 X x2 x2 X x2 x2 X x2 x2 X x2 x2 x2 two-fold expression ❋✐❣✉r❡ ✶✳✶ ✕ ▼❛♠♠❛❧✐❛♥ s❡① ❝❤r♦♠♦s♦♠❡ ❡✈♦❧✉t✐♦♥ ❛♥❞ t❤❡ r❡q✉✐r❡♠❡♥t ❢♦r ❞♦s❛❣❡ ❝♦♠♣❡♥s❛t✐♦♥✳ ✭❆✮ ▼✉t❛t✐♦♥s ✐♥ t❤❡ ❙❖❳✸ ❣❡♥❡ ❧❡❛❞ t♦ ✭❇✮ t❤❡ ❡♠❡r❣❡♥❝❡ ♦❢ ❛ s❡①✲ ❞❡t❡r♠✐♥✐♥❣ ❣❡♥❡✱ ❙❘❨✱ ♦♥ ♦♥❡ ♦❢ t❤❡ t✇♦ ♣r♦t♦✲s❡① ❝❤r♦♠♦s♦♠❡s✳ ✭❈✮ ▼❛❧❡✲s♣❡❝✐✜❝ ❣❡♥❡s ❛❝❝✉♠✉❧❛t❡ ♦♥ t❤❡ ♣r♦t♦✲❨✱ ❧❡❛❞✐♥❣ t♦ ✭❉✮ ❞❡❧❡t❡r✐♦✉s ✐♥t❡r✲s❡① ❛rr❛♥❣❡♠❡♥ts ❢♦❧❧♦✇✐♥❣ r❡❝♦♠✲ ❜✐♥❛t✐♦♥ ❡✈❡♥ts ✐♥ ♠❡✐♦s✐s✳ ✭❊✮ ❈♦♥s❡q✉❡♥t✐❛❧ s✉♣♣r❡ss✐♦♥ ♦❢ ♠❡✐♦t✐❝ r❡❝♦♠❜✐♥❛t✐♦♥ ✐♥ t❤❡ ♠❛❧❡ ❣❡r♠❧✐♥❡ ❢❛✈♦rs ✜①❛t✐♦♥ ♦❢ ❞❡❧❡t❡r✐♦✉s ♠✉t❛t✐♦♥s✱ r❡s✉❧t✐♥❣ ✐♥ t❤❡ ♣r♦❣r❡ss✐✈❡ ❞❡❣❡♥❡r❛t✐♦♥ ♦❢ t❤❡ ♣r♦t♦✲❨ ❝❤r♦♠♦s♦♠❡✳ ❆s ❣❡♥❡s ❛r❡ ❧♦st ♦♥ t❤❡ ♣r♦t♦✲❨✱ s❡❧❡❝t✐✈❡ ♣r❡ss✉r❡s ❡♥s✉r❡ t✇♦✲❢♦❧❞ ✉♣r❡❣✉❧❛t✐♦♥ ♦❢ ❝♦rr❡s♣♦♥❞✐♥❣ ❣❡♥❡s ♦♥ t❤❡ ♣r♦t♦✲❳✳ ✭❋✮ ❚❤❡ r❡s✉❧t✐♥❣ ✐♥❝r❡❛s❡ ✐♥ ❳✲❧✐♥❦❡❞ ❣❡♥❡ ❞♦s❛❣❡ ✐♥ ❢❡♠❛❧❡s ✐s ❝♦♠♣❡♥s❛t❡❞ t❤r♦✉❣❤ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥✳ ✽

■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥

✶✳✷ ❉♦s❛❣❡ ❝♦♠♣❡♥s❛t✐♦♥ ♣r♦❜❧❡♠s ❛♥❞ t❤❡ ❞✐s❝♦✈❡r②

♦❢ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥

■♠♣♦rt❛♥t❧②✱ ❛s ❣❡♥❡s ✇❡r❡ ❜❡✐♥❣ ❧♦st ♦♥ t❤❡ ❨ ❝❤r♦♠♦s♦♠❡✱ ❛ ❣❡♥❡ ❞♦s❛❣❡ ♣r♦❜✲ ❧❡♠ ❛r♦s❡ ❜❡t✇❡❡♥ ❳❳ ❢❡♠❛❧❡s ❛♥❞ ❳❨ ♠❛❧❡s✳ ❚❤✐s ♣r♦❜❧❡♠ ✇❛s ✜rst ✐❞❡♥t✐✜❡❞ ❜② t❤❡ ❏❛♣❛♥❡s❡ ❣❡♥❡t✐❝✐st ❙✉s✉♠✉ ❖❤♥♦✱ ✇❤♦ ♣r♦♣♦s❡❞ t❤❛t ❛s ❡❛❝❤ ❣❡♥❡ ❣r❛❞✉❛❧❧② ❞✐s❛♣♣❡❛r❡❞ ❢r♦♠ t❤❡ ❨ ❝❤r♦♠♦s♦♠❡✱ s❡❧❡❝t✐✈❡ ♣r❡ss✉r❡s ❡♥s✉r❡❞ t❤❛t t❤❡ ❡①♣r❡ss✐♦♥ ♦❢ ✐ts ❝♦rr❡s♣♦♥❞✲ ✐♥❣ ❝♦♣② ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ✇❛s ✐♥❝r❡❛s❡❞ ❜② t✇♦✲❢♦❧❞ ✭❋✐❣✉r❡ ✶✳✶❊✮ ❬❖❤♥♦✱ ✶✾✻✼❪✳ ❙❡✈❡r❛❧ st✉❞✐❡s ❤❛✈❡ ♣r♦✈✐❞❡❞ s✉♣♣♦rt✐♥❣ ❡✈✐❞❡♥❝❡ ❢♦r ❖❤♥♦✬s ❤②♣♦t❤❡s✐s t❤❛t ❳✲❧✐♥❦❡❞ ❣❡♥❡s ❛r❡ t✇♦✲❢♦❧❞ ✉♣r❡❣✉❧❛t❡❞ ❬❉❡♥❣ ❡t ❛❧✳✱ ✷✵✶✶❀ ❄❀ ▲✐♥ ❡t ❛❧✳✱ ✷✵✵✼❪✳ ❍♦✇❡✈❡r✱ ✇❤✐❧❡ ❛ t✇♦✲❢♦❧❞ ❡①♣r❡ss✐♦♥ ♦❢ ❳✲❧✐♥❦❡❞ ❣❡♥❡s r❡s♦❧✈❡s ❞♦s❛❣❡ ♣r♦❜❧❡♠s ✐♥ ❳❨ ♠❛❧❡s✱ ✐t ✐♠♣❧✐❡s t❤❛t ❳❳ ❢❡♠❛❧❡s ♥♦✇ ❤❛✈❡ t♦ ❝♦♣❡ ✇✐t❤ t✇♦ ❝♦♣✐❡s ♦❢ t✇♦✲❢♦❧❞ ❡①♣r❡ss❡❞ ❳✲❧✐♥❦❡❞ ❣❡♥❡s ✭❋✐❣✉r❡ ✶✳✶❋✮✳ ❈♦♥s❡q✉❡♥t❧②✱ ❢❡♠❛❧❡ ♠❛♠♠❛❧s ❡✈♦❧✈❡❞ ❛ s♣❡❝✐✜❝ ♠❡❝❤❛♥✐s♠ ❝❛❧❧❡❞ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ✭❳❈■✮✱ ❞✉r✐♥❣ ✇❤✐❝❤ ♦♥❡ ♦❢ t❤❡ t✇♦ ❳ ❝❤r♦♠♦s♦♠❡s ✐s tr❛♥✲ s❝r✐♣t✐♦♥❛❧❧② s✐❧❡♥❝❡❞ ✐♥ ❛❧❧ ❝❡❧❧s✱ ❡♥s✉r✐♥❣ t❤❛t ❡❛❝❤ ❢❡♠❛❧❡ ❝❡❧❧ ❡①♣r❡ss❡s ♦♥❡ ❝♦♣② ♦❢ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✱ ✐❞❡♥t✐❝❛❧❧② t♦ ♠❛❧❡ ❝❡❧❧s ✭❋✐❣✉r❡ ✶✳✶❋✮✳ ❚❤✐s ♣r♦❝❡ss ✇❛s ✜rst ♣✉t ❢♦rt❤ ❜② ▼❛r② ▲②♦♥ ✐♥ ✶✾✻✶ ❬▲②♦♥✱ ✶✾✻✶❪✳ ❇✉✐❧❞✐♥❣ ✉♣ ♦♥ ♣r❡✈✐♦✉s ♦❜s❡r✈❛t✐♦♥s t❤❛t ♦♥❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐s ❤❡t❡r♦♣②❦♥♦t✐❝ ✐♥ ❢❡♠❛❧❡ ❝❡❧❧s✱ ❛♥❞ t❤❛t ❢❡♠❛❧❡s ❤❡t❡r♦③②❣♦✉s ❛t s❡①✲❧✐♥❦❡❞ ❧♦❝✐ ❛✛❡❝t✐♥❣ ❝♦❛t ❝♦❧♦r s❤♦✇ ❛ ♠♦s❛✐❝ ♣❤❡♥♦t②♣❡ ✭✇✐t❤ ❤❛✐r ♣❛t❝❤❡s ♦❢ ❞✐✛❡r❡♥t ❝♦❧♦r ❛❝r♦ss t❤❡ ❜♦❞②✮✱ ▲②♦♥ ♣r♦♣♦s❡❞ t❤❛t✿ ❼ ❖♥❡ ♦❢ t❤❡ t✇♦ ❳ ❝❤r♦♠♦s♦♠❡s ✐♥ ❢❡♠❛❧❡ ❝❡❧❧s ✐s ✏❣❡♥❡t✐❝❛❧❧② ✐♥❛❝t✐✈❛t❡❞✑ ❬s✐❝❪ ❼ ❚❤✐s ✐♥❛❝t✐✈❛t✐♦♥ ❤❛♣♣❡♥s ❡❛r❧② ❞✉r✐♥❣ ❡♠❜r②♦♥✐❝ ❞❡✈❡❧♦♣♠❡♥t ❼ ❖♥❝❡ ✐♥❛❝t✐✈❛t❡❞✱ t❤❡ s❛♠❡ ❳ ❝❤r♦♠♦s♦♠❡ r❡♠❛✐♥s ✐♥❛❝t✐✈❛t❡❞ ✐♥ ❞❡s❝❡♥❞✐♥❣ ❝❡❧❧s✳ ❉❡❝❛❞❡s ♦❢ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ r❡s❡❛r❝❤ ❤❛✈❡ ♣r♦✈❡❞ t❤❛t ▼❛r② ▲②♦♥✬s ❤②✲ ♣♦t❤❡s❡s ✇❡r❡ ❝♦rr❡❝t✱ ❛♥❞ ❤❛✈❡ ❤✐t❤❡rt♦ ♣r♦✈✐❞❡❞ r❡♠❛r❦❛❜❧❡ ✐♥s✐❣❤ts ✐♥t♦ t❤✐s ♣r♦❝❡ss✱ ✇❤✐❝❤ ❡♠❡r❣❡❞ ❛s ❛ ♣❛r❛❞✐❣♠ ❢♦r t❤❡ st✉❞② ♦❢ ❡♣✐❣❡♥❡t✐❝s ✭s❡❡ s❡❝t✐♦♥ ✶✳✶✶✮✳

✶✳✸ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ❞②♥❛♠✐❝s ❞✉r✐♥❣ ❡❛r❧②

❞❡✈❡❧♦♣♠❡♥t

▼❛r② ▲②♦♥ ♣r♦♣♦s❡❞ t❤❛t ❳❈■ t❛❦❡s ♣❧❛❝❡ ❡❛r❧② ❞✉r✐♥❣ ❡♠❜r②♦♥✐❝ ❞❡✈❡❧♦♣♠❡♥t✳ ■♥✲ ❞❡❡❞✱ t❤❡ ♦❜s❡r✈❛t✐♦♥ ♦❢ ♠♦s❛✐❝ ❳✲❧✐♥❦❡❞ ❤❛✐r ❝♦❧♦r ♣❤❡♥♦t②♣❡s ✐♥ ❢❡♠❛❧❡s s✉❣❣❡sts t❤❛t t❤❡r❡ s❤♦✉❧❞ ❜❡ ❛t ❧❡❛st ♦♥❡ ❞❡✈❡❧♦♣♠❡♥t❛❧ t✐♠❡✲✇✐♥❞♦✇ ❞✉r✐♥❣ ✇❤✐❝❤ ❜♦t❤ ♠❛t❡r♥❛❧ ❛♥❞ ♣❛t❡r♥❛❧ ❳✲❧✐♥❦❡❞ ❛❧❧❡❧❡s ✭❛♥❞✱ ❜② ❡①t❡♥s✐♦♥✱ ❝❤r♦♠♦s♦♠❡s✮ ❛r❡ ❡①♣r❡ss❡❞✱ s✉❝❤ t❤❛t ❡❛❝❤ ❝❡❧❧ ✏❝❤♦♦s❡s✑ t♦ ✐♥❛❝t✐✈❛t❡ ❡✐t❤❡r ♦♥❡ ♦r t❤❡ ♦t❤❡r ❳ ❝❤r♦♠♦s♦♠❡✳ ✾

■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ ❊❛r❧② r❡s❡❛r❝❤ ❝♦♥❞✉❝t❡❞ ✐♥ ♠✐❝❡✱ ✇❤✐❝❤ ❡♠❡r❣❡❞ ❛s t❤❡ ♣r❡❞♦♠✐♥❛♥t ♠♦❞❡❧ t♦ st✉❞② ❳❈■✱ ❛♥❞ ♦♥ ✇❤✐❝❤ ✇❡ ✇✐❧❧ ❢♦❝✉s ❢♦r t❤❡ r❡st ♦❢ t❤✐s ❞✐ss❡rt❛t✐♦♥✱ r❡✈❡❛❧❡❞ t❤❛t t❤❡ ♣❛✲ t❡r♥❛❧ ❳ ❝❤r♦♠♦s♦♠❡ ✭❳♣✮ ✐s ❛❧✇❛②s ✐♥❛❝t✐✈❡ ✐♥ ❡①tr❛❡♠❜r②♦♥✐❝ t✐ss✉❡s✱ ❛ ♣❤❡♥♦♠❡♥♦♥ ❞❡s❝r✐❜❡❞ ❛s ✐♠♣r✐♥t❡❞ ❳❈■ ✭✐❳❈■✮ ❬❚❛❦❛❣✐ ❛♥❞ ❙❛s❛❦✐✱ ✶✾✼✺❀ ❲❡st ❡t ❛❧✳✱ ✶✾✼✼❪✳ ●✐✈❡♥ t❤❛t ❡♠❜r②♦♥✐❝✴s♦♠❛t✐❝ t✐ss✉❡s ✐♥st❡❛❞ s❤♦✇ ❛ r❛♥❞♦♠ ♣❛tt❡r♥ ♦❢ ❳❈■ ✭♠♦s❛✐❝ ♣❤❡♥♦✲ t②♣❡✮✱ ✐t ✇❛s ✐♥✐t✐❛❧❧② ❜❡❧✐❡✈❡❞ t❤❛t ❡♠❜r②♦♥✐❝ ❛♥❞ ❡①tr❛❡♠❜r②♦♥✐❝ ❧✐♥❡❛❣❡s ✉♥❞❡r❣♦ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ✐♥❞❡♣❡♥❞❡♥t❧②✱ ❛♥❞ ✉♥❞❡r ❞✐✛❡r❡♥t ♠♦❞❛❧✐t✐❡s✳ ▲❛t❡r✱ ✐t ✇❛s s❤♦✇♥ t❤❛t ✇❤✐❧❡ ❜♦t❤ ♠❛t❡r♥❛❧ ❛♥❞ ♣❛t❡r♥❛❧ ❳ ❝❤r♦♠♦s♦♠❡s ❛r❡ ❛❝t✐✈❡❧② tr❛♥s❝r✐❜❡❞ ❛t ③②❣♦t✐❝ ❣❡♥❡ ❛❝t✐✈❛t✐♦♥ ✭✷✲❝❡❧❧ st❛❣❡✱ ❋✐❣✉r❡ ✶✳✷❆✮ ❬❖❦❛♠♦t♦ ❡t ❛❧✳✱ ✷✵✵✺❀ P❛tr❛t ❡t ❛❧✳✱ ✷✵✵✾❪✱ ❛❧❧ ❝❡❧❧s ✐♥✐t✐❛❧❧② ✉♥❞❡r❣♦ ✐♠♣r✐♥t❡❞ ❳❈■ ✭❋✐❣✉r❡ ✶✳✷❇✮✳ ■♠♣r✐♥t❡❞ ❳❈■ ✐♥✐t✐❛t❡s ❛t t❤❡ ✹✲❝❡❧❧ st❛❣❡ s✉❝❤ t❤❛t ❜② t❤❡ ❡❛r❧② ❜❧❛st♦❝②st st❛❣❡✱ ✸✳✺ ❞❛②s ♣♦st ❝♦✐t✉♠ ✭❊✸✳✺✮✱ ❛❧❧ ❝❡❧❧s ♦❢ t❤❡ ❡♠❜r②♦ ❤❛✈❡ ❛♥ ✐♥❛❝t✐✈❡ ❳♣ ✭❋✐❣✉r❡ ✶✳✷❈✮ ❬▼❛❦ ❡t ❛❧✳✱ ✷✵✵✹❀ ❖❦❛♠♦t♦ ❡t ❛❧✳✱ ✷✵✵✹❪✳

1st _{wave of XCI: specific to the Xp}

2nd _{wave of XCI: random, either Xm or Xp}

hypoblast trophectoderm future epiblast active gene silent gene oocyte sperm zygote late blastocyst E3.5 - early blastocyst E5.5 ZGA imprinted 8-cell stage Xm Xp Xm Xp Xm Xp 4-cell stage 2-cell stage ZGA XCI imprinted XCI Xm Xp Xm Xp Xm Xp Xm Xp random XCI ICM Xp reactivation A 50% 50% B C D E F G ❋✐❣✉r❡ ✶✳✷ ✕ ❉②♥❛♠✐❝s ♦❢ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ❞✉r✐♥❣ ♠♦✉s❡ ❞❡✈❡❧♦♣♠❡♥t✳ ✭❆✮ ❋♦❧❧♦✇✐♥❣ ❢❡rt✐❧✐③❛t✐♦♥ ❛♥❞ ③②❣♦t✐❝ ❣❡♥♦♠❡ ❛❝t✐✈❛t✐♦♥ ✭❩●❆✮✱ ❜♦t❤ ❳ ❝❤r♦♠♦s♦♠❡s ❛r❡ ❡①♣r❡ss❡❞✳ ✭❇✮ ❆t t❤❡ ✹✲❝❡❧❧ st❛❣❡✱ ✐♠♣r✐♥t❡❞ ❳❈■ ✐♥✐t✐❛t❡s ❛♥❞ ✭❈✮ ❜② t❤❡ ❡❛r❧② ❜❧❛st♦❝②st st❛❣❡✱ ❛❧❧ ❝❡❧❧s ❤❛✈❡ ✐♥❛❝t✐✈❛t❡❞ t❤❡ ❳♣✳ ❚❤❡ ❳♣ r❡♠❛✐♥s ✐♥❛❝t✐✈❛t❡❞ ✐♥ ❡①tr❛❡♠❜r②♦♥✐❝ ❝❡❧❧s ✭tr♦♣❤❡❝t♦❞❡r♠ ❛♥❞ ❤②♣♦❜❧❛st✮✱ ✇❤✐❧❡ ✭❉✮ ✐t ❜❡❝♦♠❡s r❡❛❝t✐✈❛t❡❞ ♦♥❧② ✐♥ ■❈▼ ❝❡❧❧s t❤❛t ✇✐❧❧ ❣✐✈❡ r✐s❡ t♦ t❤❡ ❡♣✐❜❧❛st✳ ✭❊✮ ■♥ s✉❝❤ ❝❡❧❧s✱ r❛♥❞♦♠ ❳❈■ ♦❝❝✉rs s✉❝❤ t❤❛t ❢♦❧❧♦✇✐♥❣ ✐♠♣❧❛♥t❛t✐♦♥ ❛♥❞ ❝❧♦♥❛❧ ♣r♦♣❛❣❛t✐♦♥ ♦❢ ❳❈■ st❛t✉s✱ ✭❋✮ ❤❛❧❢ ♦❢ t❤❡ ❝❡❧❧s ♦❢ t❤❡ ❡♠❜r②♦ ♣r♦♣❡r ❤❛✈❡ ✐♥❛❝t✐✈❛t❡❞ t❤❡ ❳♣✱ ❛♥❞ t❤❡ ♦t❤❡r ❤❛❧❢ t❤❡ ❳♠✳ ✭●✮ ❘❛♥❞♦♠ ❳❈■ ❤❡♥❝❡ r❡s✉❧ts ✐♥ ❝❡❧❧✉❧❛r ♠♦s❛✐❝✐s♠ ✇✐t❤ r❡s♣❡❝t t♦ ❳✲❧✐♥❦❡❞ ❛❧❧❡❧✐❝ ❡①♣r❡ss✐♦♥✱ ✇❤✐❝❤ ❝❛♥ ❢♦r ❡①❛♠♣❧❡ tr❛♥s❧❛t❡ ✐♥t♦ ♠♦s❛✐❝ ❤❛✐r ♣❤❡♥♦t②♣❡s✳ ✶✵

■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ ❚❤❡ ❳♣ r❡♠❛✐♥s st❛❜❧② ✐♥❛❝t✐✈❛t❡❞ ✐♥ ❡①tr❛❡♠❜r②♦♥✐❝ t✐ss✉❡s ✭✐✳❡✳ tr♦♣❤❡❝t♦❞❡r♠ ❛♥❞ ❤②♣♦❜❧❛st✱ ❋✐❣✉r❡ ✶✳✷❈✮✳ ❘❡♠❛r❦❛❜❧② ❤♦✇❡✈❡r✱ t❤❡ ❳♣ ✐s r❡❛❝t✐✈❛t❡❞ s♣❡❝✐✜❝❛❧❧② ✐♥ ❛❧❧ ❝❡❧❧s ♦❢ t❤❡ ✐♥♥❡r ❝❡❧❧ ♠❛ss ✭■❈▼✮ t❤❛t ✇✐❧❧ ❣✐✈❡ r✐s❡ t♦ t❤❡ ❡♣✐❜❧❛st ✭❋✐❣✉r❡ ✶✳✷❉✮ ❬▼❛❦ ❡t ❛❧✳✱ ✷✵✵✹❀ ❖❦❛♠♦t♦ ❡t ❛❧✳✱ ✷✵✵✹❀ ❇♦r❡♥s③t❡✐♥ ❡t ❛❧✳✱ ✷✵✶✼❛❪✳ ❆s t❤❡ ❡♣✐❜❧❛st ❢♦r♠s ❛♥❞ ✐s ❢✉rt❤❡r s♣❡❝✐✜❡❞✱ ❡❛❝❤ ❝❡❧❧ ✉♥❞❡r❣♦❡s r❛♥❞♦♠ ✐♥❛❝t✐✈❛t✐♦♥ ♦❢ ❡✐t❤❡r t❤❡ ♣❛t❡r♥❛❧ ♦r t❤❡ ♠❛t❡r♥❛❧ ❳ ❝❤r♦♠♦s♦♠❡✱ ❛r♦✉♥❞ ✺✳✺ ❞❛②s ♣♦st ❝♦✐t✉♠ ✭❊✺✳✺✱ ❋✐❣✉r❡ ✶✳✷❊ ❛♥❞ ❋✮ ❬▼❛❦ ❡t ❛❧✳✱ ✷✵✵✹❀ ❖❦❛♠♦t♦ ❡t ❛❧✳✱ ✷✵✵✹❀ ❘❛st❛♥✱ ✶✾✽✷❪✳ ❙tr✐❦✐♥❣❧②✱ t❤❡ ❞❡✈❡❧♦♣♠❡♥t❛❧ ❞②♥❛♠✐❝s ❛♥❞ t✐♠✐♥❣ ♦❢ ❳❈■ ✈❛r② ❡①t❡♥s✐✈❡❧② ❛♠♦♥❣ ♣❧❛❝❡♥t❛❧ ♠❛♠♠❛❧s ❬❖❦❛♠♦t♦ ❡t ❛❧✳✱ ✷✵✶✶❀ ❊s❝❛♠✐❧❧❛✲❉❡❧✲❆r❡♥❛❧ ❡t ❛❧✳✱ ✷✵✶✶❪✳ ■♥ r❛❜✲ ❜✐ts ❛♥❞ ❤✉♠❛♥s ❢♦r ❡①❛♠♣❧❡✱ ✇❤❡r❡ ❳❈■ ❜❡❣✐♥s ❛t ❧❛t❡r ❞❡✈❡❧♦♣♠❡♥t❛❧ st❛❣❡s t❤❛♥ ✐♥ ♠✐❝❡✱ ❳✐st ❡①♣r❡ss✐♦♥ ✐s ♥♦t ✐♠♣r✐♥t❡❞ ✐♥ ❡❛r❧② ❡♠❜r②♦s ❛♥❞ ❳❈■ ❞♦❡s ♥♦t ♦❝❝✉r ❞✉r✲ ✐♥❣ ♣r❡✲✐♠♣❧❛♥t❛t✐♦♥ ❞❡✈❡❧♦♣♠❡♥t ❬❖❦❛♠♦t♦ ❡t ❛❧✳✱ ✷✵✶✶❪✳ ■♥st❡❛❞✱ ❳✐st ❘◆❆ ✐s s❡❡♥ ❛❝❝✉♠✉❧❛t✐♥❣ ♦♥ ❜♦t❤ ❤✉♠❛♥ ❳ ❝❤r♦♠♦s♦♠❡s ❬❖❦❛♠♦t♦ ❡t ❛❧✳✱ ✷✵✶✶❪✱ ❧❡❛❞✐♥❣ t♦ t❤❡✐r tr❛♥s❝r✐♣t✐♦♥❛❧ ❞❛♠♣❡♥✐♥❣ ✭r❛t❤❡r t❤❛♥ ❝♦♠♣❧❡t❡ ✐♥❛❝t✐✈❛t✐♦♥ ♦❢ ♦♥❡ ❳ ❝❤r♦♠♦s♦♠❡✮✱ ❡♥✲ s✉r✐♥❣ ❞♦s❛❣❡ ❝♦♠♣❡♥s❛t✐♦♥ ❜② t❤❡ ❡❛r❧② ❜❧❛st♦❝②st st❛❣❡ ❬❱❛❧❧♦t ❡t ❛❧✳✱ ✷✵✶✼❀ P❡tr♦♣♦✉❧♦s ❡t ❛❧✳✱ ✷✵✶✻❪✳ ❘❛♥❞♦♠ ❳❈■ ♦♥❧② ♣r♦❝❡❡❞s ❛❢t❡r t❤❡ ❧❛t❡ ❜❧❛st♦❝②st st❛❣❡ ❬❖❦❛♠♦t♦ ❡t ❛❧✳✱ ✷✵✶✶❪✳ ❚❤❡s❡ ❞✐✈❡rs❡ str❛t❡❣✐❡s ♦❢ ❳❈■ ❛r❡ ♣r♦♣♦s❡❞ t♦ r❡✢❡❝t ❞✐✛❡r❡♥❝❡s ✐♥ ❡♠❜r②♦ st❛❣✲ ✐♥❣ ❛♥❞ t✐♠✐♥❣ ♦❢ ❝❡❧❧✉❧❛r ❞✐✛❡r❡♥t✐❛t✐♦♥ ❜❡t✇❡❡♥ ❡✉t❤❡r✐❛♥ s♣❡❝✐❡s ❬▼♦♥❦ ❛♥❞ ❍❛r♣❡r✱ ✶✾✼✾❀ ▼✐❣❡♦♥✱ ✷✵✶✻❪✳

✶✳✹ ❳ ✐♥❛❝t✐✈❛t✐♦♥ ❝❛♥ ❜❡ r❡❝❛♣✐t✉❧❛t❡❞ ✐♥ ✈✐tr♦

P❧✉r✐♣♦t❡♥t ♠♦✉s❡ ❡♠❜r②♦♥✐❝ st❡♠ ❝❡❧❧s ✭♠❊❙❈s✮ ❞❡r✐✈❡❞ ❢r♦♠ t❤❡ ■❈▼ ❝❛♥ ❜❡ ❝✉❧✲ t✉r❡❞ ✐♥ ✈✐tr♦✱ ✇❤❡r❡ t❤❡② ❝❛♥ ❞✐✈✐❞❡ ❢♦r ♠❛♥② ♣❛ss❛❣❡s✳ ❙✐♠✐❧❛r❧② t♦ ✇❤❛t ✐s ♦❜s❡r✈❡❞ ✐♥ ✈✐✈♦ ❛t t❤❡ ❜❧❛st♦❝②st st❛❣❡✱ ❝✉❧t✉r❡❞ ❢❡♠❛❧❡ ♠❊❙❈s ❤❛✈❡ t✇♦ ❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡s ❛♥❞ ✉♥❞❡r❣♦ r❛♥❞♦♠ ❳❈■ ✇❤❡♥ ❞✐✛❡r❡♥t✐❛t✐♦♥ ✐s tr✐❣❣❡r❡❞ ✉♣♦♥ ❝❤❛♥❣❡s ✐♥ ❝✉❧t✉r❡ ❝♦♥❞✐t✐♦♥s ✭❋✐❣✉r❡ ✶✳✸✮✳ ❚❤❡ ❛❜✐❧✐t② ♦❢ ❢❡♠❛❧❡ ♠❊❙❈s t♦ r❡❝❛♣✐t✉❧❛t❡ ❳❈■ ✐♥ ✈✐tr♦ ❤❛s ♠❛❞❡ ❳❈■ r❡s❡❛r❝❤ ♠✉❝❤ ♠♦r❡ ❛❝❝❡ss✐❜❧❡ ❛♥❞ ❝♦♥✈❡♥✐❡♥t✱ ❛s ✐t ❞♦❡s ♥♦t r❡q✉✐r❡ ❤❛♥❞❧✐♥❣ ♣❡r✐✲✐♠♣❧❛♥t❛t✐♦♥ ❡♠❜r②♦s✱ ✇❤✐❝❤ ❛r❡ ♣❛rt✐❝✉❧❛r❧② ❝❤❛❧❧❡♥❣✐♥❣ t♦ ✇♦r❦ ✇✐t❤✳ ❋✉rt❤❡r♠♦r❡✱ ♠❊❙❈s ✐♥ ❝✉❧✲ t✉r❡ ❝❛♥ ❡❛s✐❧② ❜❡ ❣❡♥❡t✐❝❛❧❧② ♠❛♥✐♣✉❧❛t❡❞ ❛♥❞ ②✐❡❧❞ ❤✐❣❤ ❝❡❧❧ ♥✉♠❜❡rs✱ ❛❧❧♦✇✐♥❣ ❢♦r t❤❡ ✉s❡ ♦❢ s❡✈❡r❛❧ t❡❝❤♥✐❝❛❧ t♦♦❧s ❛♥❞ ❛♣♣r♦❛❝❤❡s t❤❛t ❝♦✉❧❞ ♥♦t ❤❛✈❡ ❜❡❡♥ ❡♠♣❧♦②❡❞ ✐♥ ❡❛r❧② ❡♠❜r②♦s✳ ■♥ ❢❛❝t✱ ♦✈❡r t❤❡ ♣❛st ✷✵ ②❡❛rs✱ ♠❛♥② ✐♠♣♦rt❛♥t ❞✐s❝♦✈❡r✐❡s ✐♥ t❤❡ ❳❈■ ✜❡❧❞ ❤❛✈❡ ❜❡❡♥ ♠❛❞❡ t❤❛♥❦s t♦ t❤❡ ♠❊❙❈ ✐♥ ✈✐tr♦ ♠♦❞❡❧✳ ❚❤❡ ❝♦r❡ ♦❢ ♠② ❞♦❝t♦r❛❧ r❡s❡❛r❝❤ ✇❛s ✉♥❞❡rt❛❦❡♥ ✐♥ s✉❝❤ ✐♥ ✈✐tr♦ ♠♦❞❡❧ ♦❢ ❳❈■✱ ❛♥❞ ♦♥❧② ❛ ❢❡✇ ❡①♣❡r✐♠❡♥ts ✭s❡❡ s✉❜s❡❝✲ t✐♦♥ ✻✳✷✳✷✮ ✇❡r❡ ❛❧s♦ ♣❡r❢♦r♠❡❞ ✐♥ ✈✐✈♦ ✭✐✳❡✳ ✐♥ ♠♦✉s❡ ❡♠❜r②♦s✮✳ ✶✶

■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ Xa Xa early blastocyst differentiation in vitro culture

mouse embryonic stem cells

random XCI Xi Xa 50% and ICM Xa Xi 50% ❋✐❣✉r❡ ✶✳✸ ✕ ❈✉❧t✉r❡❞ ♠♦✉s❡ ❡♠❜r②♦♥✐❝ st❡♠ ❝❡❧❧s ❛s ❛♥ ✐♥ ✈✐tr♦ ♠♦❞❡❧ ♦❢ r❛♥❞♦♠ ❳❈■✳ ▼♦✉s❡ ❡♠❜r②♦♥✐❝ st❡♠ ❝❡❧❧s ❞❡r✐✈❡❞ ❢r♦♠ t❤❡ ■❈▼ ❝❛rr② t✇♦ ❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡s✳ ❯♣♦♥ ❞✐✛❡r❡♥t✐❛t✐♦♥✱ t❤❡s❡ ❝❡❧❧s ✉♥❞❡r❣♦ r❛♥❞♦♠ ❳❈■✱ ❛♥❞ ♦♥❡ ♦❢ t❤❡ t✇♦ ❳ ❝❤r♦♠♦s♦♠❡s ✐s tr❛♥s❝r✐♣t✐♦♥❛❧❧② s✐❧❡♥❝❡❞✳

✶✳✺ ❚❤❡ ❳✲✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥t❡r ❛♥❞ t❤❡ ❞✐s❝♦✈❡r② ♦❢

❳✐st ❛s ❛♥ ❡ss❡♥t✐❛❧ ♠❡❞✐❛t♦r ♦❢ ❳❈■

❚✇❡♥t② ②❡❛rs ❛❢t❡r ▲②♦♥✬s s❡♠✐♥❛❧ ❛rt✐❝❧❡✱ t❤r♦✉❣❤ ❛♥❛❧②s✐s ♦❢ ❳✲❝❤r♦♠♦s♦♠❛❧ ❞❡❧❡✲ t✐♦♥s ❛♥❞ tr❛♥s❧♦❝❛t✐♦♥s ✐♥ ♠♦✉s❡ ❡♠❜r②♦s ❛♥❞ ❡♠❜r②♦♥✐❝ ❝❡❧❧s✱ ❛ s✐♥❣❧❡ ❳✲❧✐♥❦❡❞ ❧♦❝✉s✱ t❡r♠❡❞ t❤❡ ❳ ✲✐♥❛❝t✐✈❛t✐♦♥ ❝❡♥t❡r ✭❳✐❝✱ ❋✐❣✉r❡ ✶✳✹❆✮✱ ✇❛s ✐❞❡♥t✐✜❡❞ t♦ ❜❡ t❤❡ r❡❣✐♦♥ t❤❛t ✐s ♥❡❝❡ss❛r② ❛♥❞ s✉✣❝✐❡♥t t♦ tr✐❣❣❡r ❳❈■ ❬❘❛st❛♥✱ ✶✾✽✸❀ ❘❛st❛♥ ❛♥❞ ❘♦❜❡rts♦♥✱ ✶✾✽✺❪✳ ■♠♣♦rt❛♥t❧②✱ t❤✐s ❧♦❝✉s ❤❛s t♦ ❜❡ ♣❤②s✐❝❛❧❧② ❧✐♥❦❡❞ t♦ t❤❡ ❳✲❝❤r♦♠♦s♦♠❡ ❢♦r ❳❈■ t♦ ♦❝❝✉r✱ ❞❡♠♦♥str❛t✐♥❣ t❤❛t ❡❧❡♠❡♥ts ❝♦♠♣r✐s❡❞ ✇✐t❤✐♥ t❤❡ ❳✐❝ r❡❣✉❧❛t❡ ❳❈■ ✐♥ ❝✐s✳ ❚❤❡s❡ st✉❞✐❡s ♣❛✈❡❞ t❤❡ ✇❛② ❢♦r ❢✉t✉r❡ ❧✐♥❡s ♦❢ r❡s❡❛r❝❤ ❛✐♠✐♥❣ t♦ ✐❞❡♥t✐❢② t❤❡ ❣❡♥❡✭s✮ r❡s♣♦♥s✐❜❧❡ ❢♦r ♠❡❞✐❛t✐♥❣ ❳❈■✳ ■♥ t❤❡ ❡❛r❧② ✶✾✾✵s✱ r❡s❡❛r❝❤ ❧❡❞ ❜② ❈❛r♦❧②♥ ❇r♦✇♥ ✐❞❡♥t✐✜❡❞ ❛ ♥♦✈❡❧ ❤✉♠❛♥ ❳✲❧✐♥❦❡❞ ❣❡♥❡ ✇❤✐❝❤✱ ❝♦♥tr❛r② t♦ ♠♦st ♦t❤❡r ❳✲❧✐♥❦❡❞ ❣❡♥❡s✱ ✇❛s ❡①♣r❡ss❡❞ ❢r♦♠ t❤❡ ✐♥❛❝t✐✈❡ ❳ ❝❤r♦♠♦s♦♠❡ ❜✉t ♥♦t ❢r♦♠ t❤❡ ❛❝t✐✈❡ ♦♥❡ ❬❇r♦✇♥ ❡t ❛❧✳✱ ✶✾✾✶❪✳ ❚❤✐s ❣❡♥❡✱ ❝❛❧❧❡❞ ❳■❙❚ ✭❢♦r ❳ ✲✐♥❛❝t✐✈❡ s♣❡❝✐✜❝ tr❛♥s❝r✐♣t✮✱ ♠❛♣s ✇✐t❤✐♥ t❤❡ ❳✐❝ ❧♦❝✉s ✭❋✐❣✉r❡ ✶✳✹❇✮ ✐♥ ❜♦t❤ ♠♦✉s❡ ❛♥❞ ❤✉♠❛♥s ❛♥❞ s❤♦✇s ❝♦♥s❡r✈❛t✐♦♥ ❛t ❜♦t❤ s❡q✉❡♥❝❡ ❛♥❞ ❣❡♥❡ str✉❝t✉r❡ ❧❡✈❡❧s ❬❇r♦❝❦❞♦r✛ ❡t ❛❧✳✱ ✶✾✾✷❀ ❇r♦✇♥ ❡t ❛❧✳✱ ✶✾✾✷❪✳ ❚❤❡ ❳✐st tr❛♥s❝r✐♣t ✭✶✺❦❜ ✐♥ ♠♦✉s❡✱ ✶✼❦❜ ✐♥ ❤✉♠❛♥s ❛❢t❡r s♣❧✐❝✐♥❣✮ ✐s ❝❛♣♣❡❞ ❛♥❞ ♣♦❧②❛❞❡♥②❧❛t❡❞ ✭❋✐❣✉r❡ ✶✳✹❈✮✱ ❜✉t ❤❛s ♥♦ ♣r♦t❡✐♥✲ ❝♦❞✐♥❣ ♣♦t❡♥t✐❛❧✱ ❛s ✐t ❝♦♥t❛✐♥s ♥♦ ❝♦♥s❡r✈❡❞ ❖❘❋s ❛♥❞ ❞♦❡s ♥♦t ❛ss♦❝✐❛t❡ ✇✐t❤ t❤❡ tr❛♥s❧❛t✐♦♥ ♠❛❝❤✐♥❡r②✳ ✶✷

■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥ mouse X chromosome (171 Mb) Xic A B C centromere telomere 10 Mb 50 kb Tsix Xist Nap1l2 Ppnx Ftx Xpct Rnf12 ...AAAAAAAAA N⁷-mG 5’

Xist transcription unit

2.5 kb

3’

Xist RNA (15 Kb)

5’ capping, splicing and polyadenylation

Cdx4 Chic1 Tsx Jpx Cnbp2 ❋✐❣✉r❡ ✶✳✹ ✕ ❙❝❤❡♠❡ ♦❢ t❤❡ ❳✐❝ r❡❣✐♦♥ ❛♥❞ t❤❡ ❳✐st ❧♦❝✉s✳ ✭❆✮ ❚❤❡ ♣♦s✐t✐♦♥ ♦❢ t❤❡ ❳✐❝ ❧♦❝✉s ✭❤✐❣❤❧✐❣❤t❡❞ ✐♥ ❜❧✉❡✮ ✐s s❤♦✇♥ r❡❧❛t✐✈❡ t♦ t❤❡ ❡♥t✐r❡ ♠♦✉s❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ✭❇✮ ❚❤❡ ❳✐st ❣❡♥❡ ✭s❤♦✇♥ ✐♥ ❜❧✉❡✮ ✐s ❧♦❝❛t❡❞ ✇✐t❤✐♥ t❤❡ ❳✐❝ r❡❣✐♦♥ ❛♥❞ ✐s s✉rr♦✉♥❞❡❞ ❜② s❡✈❡r❛❧ ♣r♦t❡✐♥✲ ❛♥❞ ♥♦♥✲♣r♦t❡✐♥✲❝♦❞✐♥❣ ❣❡♥❡s ✭s❤♦✇♥ ✐♥ ❣r❡②✮✳ ❙♦♠❡ ♦❢ t❤❡s❡ ♥❡✐❣❤❜♦r✐♥❣ ❣❡♥❡s ❛r❡ ✐♠♣❧✐❝❛t❡❞ ✐♥ r❡❣✉❧❛t✐♥❣ ❳✐st ❡①♣r❡ss✐♦♥ ✭s❡❡ s❡❝t✐♦♥ ✶✳✻✮✳ ✭❈✮ ❚❤❡ ♣r❡❞♦♠✐♥❛♥t ❳✐st tr❛♥s❝r✐♣t ✐s♦❢♦r♠ ✐s ✶✺ ❦✐❧♦❜❛s❡s ❧♦♥❣ ❛♥❞ ❝♦♠♣r✐s❡s ✼ ❡①♦♥s✳ ❙✐♠✐❧❛r❧② t♦ ♦t❤❡r ❘◆❆ ♣♦❧②♠❡r❛s❡ ■■✲tr❛♥s❝r✐❜❡❞ ❣❡♥❡s✱ ❳✐st ❘◆❆ ✐s ✺✬✲❝❛♣♣❡❞ ❛♥❞ ✸✬✲♣♦❧②❛❞❡♥②❧❛t❡❞✳ ■♥st❡❛❞✱ ❳✐st ❘◆❆ ✐s r❡t❛✐♥❡❞ ✐♥ t❤❡ ♥✉❝❧❡✉s✱ ✇❤❡r❡ ✐t st❛❜❧② ❛❝❝✉♠✉❧❛t❡s ♦♥ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❢r♦♠ ✇❤✐❝❤ ✐t ✐s tr❛♥s❝r✐❜❡❞ ❬❈❧❡♠s♦♥ ❡t ❛❧✳✱ ✶✾✾✻❪✱ ❛ ♣❤❡♥♦♠❡♥♦♥ ❝♦♠♠♦♥❧② ❞❡s❝r✐❜❡❞ ❛s ✏❝♦❛t✐♥❣✑ ✭❋✐❣✉r❡ ✶✳✺✮✳ ❆❧t❤♦✉❣❤ s❡✈❡r❛❧ ❢❛❝t♦rs ✕ ♥♦t❛❜❧② t❤❡ ♥✉❝❧❡❛r ♠❛✲ tr✐① ♣r♦t❡✐♥s ❙❆❋✲❆✴❤♥❘◆P❯ ❬❍❛s❡❣❛✇❛ ❡t ❛❧✳✱ ✷✵✶✵❪ ❛♥❞ ❈■❩✶ ❬❘✐❞✐♥❣s✲❋✐❣✉❡r♦❛ ❡t ❛❧✳✱ ✷✵✶✼❀ ❙✉♥✇♦♦ ❡t ❛❧✳✱ ✷✵✶✼❪ ✕ ❤❛✈❡ ❜❡❡♥ s❤♦✇♥ t♦ ♣❧❛② ❛♥ ✐♠♣♦rt❛♥t r♦❧❡ ✐♥ ❳✐st ❧♦❝❛❧✐③❛✲ t✐♦♥✱ ❤♦✇ s✉❝❤ ❝♦❛t✐♥❣ t❛❦❡s ♣❧❛❝❡ ❤✐t❤❡rt♦ r❡♠❛✐♥s ❡♥✐❣♠❛t✐❝ ❬s❡❡ ❇r♦❝❦❞♦r✛✱ ✷✵✶✾✱ ❢♦r ❛ r❡✈✐❡✇ ♦♥ t❤❡ t♦♣✐❝❪✳ ❘❡♠❛r❦❛❜❧②✱ ❳✐st ❘◆❆ ✐♥✐t✐❛❧❧② ❞♦❡s ♥♦t s♣r❡❛❞ ✉♥✐❢♦r♠❧② ❛♥❞ ❧✐♥❡❛r❧② ❛❧♦♥❣ t❤❡ ❳ ❝❤r♦♠♦s♦♠❡✳ ■♥st❡❛❞✱ ❳✐st ✜rst ❛❝❝✉♠✉❧❛t❡s ❛t ❞✐s❝r❡t❡ ✏❡♥tr② s✐t❡s✑ ✕ ✇❤✐❝❤ s❤♦✇ s♣❛t✐❛❧ ♣r♦①✐♠✐t② t♦ t❤❡ s✐t❡ ♦❢ ❳✐st tr❛♥s❝r✐♣t✐♦♥ ✕ ❛♥❞ ♦♥❧② t❤❡♥ s♣r❡❛❞s t♦ ♠♦r❡ s♣❛t✐❛❧❧② ❞✐st❛♥t s✐t❡s ❬❊♥❣r❡✐t③ ❡t ❛❧✳✱ ✷✵✶✸❪✳ ✶✸

■♥tr♦❞✉❝t✐♦♥ ❳ ❈❤r♦♠♦s♦♠❡ ■♥❛❝t✐✈❛t✐♦♥

A

Huwe1 Xist

before XCI B initiation/establishment of XCI C after XCI

❋✐❣✉r❡ ✶✳✺ ✕ ❳✐st ❝♦❛ts t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❛♥❞ ♠❡❞✐❛t❡s ❳✲❧✐♥❦❡❞ ❣❡♥❡ s✐❧❡♥❝✐♥❣✳ ■♠❛❣❡s ❢r♦♠ ❘◆❆ ❋■❙❍ ♣❡r❢♦r♠❡❞ ♦♥ ♠❊❙❈s ❛t ❞✐✛❡r❡♥t st❛❣❡s ♦❢ t❤❡ ❳❈■ ♣r♦❝❡ss s❤♦✇ t❤❛t ✭❆✮ ❜♦t❤ ❳ ❝❤r♦♠♦s♦♠❡s ❛r❡ ✐♥✐t✐❛❧❧② ❛❝t✐✈❡ ✐♥ ♠❊❙❈s ♣r✐♦r t♦ ❳❈■✱ ✇✐t❤ t✇♦ ♥❛s❝❡♥t tr❛♥s❝r✐♣t✐♦♥ ❢♦❝✐ ❜❡✐♥❣ ❞❡t❡❝t❡❞ ❢♦r t❤❡ ❳✲❧✐♥❦❡❞ ❣❡♥❡ ❍✉✇❡✶ ✭❣r❡❡♥ s✐❣♥❛❧✮✳ ✭❇✮ ❳❈■ ✐♥✐t✐❛t❡s ✇✐t❤ t❤❡ ♠♦♥♦❛❧❧❡❧✐❝ ✉♣r❡❣✉❧❛t✐♦♥ ♦❢ ❳✐st ✭r❡❞ s✐❣♥❛❧✮✱ ✇❤♦s❡ ❘◆❆ ❝♦❛ts t❤❡ ❳ ❝❤r♦♠♦s♦♠❡ ❢r♦♠ ✇❤✐❝❤ ✐t ✐s tr❛♥s❝r✐❜❡❞✱ ❢♦r♠✐♥❣ ❛ ❞❡t❡❝t❛❜❧❡ ❞♦♠❛✐♥ ❝❛❧❧❡❞ ❛ ❳✐st✲❝❧♦✉❞✳ ✭❈✮ ❊✈❡♥t✉❛❧❧②✱ ❳✐st✲ ❝♦❛t✐♥❣ ❧❡❛❞s t♦ ❝❤r♦♠♦s♦♠❡✲✇✐❞❡ tr❛♥s❝r✐♣t✐♦♥❛❧ s✐❧❡♥❝✐♥❣ ♦❢ ❳✲❧✐♥❦❡❞ ❣❡♥❡s ✐♥ ❝✐s✱ ❛s ✐❧❧✉str❛t❡❞ ✇✐t❤ t❤❡ ❞❡t❡❝t✐♦♥ ♦❢ ❛ s✐♥❣❧❡ ❍✉✇❡✶ ♥❛s❝❡♥t tr❛♥s❝r✐♣t✐♦♥ ❢♦❝✉s✱ ❝♦♠✐♥❣ ❢r♦♠ t❤❡ ❛❝t✐✈❡ ✭✐✳❡✳ ♥♦♥ ❳✐st✲❝♦❛t❡❞✮ ❳ ❝❤r♦♠♦s♦♠❡✳ ❉❡❧❡t✐♦♥ ♦❢ t❤❡ ❳✐st ❣❡♥❡ ✐♥ ♠♦✉s❡ ❡♠❜r②♦♥✐❝ st❡♠ ❝❡❧❧s ❬P❡♥♥② ❡t ❛❧✳✱ ✶✾✾✻❪ ❛♥❞ ❡♠✲ ❜r②♦s ❬▼❛r❛❤r❡♥s ❡t ❛❧✳✱ ✶✾✾✼❪ r❡s✉❧ts ✐♥ ❢❛✐❧✉r❡ t♦ ✉♥❞❡r❣♦ ❳❈■✱ ✇❤✐❧❡ ❡❝t♦♣✐❝ ❡①♣r❡ss✐♦♥ ♦❢ ❳✐st✲❡♥❝♦❞✐♥❣ tr❛♥s❣❡♥❡s ❢r♦♠ ❛✉t♦s♦♠❡s ❧❡❛❞s t♦ ❝✐s✲❛✉t♦s♦♠❛❧ ❣❡♥❡ ✐♥❛❝t✐✈❛t✐♦♥ ❬❲✉t③ ❛♥❞ ❏❛❡♥✐s❝❤✱ ✷✵✵✵❪✳ ❆❧t♦❣❡t❤❡r✱ t❤❡s❡ st✉❞✐❡s ❞❡♠♦♥str❛t❡ t❤❛t ❳✐st ✐s ❛ ❧♦♥❣ ♥♦♥✲❝♦♥❞✐♥❣ ❘◆❆ ✭❧♥❝❘◆❆✮ ✇❤♦s❡ ❡①♣r❡ss✐♦♥ ✐s t❤❡ ❡❛r❧✐❡st ❡✈❡♥t r❡q✉✐r❡❞ t♦ ♠❡❞✐✲ ❛t❡ ❳ ❝❤r♦♠♦s♦♠❡ ✐♥❛❝t✐✈❛t✐♦♥ ✐♥ ❝✐s ✭❋✐❣✉r❡ ✶✳✺✮✳ ❍♦✇ ❳✐st ❡①♣r❡ss✐♦♥ tr✐❣❣❡rs ❝❤r♦♠♦s♦♠❡✲✇✐❞❡ ❣❡♥❡ s✐❧❡♥❝✐♥❣ ❤❛s ❜❡❡♥ t❤❡ ❝❡♥tr❛❧ q✉❡st✐♦♥ ♦❢ ♠② ❞♦❝t♦r❛❧ ✇♦r❦✱ ❛♥❞ t❤❡ ❡①✐st✐♥❣ st❛t❡ ♦❢ ❦♥♦✇❧❡❞❣❡ ❛❜♦✉t ❳✐st✬s s✐❧❡♥❝✐♥❣ ❢✉♥❝t✐♦♥ ✕ ❛t t❤❡ t✐♠❡ ■ st❛rt❡❞ ♠② P❤❉ ✕ ✇✐❧❧ ❜❡ ❛❞❞r❡ss❡❞ ✐♥ s❡❝t✐♦♥ ✶✳✶✷ ❛♥❞ s❡❝t✐♦♥ ✶✳✶✸ ♦❢ t❤✐s ❝❤❛♣t❡r✳

✶✳✻ ❳✐st ❡①♣r❡ss✐♦♥ ✐s ❧✐♥❦❡❞ t♦ ❝❡❧❧✉❧❛r ❞✐✛❡r❡♥t✐❛t✐♦♥

Pr✐♦r t♦ ❝❡❧❧✉❧❛r ❞✐✛❡r❡♥t✐❛t✐♦♥ ✭✐✳❡✳ ❡①✐t ❢r♦♠ ♣❧✉r✐♣♦t❡♥❝②✮✱ ❳✐st ✐s ❜❛r❡❧② ❡①♣r❡ss❡❞ ✐♥ ♠❊❙❈s ✭❋✐❣✉r❡ ✶✳✻❆✮✳ ❯♣♦♥ ❞✐✛❡r❡♥t✐❛t✐♦♥ ❤♦✇❡✈❡r✱ ❳✐st ❜❡❝♦♠❡s ✉♣r❡❣✉❧❛t❡❞ ✭❋✐❣✉r❡ ✶✳✻❆✮✱ str✐❝t❧② ♦♥ ♦♥❡ ♦❢ t❤❡ t✇♦ ❳ ❝❤r♦♠♦s♦♠❡s ✭s❡❝t✐♦♥ ✶✳✹✮✳ ❍♦✇ ❳✐st ❡①♣r❡ss✐♦♥ ✐s s♦ t✐❣❤t❧② r❡❣✉❧❛t❡❞ ❞✉r✐♥❣ ❞✐✛❡r❡♥t✐❛t✐♦♥✱ ❛♥❞ ❤♦✇ ✐t ✐s r❡str✐❝t❡❞ t♦ ♦♥❧② ♦♥❡ ♦❢ t❤❡ t✇♦ ❳ ❝❤r♦♠♦s♦♠❡s ❤❛s ❜❡❡♥ ❡①t❡♥s✐✈❡❧② ✐♥✈❡st✐❣❛t❡❞ ❬s❡❡ ❆✉❣✉✐ ❡t ❛❧✳✱ ✷✵✶✶❀ ✈❛♥ ❇❡♠♠❡❧ ❡t ❛❧✳✱ ✷✵✶✻❀ ●❛❧✉♣❛ ❛♥❞ ❍❡❛r❞✱ ✷✵✶✺❀ P♦♥t✐❡r ❛♥❞ ●r✐❜♥❛✉✱ ✷✵✶✶✱ ❢♦r r❡✲ ✈✐❡✇s ❜② ❡①♣❡rts ❢r♦♠ t❤❡ ✜❡❧❞❪✳ ■t ✐s ♥♦✇ ❛❝❝❡♣t❡❞ t❤❛t ❳✐st ✐s ♠❛✐♥❧② r❡❣✉❧❛t❡❞ ❛t t❤❡ tr❛♥s❝r✐♣t✐♦♥❛❧ ❧❡✈❡❧✱ t❤r♦✉❣❤ ❛❝t✐♦♥ ♦❢ ❜♦t❤ ❝✐s ❛♥❞ tr❛♥s✲r❡❣✉❧❛t♦rs✱ ✇❤✐❝❤ ✇✐❧❧ ♦♥❧② ❜❡ ❜r✐❡✢② ❝♦✈❡r❡❞ ✐♥ t❤❡ ❢♦❧❧♦✇✐♥❣ ♣❛r❛❣r❛♣❤s ✭❋✐❣✉r❡ ✶✳✻✮✳ ✶✹