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\DOI{10.5802/crchim.272}
\datereceived{2023-03-03}
\daterevised{2023-11-09}
\dateaccepted{2023-11-10}
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\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Breaking Barriers in Chemical Biology -- Toulouse 2022}
%\def\cdr@specialtitle@french{Briser les barri\`eres entre la chimique et la biologie -- Toulouse 2022}
%\makeatother

\CDRsetmeta{articletype}{research-article}

\title{Structural characterization of stem cell factors Oct4, Sox2,
Nanog and Esrrb disordered domains, and a method to detect
phospho-dependent binding partners}

\alttitle{Caract\'{e}risation structurale des domaines
d\'{e}sordonn\'{e}s des facteurs de pluripotence Oct4, Sox2, Nanog et
Esrrb, et une m\'{e}thode pour d\'{e}celer leurs partenaires
phospho-d\'{e}pendants potentiels}

\author{\firstname{Chafiaa} \lastname{Bouguechtouli}}
\address{Universit\'{e} Paris-Saclay, CEA, CNRS, Institute for
Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France}
\curraddr[C. Bouguechtouli]{Structural Motility, Institut Curie, Paris
Universit\'{e} Sciences et Lettres, Sorbonne Universit\'{e}, CNRS
UMR144, 75005 Paris, France}
%\email{chafiaa.bouguechtouli@curie.fr}

\author{\firstname{Rania} \lastname{Ghouil}\CDRorcid{0000-0003-1628-0249}}
\addressSameAs{1}{Universit\'{e} Paris-Saclay, CEA, CNRS, Institute for
Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France}
%\email{ran.ghouil@gmail.com}

\author{\firstname{Ania} \lastname{Alik}}
\addressSameAs{1}{Universit\'{e} Paris-Saclay, CEA, CNRS, Institute for
Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France}
\curraddr[A. Alik]{Universit\'{e} de Paris, Institut Cochin, CNRS
UMR8104, INSERM U1016, Paris, France}
%\email{Alik.Ania@hotmail.fr}

\author{\firstname{Florent} \lastname{Dingli}\CDRorcid{0000-0002-7715-2446}}
\address{Institut Curie, PSL Research University, Centre de Recherche,
CurieCoreTech Spectrom\'{e}trie de Masse Prot\'{e}omique, Paris cedex
05, France}
%\email{Florent.Dingli@curie.fr}

\author{\firstname{Damarys}\nobreakauthor \lastname{Loew}\CDRorcid{0000-0002-9111-8842}}
\addressSameAs{2}{Institut Curie, PSL Research University, Centre de
Recherche, CurieCoreTech Spectrom\'{e}trie de Masse Prot\'{e}omique,
Paris cedex 05, France}
%\email{Damarys.Loew@curie.fr}

\author{\firstname{Francois-Xavier} \lastname{Theillet}\CDRorcid{0000-0002-3264-210X}\IsCorresp}
\addressSameAs{1}{Universit\'{e} Paris-Saclay, CEA, CNRS, Institute for
Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France}
\email[F.-X. Theillet]{francois-xavier.theillet@cnrs.fr}

\shortrunauthors

\keywords{\kwd{Pluripotency transcription factors}\kwd{Intrinsically
disordered proteins}\kwd{Post-translational
modifications}\kwd{Kinases}\kwd{NMR}\kwd{Proteomics}\kwd{Quantitative
mass spectrometry}}

\altkeywords{\kwd{Facteurs de transcription de
pluripotence}\kwd{Prot\'{e}ines intrins\`{e}quement
d\'{e}sordonn\'{e}es}\kwd{Modifications
post-traductionnelles}\kwd{Kinases}\kwd{RMN}
\kwd{Prot\'{e}omique}\kwd{Spectrom\'{e}trie de masse quantitative}}

\thanks{CNRS and CEA-Saclay, French Infrastructure for Integrated
Structural Biology (FRISBI, \url{https://frisbi.eu/}, grant number
ANR-10-INSB-05-01) and French National Research Agency (ANR; research
grants ANR-14-ACHN-0015 and ANR-20-CE92-0013), IR INFRANALYTICS FR2054,
``R\'{e}gion Ile-de-France'' and ``Fondation pour la Recherche
M\'{e}dicale''}

\begin{abstract} 
The combined expression of a handful of pluripotency transcription
factors (PluriTFs) in somatic cells can generate induced pluripotent
stem cells (iPSCs). Here, we report the structural characterization of
disordered regions contained in four important PluriTFs, namely Oct4,
Sox2, Nanog and Esrrb. Moreover, many post-translational modifications
(PTMs) have been detected on PluriTFs, whose roles are not yet
characterized. To help in their study, we also present a method (i)~to
produce well-characterized phosphorylation states of PluriTFs, using
NMR analysis, and (ii)~to use them for pull-downs in stem cell extracts
analyzed by quantitative proteomics to detect potential Sox2 binders.
\looseness=-1
\end{abstract} 

\begin{altabstract} 
L'expression de facteurs de transcription de la pluripotence (PluriTF)
dans des cellules somatiques peut g\'{e}n\'{e}rer des cellules souches
pluripotentes induites. Nous pr\'{e}sentons ici la caract\'{e}risation
structurale des r\'{e}gions d\'{e}sordonn\'{e}es de quatre PluriTFs
importants : Oct4, Sox2, Nanog et Esrrb. De nombreuses modifications
post-traductionnelles ont \'{e}t\'{e} d\'{e}tect\'{e}es sur les
PluriTFs, dont les r\^{o}les sont inconnues. Pour aider \`{a} leur
\'{e}tude, nous proposons une m\'{e}thode (i)~pour produire des
\'{e}tats de phosphorylation bien d\'{e}finis des PluriTFs analys\'{e}s
par RMN, et (ii)~les utiliser pour des pr\'{e}cipitations par
affinit\'{e} d'extraits de cellules souches, analys\'{e}es par
prot\'{e}omique quantitative pour identifier des partenaires
potentiels.
\end{altabstract} 

\maketitle
{\vspace*{.5pc}}

\twocolumngrid

\end{noXML}

\section{Introduction} \label{sec1}

The possibility of reprogramming somatic cells to an induced
pluripotency state was revealed in the 2000s, giving great hopes in the
fields of Biology and Medicine~\cite{1,2,3}. Induced pluripotent stem
cells \mbox{(iPSCs)} and embryonic stem cells (ESCs) are characterized
by the active state of a pluripotency network, whose core comprises the
pluripotency transcription factors (PluriTFs) Oct4, Sox2, Nanog and
Esrrb (OSNE). These bind to enhancer sequences and thus activate or
repress, or even ``bookmark'' during mitosis, a wealth of genes related
to pluripotency or cell differentiation~\cite{4,5,6,7,8,9}.
Consistently, their misregulation correlates with cancer malignancy and
stemness~\cite{10,11,12,13,14}.

Comprehensive structural descriptions of OSNE are still missing to the
best of our knowledge. The folded DNA-binding domains (DBDs) of OSNE
have been structurally characterized in complex with their DNA target
sequences~\cite{15,16,17,18,19} together with the ligand-binding domain
(LBD) of Esrrb~\cite{20}. Recent studies have depicted even splendid
structures of Oct4's and Sox2's DBDs bound to nucleosomes, hence
deciphering their ``pioneer factor''
abilities~\cite{21,22,23,24,25,26,27,28,29}. Another structure of Sox2
bound to the importin Imp$\upalpha$3 has also been published, showing how
its two Nuclear Localization Sequences (NLSs) flanking the DBD are
involved in Sox2 nuclear import~\cite{30}. The other segments of OSNE
have been predicted to be intrinsically disordered regions of proteins
(IDRs)~\cite{31}, i.e., they should have no stable tertiary fold when
isolated~\cite{32,33,34,35,36}.

These IDRs appear to have important roles in binding partners involved
in epigenetic reprogramming, chromatin reorganization, and in
recruiting transcription or repression
machineries~\cite{5,37,38,39,40,41}. These functions are poorly
understood, and, to the best of our knowledge, no experimental
characterization of the structural behavior of these regions in N- and
C-terminal of DBDs has been released yet. Recent studies have shown
that C-terminal regions of Oct4 and Sox2 are important for their
reprogramming capacities~\cite{42,43}, notably by contributing to the
engagement in molecular phase-separated condensates with the Mediator
complex~\cite{44}. More generally, the activating or repressive
activities of IDRs of transcription factors (TFs) have been scarcely
studied at the structural level: these segments are thought to contain
hydrophobic patches flanked by acidic amino acids, which favors
DNA-binding specificity, phase separation, and low-specificity
interactions, notably with the Mediator subunit
Med15~\cite{44,45,46,47,48,49,50,51,52,53}; more specific interactions
have been described in some cases~\cite{45,54,55}.

Post-translational modifications (PTMs) add a layer of complexity by
often regulating IDRs' interactions~\cite{32,33,56,57} and notably TFs'
activity \cite{58,59,60,61}. PTMs are classical carriers of cell
signaling by{\break} regulating the stability and the interactions of
proteins. An increasing number of PTMs have been described on OSNE's
IDRs in the recent years
\cite{5,37,38,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77}, notably
phosphorylation by cyclin-dependent kinases
(CDKs)~\cite{66,78,79,80,81,82,83,84,85,86,87} or by
mitogen-{\ubreak}activated protein kinases (MAPKs)~\cite{83,88,89}, or
their complementary Ser/Thr O-GlcNAcylation by
OGT~\cite{65,90,91,92,93,94,95,96}.

In order to prompt future studies on this topic, we describe here a
feasibility study (i)~for producing well-characterized samples made of
post-translationally modified IDRs of PluriTFs and (ii)~to use these as
baits in pull-down assays for detecting PTMs' related binding partners.
Hence, we characterized some of the phosphorylation reactions of Esrrb
and Sox2 by p38$\upalpha$/$\upbeta$, Erk2 and Cdk1/2. Then, we showed that
biotinylated chimera of Sox2 and Esrrb coupled to an AviTag peptide
could be attached to streptavidin-coated beads. Finally, we loaded
truncated segments of the C-terminal IDR of Sox2 (phosphorylated or
not) on these beads, and exposed them to extracts of mouse ESCs (mESCs)
in pull-down assays, which we analyzed using quantitative mass
spectrometry-based proteomics. Among the quantified (phospho-)Sox2
binders, we verified the phospho-dependent interaction between the
proline cis-trans isomerase Pin1 and Sox2 using NMR spectroscopy.

\section{Material and methods} \label{sec2}
\subsection{Production of recombinant fragments of{\hfill\break} Oct4,
Sox2, Nanog and Esrrb} \label{sec2.1}

We used human protein sequences, unless specified. Codon-optimized (for
expression in \textit{Escherichia coli}) genes coding for human
Oct4(aa1--145) and Oct4(aa286--380) were synthesized in the context of
larger genes coding for Tev--Oct4(aa1--145)--Tev--GB1 and
Tev--Oct4(aa286--380)--Tev--GB1 by Genscript and{\break} cloned into
pET-41a(+) vector between SacII and HindIII restriction sites, hence
permitting the \mbox{expression} of
GST--His6--Tev1--Oct4(aa1--145)--Tev2--GB1 and
GST--His6--Tev1--Oct4(aa286--380)--Tev2--GB1; Tev1 and Tev2 are
the heptapeptide ENLYFQG cleavage site of the TEV protease, Tev2 is
separated by GAGGAGG from GB1 (T2Q variant of the immunoglobulin
binding domain B1 of the protein~G from group~G
Streptococcus~\cite{97,98}). The C-terminal GB1 tag was added to avoid
any C-terminal proteolysis of the IDR of interest during the expression
and the first purification steps; we did not test constructs without
this supplementary folded domain, whose necessity for the stability of
the IDR is thus not proven.

The same rationale (cDNA synthesis, cloning, vectors, chimera
constructs) was used for producing
{\fontsize{9.8}{11.8}\selectfont Nanog(aa154--305)},
{\fontsize{9.8}{11.8}\selectfont Nanog(aa154--215)},
{\fontsize{9.8}{11.8}\selectfont Nanog(aa154--{\ubreak}272)},
{\fontsize{9.7}{11.7}\selectfont Nanog(aa154--305\_C185A-C227A-C243A-C251A)},{\break}
{\fontsize{9.7}{11.7}\selectfont Nanog(aa154--272\_C185A-C227A-C243A-C251A)},~and
a very similar rationale (chimera constructs missing the C-terminal
Tev2--GB1) for Sox2(aa1--42), Sox2{\ubreak}(aa115--317\_C265A),
Sox2(aa115--187), Sox2(aa115--236), Sox2(aa115--282\_C265A),
Esrrb(aa1--102\_C12A-{\ubreak}C72A-C91A), Esrrb(aa1--102\_C12A-C91A),
Nanog{\ubreak}(aa1--85) (this latter was cloned in the MfeI/HindIII
restriction sites from pET-41a(+)).

The recombinant production and the purification of the protein
constructs followed the procedures described previously~\cite{99},
using the soluble fraction of bacterial lysates, except for the
constructs containing the Sox2 C-terminal fragments. These latter
constructs were recovered from the insoluble fractions of the lysates
and resolubilized in 8~M urea; these were submitted to a His-tag
purification in urea, and the last size-exclusion chromatography (SEC)
had to be carried out in 2~M urea, which avoided clogging of the column
and permitted obtaining regular elution peak widths (these were
otherwise extremely broad, up to 100~mL for the longest
Sox2(aa115--317\_C265A) construct). The samples were concentrated and
stored at ${-}$20~{\textdegree}C, and thawed just before the NMR
experiments. The Sox2 samples containing 2~M urea were submitted to
2--3 cycles of concentration/dilution in Hepes at 20~mM, NaCl at 75~mM
to generate samples in urea at 0.25 or 0.125~M.

All purification steps were carefully carried out at
\mbox{4~{\textdegree}C}; protein eluates from every purification step
were immediately supplemented with protease \mbox{inhibitors}
(EDTA-free cOmplete, Roche) (together with DTT at 10~mM for
cysteine-containing protein constructs), before being submitted to a
concentration preparing the next purification step.

Chimera constructs of Sox2's and Esrrb's IDR fragments containing a
15-mer peptide AviTag GLNDIFEAQKIEWHE were produced using procedures
similar to those described earlier for OSNE constructs. The construct
Sox2(aa234--317)--AviTag--His6 was soluble and did not require to be
purified in{\break} urea.

More details about the production of OSNE peptides are given in the
Supplementary Material.
{\vspace*{-.2pc}}

\subsection{Production of the biotin ligase BirA and{\hfill\break}
specific biotinylation of the AviTag--peptide{\hfill\break} chimera}
\label{sec2.2}

The biotin ligase BirA was produced using recombinant production in
\textit{E.~coli} BL21(DE3)Star transformed with a pET21-a(+) plasmid
containing a gene coding for BirA cloned at EcoRI and HindIII
restriction sites. pET21a-BirA was a gift from Alice Ting (Addgene
plasmid \#20857)~\cite{100}. The expression was carried out overnight
at 20~{\textdegree}C in a Luria--Bertani culture medium. The construct
contained a His6 tag in C-terminal and was purified using a two-step
purification procedure including a His-trap followed by a SEC. Details
about the production of BirA are given in the Supplementary Material.

The biotinylation was executed using a rationale inspired by a
published protocol~\cite{101}, at room temperature during 90~min, in
samples containing the AviTag--chimera of interest at 100~$\upmu$M and
BirA at 0.7~$\upmu$M in a buffer containing ATP at 2~mM, biotin at
600~$\upmu$M, MgCl$_2$ at 5~mM, DTT at 1~mM, HEPES at 50~mM, NaCl at
150~mM, protease inhibitors (final concentration $1{\times}$, EDTA-free
cOmplete, Roche), at pH~7.0. To remove some possible proteolyzed
peptides and BirA, the biotinylated constructs were purified using a
SEC in a column (Superdex 16/60 75~pg, Cytiva) preequilibrated with a
buffer containing phosphate at 20~mM, NaCl at 150~mM at pH~7.4 (buffer
called thereafter Phosphate Buffer Saline, PBS). The eluted fractions
of interest were concentrated and stored at ${-}$20~{\textdegree}C.
{\vspace*{-.2pc}}

\subsection{Assignment of NMR signals from OSNE{\hfill\break} fragments
and structural propensities} \label{sec2.3}

The assignment strategy was the same as in previous reports from our
laboratory~\cite{99}. The ${}^{15}$N relaxation data were recorded and
analyzed according to the methods described in previous
reports~\cite{102}. Details are given in the Supplementary Material.

Disorder prediction was calculated using the ODINPred website
(\url{https://st-protein.chem.au.dk/odinpred})~\cite{103}. Experimental
secondary structure{\break} propensities of unmodified OSNE peptides
were obtained using the neighbor-corrected structural propensity
calculator ncSCP~\cite{104,105} (\url{http://www.protein-nmr.org/},
\url{https://st-protein02.chem.au.dk/ncSPC/}) from the experimentally
determined, DSS-{\ubreak}referenced C$\upalpha$ and C$\upbeta$ chemical
shifts as input, with a correction for Gly--Pro motifs (${-}$0.77~ppm
instead of ${-}$2.0~ppm)~\cite{106}. We also used the $\updelta$2D method
to get requested verifications of the experimental secondary structure
propensities~\cite{107}. Some signals were too weak in 3D spectra from
Sox2(aa115--317\_C265A) recorded at 950 MHz, and their chemical shifts
were not defined. In these cases, chemical shifts from 3D spectra of
Sox2(115-236) or His6--AviTag--Sox2(aa234--317\_C265A) were used to
complete the lists of chemical shifts used to calculate the chemical
shift propensities shown in Figure~\ref{fig2}.
{\vspace*{2pt}}

${}^1\text{H}_{\mathrm{N}}/{}^{15}\text{N}/{}^{13}\text{Ca}/
{}^{13}\text{Cb}/{}^{13}\text{CO}$ NMR assignments of OSNE peptides,
together with the corresponding experimental details, have been
deposited in the Biological Magnetic Resonance Data Bank (BMRB) with
accession numbers
\href{https://bmrb.io/data\_library/summary/index.php?bmrbId=51534}
{51534} (Sox2\_aa1--42),
\href{https://bmrb.io/data\_library/summary/index.php?bmrbId=51717}
{51717} (Esrrb\_aa1--102),
\href{https://bmrb.io/data\_library/summary/index.php?bmrbId=51756}
{51756} (Oct4\_aa1--145),
\href{https://bmrb.io/data\_library/summary/index.php?bmrbId=51758}
{51758} (Oct4\_aa286--360),
\href{https://bmrb.io/data\_library/summary/index.php?bmrbId=51782}
{51782} (His6--AviTag--Sox2\_aa234--317\_C265A), and
\href{https://bmrb.io/data\_library/summary/index.php?bmrbId=51780}
{51780} (Nanog\_aa1--85).

\subsection{NMR monitoring of phosphorylation{\hfill\break} reactions
and production of phosphorylated peptides} \label{sec2.4}

We performed the phosphorylation kinetics presented in
Figure~\ref{fig4}a using commercial recombinant kinases GST--p38$\upbeta$
at 10~$\upmu$g/mL (Sigma-Aldrich, ref.~B4437), GST--Erk2 at
20~$\upmu$g/mL (Sigma-Aldrich, ref.~E1283), GST--Cdk1/CyclinA2 at
20~$\upmu$g/mL (Sigma-Aldrich, stock ref.~C0244), and
GST--Cdk2/CyclinA2 at 20~$\upmu$g/mL (Sigma-Aldrich, ref.~C0495). Then,
we used kinases produced in-house in \textit{E.~coli}, using plasmids
containing optimized genes coding for p38$\upalpha$(aa1--360,
full-length) and Erk2(aa8--360); these were produced, activated, and
purified in house as described previously~\cite{99}; in-house
p38$\upalpha$ was used at 40~$\upmu$g/mL for the experiments shown in
Figure~\ref{fig4}. Indeed, the limited activities and high costs of
commercial kinases motivated us to develop in-house capacities in
kinase production. p38$\upalpha$ was the most accessible to produce among
the MAPKs and CDKs; we produced it and activated it using recombinant
MKK6.

Phosphorylation reactions were carried out using ${}^{15}$N-labeled
IDRs at 50~$\upmu$M, in Hepes 20~mM, NaCl 50~mM, DTT or TCEP at 4~mM,
ATP 1.5~mM, MgCl$_2$ at 5~mM, protease inhibitors (Roche), 7.5\%
D$_2$O, pH~6.8 at 25~{\textdegree}C in 100~$\upmu$L using 3~mm diameter
Shigemi tubes. We monitored the phosphorylation kinetics by recording
time series of ${}^{1}$H--${}^{15}$N SOFAST-HMQC spectra at 600 or
700~MHz, and by quantifying the NMR signal intensities of the disappearing
unphospho- and appearing phospho-residues. We applied the methods that
we described in earlier publications~\cite{108,109,110,111}. More
details are given in the Supplementary Material.

\subsection{Pull-down assays} \label{sec2.5}

The mESCs extracts were obtained from mESCs cultured in the conditions
previously described~\cite{112}. Homogeneous extracts were obtained
using DNA shearing by sonication in the presence of benzonase, as
described by Gingras and colleagues~\cite{113}.

The pull-down assays were executed using 25~$\upmu$L of
streptavidin-coated magnetic beads (Magbeads streptavidine, Genscript)
loaded with 1~nmol of the biotinylated bait-peptides of interest. These
were incubated for one hour at room temperature with mESCs extracts,
washed in PBS and eluted using a $2{\times}$ Laemmli buffer. Details
are given in the Supplementary Material.

\subsection{Mass spectrometry-based proteomics{\hfill\break} analysis
of pull-down assays} \label{sec2.6}

The pull-down samples were treated on-beads by trypsin/LysC (Promega).
The resulting peptides were loaded and separated on a C18 column for
online liquid chromatography performed with an RSLCnano system
(Ultimate 3000, Thermo Scientific) coupled to an Orbitrap Fusion
Tribrid mass spectrometer (Thermo Scientific). Maximum allowed mass
deviation was set to 10 ppm for monoisotopic precursor ions and 0.6~Da
for MS/MS peaks. The resulting files were further processed using
myProMS v3.9.3 (\url{https://github.com/bioinfo-pf-curie/myproms};
Poullet et~al.~\cite{7a}). False-discovery rate (FDR) was
calculated using Percolator~\cite{8a} and was set to 1\% at the peptide
level for the whole study. Label-free quantification was performed
using extracted-ion \mbox{chromatograms} (XICs) of peptides, computed
with MassChroQ~\cite{9a} v.2.2.1. The complete details are given in the
Supplementary Material.

\subsection{Recombinant production of Pin1 and NMR analysis of its
interaction with Sox2 or{\hfill\break} phospho-Sox2} \label{sec2.7}

The plasmid containing the gene coding for the Pin1--WW domain was a
kind gift from Isabelle Landrieu. The production was executed according
to the previously published protocol~\cite{114}. NMR analysis of the
binding with phospho-Sox2(aa115--240) was performed at 283~K and pH~7.0
with the GST--Pin1--WW construct and ${}^{15}$N-labeled
Sox2(aa115--240) mixed in stoichiometric proportions, either at 50 or
10~$\upmu$M for non-phospho- and phospho-Sox2, respectively. The
details on the NMR acquisition, processing, and analysis are given in
the Supplementary Information.

\section{Results} \label{sec3}
\subsection{Structural characterization of the N- and{\hfill\break}
C-terminal regions of Oct4} \label{sec3.1}

We produced and purified protein constructs containing the fragments of
human Oct4(aa1--145) and Oct4(aa286--360), which were both predicted to
be mostly disordered (Supplementary material~3.2.3). The 2D
${}^{1}$H--${}^{15}$N HSQC NMR spectra showed cross-peaks in the region
where random coil peptides resonances are usually found
(Figure~\ref{fig1}). We assigned the backbone NMR signals of
${}^{1}\text{H}_{\mathrm{N}}$, ${}^{15}$N, ${}^{13}\text{C}\upalpha$,
${}^{13}\text{C}\upbeta$, ${}^{13}$CO for both segments, which permitted
to calculate experimentally derived secondary structure propensities
(Figures~\ref{fig1}c, f, Supplementary Figure~S4). We did not
find any sign of a stable secondary structure, the highest
$\upalpha$-helical propensities reaching about 25\% in short stretches of
about 5 consecutive amino acids. Hence, we verified experimentally that
these N- and C-terminal fragments of human Oct4 are IDRs. We noticed
that one motif RTWLSF (aa33--38) generates cross-peaks out of the
random coil area in the 2D ${}^{1}$H--${}^{15}$N HSQC, and its chemical
shifts reveal the strongest $\upalpha$-helical propensity (about 25\%
according to two distinct software products, see Figures~\ref{fig1}c,
f, Supplementary Figure~S4). This might typically correspond
to an interaction site: IDRs' binding motifs often adopt secondary
structure in complexes, whose formation is energetically favored by
local conformational preferences for the bound structure in the free
state~\cite{57,106,115}.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}(a,d)~2D ${}^{1}$H--${}^{15}$N HSQC spectra of the
N- and C-terminal IDRs of human Oct4, the labels indicating the
assignments; the grey areas show the spectral regions where random coil
amino acids resonate usually; (b,e)~primary structures of Oct4; dark
and light colors indicate the folded and disordered domains,
respectively; blue and red sticks indicate the positions of Glycines
and Prolines, respectively; (c,f)~secondary structure propensities
calculated from the experimental chemical shifts of the peptide
backbone C$\upalpha$ and C$\upbeta$, using the ncSPC
algorithm~\cite{104,105}.}
{\vspace*{-.65pc}}
\end{figure*}

We can highlight the fact that Oct4's IDRs contain a high density of
prolines, which are not directly detectable in the present
${}^{1}\text{H}_{\mathrm{N}}$-detected experiments, even though most of
the ${}^{13}\text{C}\upalpha$, ${}^{13}\text{C}\upbeta$, and ${}^{13}$CO
resonances were characterized via HNCAB and HNCO experiments. We have
shown previously that the ${}^{13}$C-detected experiments
${}^{13}\text{C}\upalpha\,{}^{13}\text{CO}$ permitted observing all these
Pro residues in Oct4(aa1--145)~\cite{99}, whose chemical shifts were
those of random coil \mbox{peptides}.

\subsection{Structural characterization of the N- and{\hfill\break}
C-terminal regions of Sox2} \label{sec3.2}

We produced and purified peptide fragments of human Sox2, namely
Sox2(aa1--42), Sox2(aa115--187), Sox2(aa115--236), Sox2(aa115--282),
Sox2(aa234--317\_{\ubreak}C265A), and Sox2(aa115--317\_C265A). We also
produced and purified chimera peptides His6--AviTag--Sox2(aa115--240)
and His6--AviTag--Sox2(aa234--317\_{\ubreak}C265A). These were all
predicted to be disordered (Supplementary material~3.3.3).

We had solubility issues with all of them but Sox2(aa1--42),
Sox2(aa234--317\_C265A), and His6--AviTag--Sox2(aa234--317\_C265A). We
had to recover these troublesome peptides from the insoluble fraction
of the bacterial extract after overexpression at 37~{\textdegree}C. We
even had to carry out our final SEC purification step in a buffer
containing urea at 2~M (at \mbox{4~{\textdegree}C}) for
Sox2(aa115--236), Sox2(aa115--282), His6--AviTag--Sox2(aa115--240),
and Sox2(aa115--317\_{\ubreak}C265A). The assignments of these latter
constructs were achieved in 0.25--0.5~M urea, after executing 2 to 3
concentration--dilution steps.\ Aggregates were forming during the
acquisition, which made the assignment rather painful. This behavior
correlated with liquid--liquid phase separation (LLPS) propensities
(Supplementary Figure~S5), which we observed a few months before such a
behavior was reported by Young and collaborators~\cite{44}. The
assignment of Sox2(aa115--317\_C265A) was possible only at 950~MHz with
the help of the previously assigned smaller fragments Sox2(aa115--236)
and His6--AviTag--Sox2(aa234--317\_C265A). Some stretches of amino
acids were particularly difficult to observe in 3D spectra, e.g., the
region aa160--185, because of an apparent fast T2 relaxation. We may
investigate these phenomena in later reports.

We observed cross-peaks in the 2D ${}^{1}$H--${}^{15}$N HSQC NMR spectra
that were all resonating in the spectral region of random coil
peptides' resonances (\mbox{Figure}~\ref{fig2}). The spectra of the
short fragments of the C-terminal region of Sox2 were exactly
overlapping with those of Sox2(aa115--317\_C265A) (Supplementary
Figure~S1). This shows that all these fragments have very similar local
conformational behaviors, a phenomenon regularly observed with IDRs. We
assigned the backbone NMR signals of ${}^{1}\text{H}_{\mathrm{N}}$,
${}^{15}$N, ${}^{13}\text{C}\upalpha$, ${}^{13}\text{C}\upbeta$,
${}^{13}$CO for Sox2(aa1--42), Sox2(aa115--236),
His6--AviTag--Sox2(aa234--317\_C265A) and partially for
Sox2(aa115--317\_C265A). We aggregated the lists of chemical shifts of
the C-terminal fragments and used them to calculate the experimental
secondary structure propensities (Figures~\ref{fig2}c, f and
Supplementary Figure~S4). This confirmed the absence of any stable
secondary structure elements in Sox2 N- and C-terminal region. The
C-terminal region is poorly soluble below 0.25~M urea; this should not
affect a stable fold, so we can affirm that these regions of Sox2 are
experimentally proven IDRs.

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}(a,d)~2D ${}^{1}$H--${}^{15}$N HSQC spectra of the
N- and C-terminal IDRs of human Sox2, the labels indicating the
assignments; the grey areas show the spectral regions where random coil
amino acids resonate usually; (b,e)~primary structures of human Sox2;
dark and light colors indicate the folded and disordered domains,
respectively; (c,f)~secondary structure propensities calculated from
the experimental chemical shifts of the peptide backbone C$\upalpha$ and
C$\upbeta$, using the ncSPC algorithm~\cite{104,105}.}
{\vspace*{-.35pc}}
\end{figure*}

\subsection{Structural characterization of the{\hfill\break} N-terminal
region of Nanog} \label{sec3.3}

We produced and purified the N-terminal peptide fragment of human
Nanog(aa1--85). All our attempts to purify C-terminal regions of Nanog
failed, even after alanine-mutation of cysteines in Nanog(aa154--305),
Nanog(aa154--272), and Nanog(aa154--215). We managed to resolubilize
our construct GST--His6--Tev--Nanog(aa154--305) from the insoluble
fraction of the bacterial extract, to partially purify it and cleave it
using the TEV protease. However, the resulting Nanog(aa154--305)
peptide was barely soluble in a detergent (NP-40 at 2\%~v/v), and not
in high-salt buffers, or not even in the presence of urea at 4~M. The
10 tryptophane residues are probably playing a role in this behavior,
in the context of a primary structure containing not enough hydrophobic
amino acids favoring stable folds.

The cross-peaks of Nanog(aa1--85) in the 2D ${}^{1}$H--${}^{15}$N HSQC
NMR spectrum were all in the spectral region of random coil peptides'
resonances (Figure~\ref{fig3}). The assignment of the backbone NMR
signals of ${}^{1}\text{H}_{\mathrm{N}}$, ${}^{15}$N,
${}^{13}\text{C}\upalpha$, ${}^{13}\text{C}\upbeta$, ${}^{13}$CO permitted
calculating experimental secondary structure propensities, which were
low through the whole peptide (Figure~\ref{fig3}b, Supplementary
Figure~S4). Thus, we confirmed that this Nanog N-terminal is an IDR.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}(a,d)~2D ${}^{1}$H--${}^{15}$N HSQC spectra of the
N-terminal IDRs of human Nanog and Esrrb, the labels indicating the
assignments; the grey areas show the spectral regions where random coil
amino acids resonate usually; (b,e)~primary structures of human Nanog
and Esrrb; dark and light colors indicate the folded and disordered
domains, respectively; orange and black sticks indicate the positions
of cysteines and tryptophanes, respectively; (c,f)~secondary structure
propensities calculated from the experimental chemical shifts of the
peptide backbone C$\upalpha$ and C$\upbeta$, using the ncSPC
algorithm~\cite{104,105}.}
{\vspace*{.45pc}}
\end{figure*}

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}(a)~Overlay of 2D ${}^{1}$H--${}^{15}$N HSQC
spectra of Esrrb(aa1--102\_C12A-C72A-C91A) before (black) and after
(grey and phosphosites colored in red/purple/blue) phosphorylation by
p38$\upbeta$; (b)~residue specific time courses of the phosphorylation of
Esrrb(aa1--102) executed by commercial kinases p38$\upbeta$, Erk2, Cdk2
or Cdk1, as measured in time series of 2D ${}^{1}$H--${}^{15}$N
SOFAST-HMQC spectra recorded during the reaction; (c)~overlay of 2D
${}^{1}$H--${}^{15}$N HSQC spectra of AviTag-Sox2(aa115--240) before
(black) and after (red) phosphorylation by p38$\upalpha$; the inset at the
top-right shows the residue specific phosphorylation build-up curves,
as measured in time series of 2D ${}^{1}$H--${}^{15}$N SOFAST-HMQC
spectra; (d)~same as~(c) with AviTag--Sox2(aa234--317\_C265A).}
\end{figure*}

\subsection{Structural characterization of the{\hfill\break} N-terminal
region of Esrrb} \label{sec3.4}

We produced and purified the N-terminal fragment of human
Esrrb(aa1--102), which was predicted to be disordered (Supplementary
material~3.5.3), in the alanine-mutated versions
Esrrb(aa1--102\_{\ubreak}C12A-C72A-C92A) and Esrrb(aa1--102\_C12A-C92A).
This was a strategic choice to attenuate the formation of disulfide
bonds; the wild-type N-terminal fragments might however be workable
too.\ Mutating cysteines permitted working in more comfortable
conditions and maintaining our construct monomeric for longer periods
of time in the next phosphorylation and biotinylation experiments.
Cysteines are indeed highly solvent-accessible in IDRs and they are
consequently difficult to keep in their thiol, non-disulfide forms,
even in the presence of fresh DTT or TCEP at neutral pH. We also
produced chimera constructs
Esrrb(aa1--102\_C12A-C72A-C92A)--AviTag--His6 and
Esrrb(aa1--102\_C12A-C72A)--AviTag--His6.

All the 2D ${}^{1}$H--${}^{15}$N HSQC NMR spectra revealed cross-peaks
in the spectral region of random coil peptides (Figure~\ref{fig3}).
These spectra are overlapping to a large extent, confirming the weak
influence of the mutations of cysteines: the mutation Cys72Ala has
almost no consequences on the chemical shifts, below 0.05~ppm even for
the neighboring amino acids (Supplementary Figure~S2a,b); the mutation
Cys91Ala has more impact, with chemical shifts perturbations of about
0.1~ppm for the next 5 amino acids (Supplementary Figure~S2c,d), which
is at least partially due to the fact that the Ala substitution favors
an increase in local $\upalpha$-helicity (about 25\%, see Supplementary
Figure~S2e,f). This N-terminal fragment of human Esrrb is thus an IDR,
according to the calculated secondary structure propensities
(Figure~\ref{fig3}f, Supplementary Figures~S2e,f, S4).

\subsection{Phosphorylation of Esrrb and Sox2 by p38$\upalpha$, Erk2,
Cdk1/2 as monitored by NMR{\hfill\break} spectroscopy} \label{sec3.5}

We reported recently the site-specific phosphorylation kinetics of Oct4
by p38$\upalpha$ using ${}^{13}$C-direct NMR detection~\cite{99}. Here,
we used more standard ${}^{1}$H-detected/${}^{15}$N-filtered
experiments to rapidly characterize the site preferences of MAPKs and
CDKs on Esrrb and Sox2, which we thought to use as baits for performing
phospho-dependent pull-down assays (see below).

To start with, we used commercial aliquots of MAPKs, namely p38$\upbeta$
and Erk2, and CDKs, namely Cdk1/CyclinA2 and Cdk2/CyclinA2, on
Esrrb(aa1--102). We observed the progressive phosphorylation of its
three Ser--Pro motifs (at Ser22, Ser34 and Ser58) in agreement with the
consensus motifs of these kinases~\cite{116,117}. The kinetics reveal a
classical distributive mechanism, where phosphorylation sites are
processed independently according to their respective
$\text{k}_{\mathrm{cat}}$ and $\text{K}_{\mathrm{M}}$. Ser22 is the
preferred target in all cases, while Ser34 is the least processed by
CDKs, if at all: the commercial CDKs are poorly active in our hands,
which we have verified with a number of other targets for years in the
laboratory; this makes it \mbox{difficult} to distinguish between sites
that are only mildly disfavored or those that are more stringently
ignored by CDKs in NMR-monitored assays.

Then, we used a potent home-made activated p38$\upalpha$ on
His6--AviTag--Sox2(aa115--240) and His6--AviTag--Sox2(aa234--317). It
phosphorylated all the Ser/Thr--Pro motifs of these two peptides, and
also T306 in a PGT$_{306}$AI context, which shows a favorable proline
in position ${-}$2~\cite{118}, and a less common S212 in a MTS$_{212}$SQ
context.

Hence, we were able to generate AviTag--IDR chimera in well-defined
phosphorylated states. To produce phosphorylated ${}^{14}$N-AviTag--IDR
dedicated to pull-down assays, we executed the same protocol on 
${}^{14}$N-peptides, while NMR-monitoring in parallel ``identical'' but
${}^{15}$N-labeled peptides. This allowed us to produce a well-defined
phosphorylation state of the ${}^{14}$N-AviTag--IDR for the next
experiments.

\subsection{Structural characterization of the{\hfill\break}
AviTag--peptide chimera and its{\hfill\break} biotinylated versions}
\label{sec3.6}

We aimed at detecting new partners of OSNE using pull-down assays. We
thought to use chimera containing GST at the N-terminus, which appeared
as a convenient approach: vectors integrating a GST-coding DNA sequence
for overexpression in \textit{E.~coli} are available and of common use;
glutathione-coated beads are also accessible and permit efficient and
specific binding of GST-containing chimera peptides. However, we were
unsatisfied by the performances of the method: GST binding to
glutathione-coated beads is slow at low temperature (necessary to avoid
IDR proteolysis); moreover, it appeared that GST--IDRs chimera are
hampered by the IDRs' ``molecular cloud'' and are even weaker and
slower to bind to the beads. Our attempts to bind GST--IDRs to the
beads were thus resulting in poor yields, which were not very
reproducible. In the context of our aims, i.e., to establish a method
allowing quantitative detection of IDRs' binding partners, this
unsatisfying lack of reproducibility was only foreboding supplementary
variable parameters.

Thus, we decided to switch to another strategy: the use of the
specifically biotinylated 15-mer peptide tag called AviTag~\cite{101}.
This is efficiently and specifically biotinylated by biotin ligase BirA
(Figure~\ref{fig5}d), which permits high-affinity binding to
streptavidin-coated beads. We designed AviTag--IDR chimera, with the
AviTag in N-terminal position for Sox2's IDR constructs, and in
C-terminal for Esrrb's IDR constructs. We characterized the AviTag and
its impact on the IDRs of interest using NMR: the AviTag is unfolded
and it does not affect the Sox2 and Esrrb fragments, according to the
observed negligible chemical shift perturbations
(Figure~\ref{fig5})---the GST Tag provoked also only very weak chemical
shift changes on Esrrb(aa1--102) (Supplementary Figure~S3). The
biotinylated AviTag peptide appears to be slightly less mobile than the
common IDPs on the ps--ns timescale, according to the heteronuclear
${}^{15}$N--$\{{}^{1}\text{H}\}$ NOEs (Figure~\ref{fig5}b).

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}(a)~2D ${}^{1}$H--${}^{15}$N HSQC spectrum of
${}^{15}$N-His6-AviTag-Sox2(aa234--317\_C265A), the blue labels
indicated the assigned signals from the AviTag residues; (b)~secondary
structure propensities calculated from the experimental chemical shifts
of the peptide backbone C$\upalpha$ and C$\upbeta$, using the ncSPC
algorithm~\cite{104,105}; the residue specific
${}^{15}$N--$\{{}^{1}\text{H}\}$ NOEs, ${}^{15}$N-R1 (grey) and
${}^{15}$N-R2 (black) measured at 600 MHz are shown below (the profiles
show values averaged over three consecutive residues); (c)~overlay of
2D ${}^{1}$H--${}^{15}$N HSQC spectra of the
Esrrb(aa1--102\_C12A-C72A-C91A)--AviTag--His6 before (black) et after
(magenta) biotinylation by BirA; the NMR signals from the residues
neighboring the biotinylation site are indicated, which permit the
quantification of the biotinylated population; (d)~scheme of the
reaction of AviTag biotinylation executed by the ATP-dependent BirA.}
\end{figure*}

We observed that the biotinylation of the AviTag provokes weak but
distinguishable chemical shift perturbations for the close neighbors of
the biotinylated lysine, but had no effect on the peptides of interest
(Figure~\ref{fig5}c). It generated also the appearance of a HN-ester
NMR signal, similar to that of acetylated lysine~\cite{108,119}. Hence,
we could quantify and monitor the reaction advancement using NMR, and
determine the incubation time that was necessary and sufficient to
obtain a complete biotinylation of our chimera AviTag--IDRs (see
Section~\ref{sec2.2}). This was one among many optimization steps
permitting the production of sufficient quantities of intact IDRs for
the pull-down assays.

Next, we tested the binding of the biotinylated AviTag--IDRs on
streptavidin-coated beads. This produced very satisfying results, i.e.,
stoichiometric, specific binding in one hour with no leakage
(Supplementary Figure~S6). This approach was thus selected for the
pull-down assays.
{\vspace*{-.5pc}}

\subsection{Use of AviTag--Sox2 for pull-down assays{\hfill\break} from
mESCs extracts} \label{sec3.7}
{\vspace*{-.2pc}}

We prepared the four peptides AviTag--Sox2(aa115--240) and
AviTag--Sox2(aa234--317) in their non-phosphorylated and phosphorylated
versions, using p38$\upalpha$ to execute the phosphorylation reactions
(Figure~\ref{fig6}a). These peptides were also biotinylated, and later
bound to streptavidin-coated beads, which we used as baits for
pull-down assays in extracts from mESCs (Figure~\ref{fig6}b).
Importantly, an additional SEC was carried out between every step to
discard proteolyzed peptides, the enzymes (kinases of BirA) and their
contaminants. We performed the pull-down assays with the four samples
in parallel with the same cell extract, in duplicate, and then analyzed
the bound fractions using quantitative LC-MS/MS analysis (see 
Supplementary material~1.6 for full description). Hence, we could
detect and evaluate the relative quantities of proteins retained by the
four AviTag--peptides (Figure~\ref{fig6}). On paper, this presents the
important advantage of removing false-positive binders, which can
interact unspecifically with the streptavidin-coated beads.

\begin{figure*}
\includegraphics{fig06}
\caption{\label{fig6}Differential interactomics of Sox2 constructs upon
p38$\upalpha$ phosphorylation. (a)~We have produced
unmodified/phospho-Sox2 truncations carrying N-ter biotinylation on the
AviTag, and later attached on streptavidin-magnetic beads. (b)~We have
generated mESCs nuclear extracts for pull-down assays using our Sox2
constructs as baits. (c)~The volcano plots of the log2 ratios, showing
a quantitative analysis of the proteins present at the end of the
pull-down assays; the dashed line indicates the threshold of $p$-value
${<}$ 0.05; we highlighted interesting partners in: \underline{blue:}
transcription factors associating with Sox2(aa115--240);
\underline{magenta:} phospho-dependent partners of Sox2(aa115--240) and
or Sox2(aa234--317). Pull-downs have been performed in duplicates,
using 15 million cells per sample (extract protein conc.: 5~mg/mL), and
1~nmol of bait protein. Experimental conditions may be improved (higher
number of replicates, cells, washing conditions,\,\ldots).}
\end{figure*}

We present here results that should be interpreted carefully: we
produced only duplicates for every condition, using one single cell
extract. To deliver trustful information, the common standards in the
field recommend 3 to 5 replicates. Here, we considered only proteins
with at least 2 distinct peptides across the 2 replicates
(Figure~\ref{fig6}c). We observed a two-fold change or more (FC ${>}$
2) of some TFs pulled out by AviTag--Sox2(aa115--240), among which the
\mbox{PluriTFs} Oct4 and Klf5 are significantly enriched 
(${>}$4~peptides, $p$-value ${<}$ 0.02). Also, we found out that
pSox2(aa115--240) was pulling out the three isoforms of CK1 (${>}$3
peptides, $p$-values ${<}$ 10$^{-5}$) and the proline isomerase Pin1
(${>}$2 peptides, $p$-values ${<}\ 2 \times 10^{-4}$). To confirm the value
of the method and of the detected interactions, one should at least
test them using an orthogonal approach, e.g., NMR spectroscopy of
purified proteins.\looseness=-1

\subsection{NMR characterization of the interaction{\hfill\break}
between Pin1 and phospho-Sox2} \label{sec3.8}

We decided to test the interaction between pSox2(aa115--240) and Pin1.
We recorded ${}^{1}$H--${}^{15}$N NMR spectra of ${}^{15}$N-labeled
Sox2(aa115--240) or pSox2(aa115--240) alone or in the presence of the
Pin1--WW domain (natural abundance peptide, i.e., 0.6\% ${}^{15}$N,
99.4\% ${}^{14}$N, hence ``NMR-invisible'' in ${}^{15}$N-filtered
experiments). We observed localized losses in signal intensities for
the residues neighboring the three pSox2(aa115--240) phosphosites when
mixed with Pin1 (Figure~\ref{fig7}b); in contrast, no significant
differences showed up in the spectra obtained with non-phosphorylated
Sox2(aa115--240) in the absence or presence of Pin1--WW
(Figure~\ref{fig7}a). Hence, these signal losses are the typical signs
of a position-specific interaction between an IDR and a folded protein
in the intermediate or slow NMR timescale ($\upmu$s--s), which
corresponds to submicromolar affinities for this type of molecules.
Interactions with this range of affinity can therefore be detected
using the presented pull-down assay approach.

\begin{figure*}
\includegraphics{fig07}
\caption{\label{fig7}(a)~Overlay of 2D ${}^{1}$H--${}^{15}$N HSQC
spectra of ${}^{15}$N-Sox2(aa115--240) alone at 50~$\upmu$M (black) or
mixed with Pin1 in isotopic natural abundance and in stoichiometric
amounts (grey); inset up-right: residue specific NMR signal intensity
ratios as measured in the two HSQC spectra. (b)~Overlay of 2D
${}^{1}$H--${}^{15}$N HSQC spectra of ${}^{15}$N-phosphoSox2(aa115--240)
alone at 10~$\upmu$M (red) or mixed with the Pin1--WW domain in
stoichiometric amounts (grey); insets, up-right: residue specific NMR
signal intensity ratios as measured in the two HSQC spectra (in the
absence/presence of Pin1--WW domain).}
{\vspace*{-.35pc}}
\end{figure*}

\section{Discussion} \label{sec4}

The structural biochemistry analysis reported here can be applied to a
large list of transcription factors (TFs). These are essential actors
of cell signaling: they are key elements for inducing or maintaining
pluripotency or differentiation, for cell proliferation or cell cycle
arrest, by activating or repressing gene transcription~\cite{61,120}.
About 90\% of the ${\sim}$1600 TFs are predicted to contain large
disordered segments (${>}$30 consecutive amino acids), which is
particularly true for PluriTFs~\cite{31,121}. A correlation exists
actually between TFs and predicted IDRs in all kingdoms of
life~\cite{45,122,123}. The IDRs of these TFs recruit transcription
co-factors or the transcription machinery, which is still not very well
characterized in detail~\cite{44,45,46,47,48,49,50,51,52,53,54,55}. 

Indeed, the fine understanding of TFs' interactions via their IDRs
appears to be hampered by the nature of these interactions, which are
characterized by weak affinities and multivalency. Moreover, possible
redundancy between TFs can emerge from coacervation. Post-translational
modifications (PTMs), which can switch on or off IDRs' interactions,
are a supplementary source of confusion when searching for binding
partners. Among the difficulties, we should also mention the basic
biochemistry issues: IDRs are difficult to produce and manipulate in
vitro, because they are prone to degradation or aggregation. Here, we
have tried to demonstrate the feasibility and interest of some
biochemical and spectroscopic approaches to better characterize IDRs of
TFs, their phosphorylation and the associated binding partners.

Using a residue specific NMR analysis, we have shown experimentally
that the pluripotency TFs OSNE contained IDRs. None of these regions do
show any strong secondary structure propensity, which often reveals
functional binding sites. Faint ${\sim}$20\% helicities were detected by
two independent algorithms (ncIDP and $\updelta$2D~\cite{104,105,107}) on
5--6 amino acid stretches, which are indicated on Figures~\ref{fig1}
and~\ref{fig2} for Oct4 and Sox2. Altogether, the analyzed peptides
show local conformational behaviors close to that of a random coil,
without any obvious structural elements except the Oct4 stretch between
amino acids 33 and 38. Finally, we shall highlight the high propensity
for liquid--liquid phase separation of the fragments of Sox2 containing
the region aa115--236.

Like other TFs, pluripotency TFs OSNE are post-translationally modified
(see Section~\ref{sec1}), notably by CDKs and
MAPKs~\cite{66,78,79,80,81,82,83,84,85,86,87,88}. These two classes of
kinases are fundamental actors in all aspects of eukaryotic cellular
life, and understanding their activity and regulation in pluripotency
or differentiation is of high significance. Interesting questions are
still pending: what is the phosphorylation status of OSNE's IDRs in
pluripotent cells, what is the impact on their interaction networks,
and how does it affect pluripotency or differentiation? The inhibition
of MAPK Erk signaling is necessary to maintain pluripotency in the
standard culture conditions of ESCs and iPSCs~\cite{66,82,83,84}, while
these cells show a high CDK activity~\cite{8,86,87}; these two kinases
family have the same core consensus sites (Ser/Thr--Pro) motifs, which
are abundant in OSNE's IDRs and whose phosphorylation has apparently
consequences for initiating differentiation~\cite{73,78,80,83,85}. We
have shown that we could produce well-defined phosphorylation status of
these peptides, using recombinant kinases and NMR analysis, which makes
it possible to study their interactions {in vitro}. We have also
demonstrated our capacity to use these peptides as baits in pull-down
assays for detecting potential new binding partners.

However, the detected interactions between
phospho-Sox2(aa115--240)--pS212--pS220--pT232 and Pin1 or CK1
correspond to widespread, degenerate interactions, whose biological
significance may be questionable~\cite{124,125,126}. This is one of the
major drawbacks in the field of IDRs' studies: they participate in
multiple, degenerate and transient interactions of weak affinities,
which can be easily released during the washes of our pull-down assays.
In this regard, the ``proximity labeling'' approaches (BioID, APEX and
their derivates) appeared recently to be quite adapted to transient
interactions: these methods, developed in the last ten years, use
chimera constructs containing enzymes that transfer chemical groups to
their intracellular neighbors, which can be later identified by mass
spectrometry~\cite{127,128,129,130}. IDRs are very flexible,
solvent-exposed and establish a lot of poorly specific transient
interactions. This was raising concerns about the possible production
of many false positives if one used proximity labeling methods to
detect IDRs' binding partners. This has been partially confirmed by a
recent study, but this bias appears to be limited~\cite{131}.
Interactomes of 109 TFs have actually been described using Bio-ID and
affinity purification MS, showing the complementarity between proximity
labeling and the pull-down approach proposed here~\cite{132}. Yeast
two-hybrid, which can detect ${\sim}$20~$\upmu$M affinity interactions,
and novel phage display approaches will also help in this
task~\cite{133,134,135,136}.

Another difficulty in studying IDRs of TFs is their propensity to
coacervate~\cite{44,45,46,47,48,49,50,51,52,53,54,55}. Here, we have
tried to use Sox2 as a bait in pull-down assays, a protein that has
been later recognized to favor liquid--liquid phase
separation~\cite{44,137}. We met this difficulty during the production
steps, which forced us to purify most of the Sox2 constructs in urea at
2~M. We could straightforwardly observe liquid--liquid phase separation
of Sox2(aa115--317) at 4~$\upmu$M using differential interference
contrast (DIC) microscopy in the presence of Ficoll-70 (Supplementary
Figure~S5), but also progressive aggregation and low solubility
thresholds while working with our purified samples. These are clear
limiting factors for sample production and NMR characterization, which
will hamper a number of other studies on IDRs of TFs. This might also
affect the results of pull-down assays: we noticed an enrichment in TFs
in the samples obtained from pull-downs using Sox2(aa115--240) as a
bait, which has a much higher coacervation propensity than
Sox2(aa234--317).\ Is it possible to generate local surface
liquid--liquid phase separation on the surface of streptavidin-coated
beads? This might be at the same time a blessing and a curse for future
studies, by helping the formation of biologically significant
assemblies, or by favoring unspecific, non-native
macromolecules{\break} interactions.

A final bottleneck in the studies of these IDRs is the capacity to
produce post-translationally modified samples. The commercial enzymes
are not very well adapted to our NMR studies, because of the required
quantities. Here, we have used in-house production of kinase
p38$\upalpha$. Since we carried out the present work, we have developed
our capacities in producing activated Erk2, Cdk2/CyclinA1 and
Cdk1/CyclinB1. These will be part of our future studies. TFs are indeed
quite adapted to NMR investigations: they are 300 to 500 residues long
and contain large IDRs (${\sim}$100 amino acids) separating small folded
domains (also ${\sim}$100 amino acids)~\cite{31,45}. Their structural
characterization would permit understanding a number of cell-signaling
mechanisms at the atomic scale, and possibly identifying new
therapeutic targets, even though this class of proteins is notoriously
difficult to inhibit~\cite{60,138,139}.

\section{Conclusion} \label{sec5}

We have applied NMR techniques to carry out a primary analysis of the
pluripotency transcription factors Oct4, Sox2, Nanog and Esrrb, in
particular of their intrinsically disordered regions. We have shown
experimentally that they did not adopt a stable fold when isolated, and
that we were able to conduct a residue-specific analysis. This relies
on the delicate production and purification of these peptides, which
are prone to proteolysis and aggregation; producing them in a
well-defined PTM status was an even more arduous challenge. We have
demonstrated the feasibility of these tasks using recombinant kinases
and NMR analysis. We have also evaluated the usefulness of such protein
constructs as baits in pull-down assays to detect new binding partners
of IDRs. These characterizations and the associated methods provide
firm basis for future investigations on transcription factors. The
proposed experimental scheme is thus a promising methodology that still
needs to be developed and to prove its merits in revealing novel and
significant interactions.

\section*{Declaration of interests} 

The authors do not work for, advise, own shares in, or receive funds
from any organization that could \mbox{benefit} from this article, and
have declared no affiliations other than their research organizations.

\section*{Funding}

\looseness=-1
This work was supported by the CNRS and the CEA-Saclay, by the French
Infrastructure for Integrated Structural Biology
(\url{https://frisbi.eu/}, grant number ANR-10-INSB-05-01, Acronym
FRISBI) and by the French National Research Agency (ANR; research
grants ANR-14-ACHN-0015 and ANR-20-CE92-0013). Financial support from
the IR INFRANALYTICS FR2054 for conducting the research is also
gratefully acknowledged. This work was also supported by grants from
the ``R\'{e}gion Ile-de-France'' and ``Fondation pour la Recherche
M\'{e}dicale'' (DL and LSMP).

\CDRGrant[FRISBI]{ANR-10-INSB-05-01}
\CDRGrant[ANR]{ANR-14-ACHN-0015}
\CDRGrant[ANR]{ANR-20-CE92-0013}
\CDRGrant[IR INFRANALYTICS]{FR2054}

\section*{Acknowledgements}

We thank Thaleia Papadopoulou, Amandine Molliex and Navarro Pablo for
providing mESCs extracts and for fruitful discussions. We thank Nadia
Izadi-Pruneyre for providing the bench space necessary to carry out the
pull-down experiments with fresh mESCs extracts. We thank Romain LeBars
and the microscopy facility of I2BC Imagerie-Gif for the preliminary
tests on liquid--liquid phase separation. We thank the IR-RMN staff
(now part of Infranalytics), notably Nelly Morellet, Fran\c{c}ois
Giraud and Ewen Lescop, for their reactivity, their support, and their
long-standing, efficient care of the 950~MHz spectrometer. We thank
Marie Sorin, Baptiste Nguyen and Benjamin Bacri, who contributed to
this study during their Master1-Master2 internships.

\back{}

\section*{Supplementary data}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-272-suppl.pdf}}

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