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Published in final edited form as:

Nature. 2020 April ; 580(7803): 402–408. doi:10.1038/s41586-020-2188-x.

A reference map of the human binary protein interactome

A full list of authors and affiliations appears at the end of the article.

Abstract
Global insights into cellular organization and genome function require comprehensive
understanding of the interactome networks that mediate genotype-phenotype relationships1,2.
Here, we present a human “all-by-all” reference interactome map of human binary protein
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interactions, or “HuRI”. With ~53,000 high-quality protein-protein interactions (PPIs), HuRI has
approximately four times more such interactions than high-quality curated interactions from small-
scale studies. Integrating HuRI with genome3, transcriptome4, and proteome5 data enables the
study of cellular function within most physiological or pathological cellular contexts. We
demonstrate the utility of HuRI in identifying specific subcellular roles of PPIs. Inferred tissue-
specific networks reveal general principles for the formation of cellular context-specific functions
and elucidate potential molecular mechanisms underlying tissue-specific phenotypes of Mendelian

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*
Correspondence and requests for materials should be addressed to M.A.C., F.P.R., M.V., or D.E.H.
[email protected]; [email protected]; [email protected]; [email protected].
Author contributions
The project was conceived and supervised by G.D.B., J.T., D.E.H., M.V., F.P.R. and M.A.C. The Y2H assay versions were developed
and benchmarked by K.S., A.D.R., and Q.Z. with help from K.L., B.E.B. and D.B. hORFeome v9.1 was generated by K.L., D.-K.K.,
K.S., W.B., M.D., D.B., D.M., and T.H. with help from A.G.C., A.Dr., A.M., S.R., Y.S., G.M.S., J.-C.T. and X.Y. The preparation of
Y2H destination clones by en masse gateway cloning and yeast transformations were performed by D.-K.K., K.S., A.G.C., J.J.K.,
R.L., D.M. with help from M.G., D.S., S.S., B.T., C.C., G.H., N.v.L., A.R., and J.W. The Y2H screens were performed and the data
analyzed by K.L., K.S., B.E.B., M.D., A.Dr., M.F.H., C.Pol., S.S., B.T., A.T., and T.H. with help from W.B., T.C., B.C., A.De., D.B.,
S.-F.C., A.M., D.M., J.Ras., A.S.M., Y.S. and Y.W. The validation experiments were performed and the data analyzed by T.C., A.De.,
I.L., S.G.C., and T.H. with help from K.L., L.L., K.S., D.B., S.D.R., Y.J., Y.K., S.R. and J.T. Sequencing and analysis of the
sequencing data was performed by W.B., A.G.C., M.G., N.K., J.K., J.C.M., Y.S. and T.H. with help from K.L., D.-K.K., M.B., C.C.,
A.G., R.L., A.R., M.T., and J.W. Integrative downstream analyses were performed by K.L., D.-K.K., L.L., F.J.C.-L., I.A.K., and
C.Pon. with help from B.C., O.B., G.C., D.D.R., M.D.-F., F.G., G.H., J.N.P., T.R., E.S., E.Y.-L., Y.X., P.A. and J.D.L.R. Follow-up
experiments and analysis were performed by K.L., D.-K.K., K.S., R.B., D.C., S.D., A.De. and A.Y. with help from L.L., T.C., C.B.-C.,
G.C., C.D.A., H.E., L.G., E.H., S.La. and R.J.W. supervised by S.G., J.Rak. and V.T. The web portal was developed by M.W.M. with
help from K.L., M.H., T.H., M.A.C. and G.D.B. The paper was written by K.L., D.-K.K., L.L., K.S., D.E.H., M.V., F.P.R., and M.A.C.
with help from F.J.C.-L., A.G.C., G.C., S.G., I.A.K., T.H, and A.Y. Authors other than co-first and co-corresponding are listed
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alphabetically and contributed equally within their group.

Competing interests J.C.M. is a founder and CEO of seqWell, Inc; F.P.R. and M.V. are shareholders and scientific advisors of
seqWell, Inc.
Extended data is linked to the online version of the paper.
Supplementary information is linked to the online version of the paper.
Online content
Methods, supplementary notes, additional references, and supplementary tables are available online. Detailed sample size information
for each figure panel is provided in Supplementary Table 29.
Data availability
HuRI, Lit-BM, and all previously published human interactome maps from CCSB are available at http://interactome-atlas.org. The PPI
data from this publication is also available through IntAct (https://www.ebi.ac.uk/intact/) with the identifier IM-25472. All HuRI-
related networks from this study are available at NDExbio.org (https://tinyurl.com/networks-HuRI-paper). The raw and analyzed
proteomic data were deposited in the PRIDE repository (https://www.ebi.ac.uk/pride/) with the accession number PXD012321.
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diseases. HuRI represents a systematic proteome-wide reference linking genomic variation to

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phenotypic outcomes.

The reference human genome sequence has enabled systematic study of genetic6 and
expression4 variability at the organism6, tissue4, cell type7 and single cell level8. Despite
advances in sequencing genomes, transcriptomes, and proteomes, we still understand little
about the cellular mechanisms that mediate phenotypic and tissue or cell type variability. A
mechanistic understanding of cellular function and organization emerges from studying how
genes and their products, primarily proteins, interact with each other, forming a dynamic
interactome that drives biological function. Analogous to the reference human genome
sequence9,10, a reference map of the human protein interactome, generated systematically
and comprehensively, is needed to provide a scaffold for the unbiased proteome-wide study
of biological mechanisms, generally and within specific cellular contexts.
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It remains infeasible to assemble a reference interactome map by systematically identifying

endogenous PPIs in thousands of physiological and pathological cellular contexts11,12.
However, systematic affinity purification of exogenously expressed bait proteins in
immortalized-cell contexts13 as well as binary PPI detection assays in cell models2,14 have
generated biophysical human protein interactome maps of demonstrated high functional
relevance. Specifically, yeast two-hybrid (Y2H) represents the only binary PPI assay that can
be operated at sufficient throughput to systematically screen the human proteome for binary
PPIs. Using Y2H followed by validation in orthogonal assays, we previously generated HI-
II-14 consisting of ~14,000 PPIs involving 4,000 proteins from screening ~40% of the
genome-by-genome search space2. In contrast to curated interactions from hypothesis-driven
small-scale studies and protein complex interactome maps favoring highly expressed
proteins, HI-II-14 covers the proteome more uniformly and is relatively free from
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ascertainment and expression biases.

To increase interactome coverage and generate a reference map of human binary PPIs, we
expanded the ORFeome collection to encompass ~90% of the protein-coding genome. We
screened this search space a total of nine times with a panel of three Y2H assay versions
(Fig. 1a, b). The resulting PPI map quadruples the number of identified PPIs overall, and
upon integration with genome, transcriptome and proteome resources enables biological
discovery across most cellular contexts, offering a reference map of the binary human
protein interactome.

Generation and characterization of HuRI

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The newly established human ORFeome v9.1 covers 17,408 protein-coding genes (Extended
Data Fig. 1a, b, Supplementary Table 1, 2), forming a search space (Space III) that
encompasses over 150 million pairwise combinations (Fig. 1a). This search space more than
doubles the space screened to generate HI-II-14 and represents the most comprehensive
search space systematically screened for human PPIs. Limitations in PPI assay sensitivity
can be overcome by using different assays15,16 or different versions of the same assay17,18.
To maximize sensitivity we used three Y2H assay versions (Fig. 1b, Supplementary Table 3,
4) that showed good sensitivity and low false positive rates when benchmarked against gold-

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standard positive and random reference sets (PRSv1 and RRSv1, respectively)15, while
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detecting complementary PPI sets (Extended Data Fig. 1c, d, Supplementary Table 5). We
assessed assay performance using a test space of ~2,000 by ~2,000 human genes2 (Extended
Data Fig. 1e, Supplementary Table 6). The Y2H versions were complementary, in that three
screens for each of three versions doubled the number of detected PPIs and proteins relative
to nine screens using a single version (Extended Data Fig. 1f, Supplementary Table 7).

To map the reference interactome, we performed nine screens of Space III, followed by
pairwise verification by quadruplicate retesting and sequence confirmation. PPIs verified by
two orthogonal binary PPI assays, MAPPIT19 and GPCA20, were recovered at rates on par
with high-confidence binary PPIs from the literature (each having ≥2 pieces of experimental
evidence, with at least one from a binary assay type; Lit-BM) over a large range of score
thresholds (Fig. 1c, Extended Data Fig. 1g, h, Supplementary Table 8). Each additional
screen identified novel PPIs and proteins, with the largest gains obtained by switching assay
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versions (Fig. 1d, Extended Data Fig. 1i). The dataset, versioned HI-III-20 (Human
Interactome obtained from screening Space III, published in 2020), contains 52,569 verified
PPIs involving 8,275 proteins (Supplementary Table 9). Although our knowledge of the
interactome remains incomplete, we refer to HI-III-20 as a reference map of the human
binary protein interactome (HuRI) given its systematic nature, extensive coverage, and scale.

Molecular mechanisms can be more readily inferred from direct than indirect PPIs, yet the
fraction of PPIs reported in various human protein interactome maps that are direct remains
unknown. Structures from protein complexes with at least three subunits21 show that, within
those complexes, more PPIs in HuRI correspond to direct biophysical contacts than do PPIs
from Lit-BM (90% vs. 81%, P = 0.019, two-sided Fisher’s exact test, n = 121 and 410 for
HuRI and Lit-BM, respectively) or from protein complex interactome maps (< 50%, P <
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0.001 for all tested maps, two-sided Fisher’s exact test; Fig. 1e, Supplementary Table 10).
Combining HuRI with all previously published systematic screening efforts at CCSB yields
64,006 binary PPIs involving 9,094 proteins (HI-union; Supplementary Table 11), which is
approximately five-fold more PPIs than the entirety of high-quality binary PPIs curated from
the literature (Fig. 1f, Extended Data Fig. 2, Supplementary Table 12–14). The union of Lit-
BM and HI-union represents the most complete collection of high-quality direct PPI data
available to date (http://interactome-atlas.org).

Complementarity of the three Y2H versions might stem from steric constraints that differ
between protein fusions used in the assays. Integrating HuRI with structures of PPIs21, we
observed reduced sensitivity of Y2H assays where the interaction interface was close (<
20Å) to whichever terminus was fused to the Gal4 activation domain (AD; Extended Data
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Fig. 3a, b, Supplementary Table 15). PPIs found in multiple screens had larger interaction
interfaces (P = 0.03, two-sided permutation test, n = 234) and corresponded more often to
direct PPIs within, rather than between, protein complexes (P = 3 × 10−18, two-sided
Fisher’s exact test, n = 1,817), however, HuRI PPIs found in a single screen were observed
to have precision as high as those found in multiple screens (Extended Data Fig. 3c–g, 4,
Supplementary Table 16–18, Supplementary Note 1). These results reinforce previous
observations12,22 that the protein interactome might be dominated by weaker and more
transient PPIs that are difficult to detect, as indicated by the fact that the majority of PPIs in

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HuRI were found in only one screen (Extended Data Fig. 3h, i). This is reflected in an
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increased estimate of the binary protein interactome size (Extended Data Fig. 3j), so that
HuRI is estimated to represent 2–11% of the binary protein interactome23 (Supplementary
Note 2).

Functional relationships in HuRI

Based on the observation that HuRI is enriched in direct PPIs, we also hypothesize that
proteins in HuRI with similar interaction interfaces tend to share interaction partners. For
example, retinoic acid receptors RXR-γ and -β (Extended Data Fig. 5a, left panel) share
previously reported interaction partners involving binding to retinoic acid receptor RAR
types24 and oxysterol receptors NR1 group H types25. We derived a profile similarity
network (PSN) from HuRI (Supplementary Table 19), and found that the number of pairs of
proteins in HuRI with similar interaction profiles is higher than random (P < 0.01, one-sided
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empirical test; Extended Data Fig. 5b) and proteins of overall higher sequence identity tend
to exhibit higher interaction profile similarities (P < 0.01, one-sided empirical test; Extended
Data Fig. 5c). We also found that proteins with a tendency to share interaction partners often
have interaction interfaces that are similar, as opposed to complementary, and therefore tend
not to interact with one another, except where proteins originate from a common ancestor
that could self-interact26 (Extended Data Fig. 5d). Indeed, only 5% of the protein pairs
found to interact in HuRI share more than 10% of their interaction partners. Both HuRI PPIs
and the PSN are enriched for links between proteins of similar function (P < 0.01, one-sided
empirical test; Fig. 2a, Supplementary Table 20, Extended Data Fig. 5e, f) and both contain
more functional modules27 than our previously published interactome maps2,14 (Fig. 2b,
Extended Data Fig. 5g). Thus, HuRI and the PSN are complementary maps of functional
relationships between proteins.
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As shown above, global sequence identity between two proteins can be indicative of shared
interaction interfaces, however, it likely fails to identify pairs of proteins whose shared
interaction interface is small. Indeed, 50% (502) of all protein pairs in HuRI with interaction
profile similarities ≥ 0.5 exhibit ≤ 20% sequence identity, so that functional relationships
identified by the PSN are not necessarily identifiable by sequence identity. One such protein
pair is the endoplasmic reticulum (ER) transmembrane protein TMEM258 and the
uncharacterized protein C19ORF18, which have only 10% sequence identity but share 80%
of their interactors (Extended Data Fig. 5a, right panel). TMEM258 catalyzes the first step in
N-glycosylation of proteins in the ER and might play a role in protein translocation across
the ER28. Roles in protein transport and ER function have also been ascribed to two of the
four shared interaction partners, ARL6IP129 and IER3IP130, suggesting that C19ORF18 and
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potentially the other two shared interaction partners (MFSD6 and AC012254.2) contribute to
ER-related functions of protein maturation and transport.

Uncharted disease-related interactions

Unlike Lit-BM, HuRI was generated by systematically testing protein pairs for interaction.
While Lit-BM is highly biased towards the most studied genes2, HuRI covers the genome-
by-genome space more uniformly and at increased depth compared to Lit-BM and our

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previous screening efforts (Fig. 3a, Extended Data Fig. 5h). Interestingly, we find that the
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agreement between Lit-BM and HuRI is highest among the best-studied genes, where Lit-
BM is most complete and where ~40% of the PPIs in HuRI have been previously identified
(Fig. 3b). HuRI substantially expands the number of biomedically interesting genes for
which high-quality direct PPI data is available (Fig. 3c, Extended Data Fig. 5i), and finds
new interaction partners for these genes in previously uncharted regions of the protein
interactome (Fig. 3d, Extended Data Fig. 5j).

Essential proteins were often found to have significantly more interaction partners31.
However, correlation between two variables does not necessarily imply causality, especially
where there are other confounding variables (Extended Data Fig. 5k). We find that protein
popularity (measured by publication count) and endogenous expression level strongly
correlate with: i) each other; ii) the number of interaction partners (‘degree’) in protein
complex and literature-curated protein interaction networks but not in HuRI; as well as iii)
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gene properties such as essentiality, age32, fitness effect33 and loss-of-function intolerance6
(Extended Data Fig. 5l, Supplementary Table 21). After correcting for popularity and
expression level, we found substantially reduced correlations between interaction degree and
other gene properties, including gene essentiality (Fig. 3e, Extended Data Fig. 5m, n). In line
with previous observations34, this suggests that correlations between degree and essentiality,
age, loss-of-function intolerance, and fitness effects are confounded by underlying
expression and study biases, and thus may not reflect causal relationships. These results
highlight the value of HuRI as a uniformly-mapped reference for the study of systems
properties of genes and networks.

Compartment-specific roles of PPIs

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Proteins are localized to specific compartments, carrying out functions that depend both on
the subcellular environment and the local PPI network. Despite available proteome-wide
datasets on the localization of individual proteins5, experimental determination of cellular
localization-specific PPI networks remains challenging. We find that proteins localizing to a
diverse range of subcellular compartments are evenly represented in HuRI (Extended Data
Fig. 6a), suggesting that cellular localization-specific PPI networks can be inferred for many
different cellular compartments via integration of HuRI with available protein localization
data.

Extracellular vesicles (EVs) have been studied intensively using proteomics approaches35.
However, our understanding of the molecular mechanisms that lead to protein recruitment
into EVs and subsequent secretion remains limited. The subnetwork of interactions between
EV proteins (Extended Data Fig. 6b) shows significantly higher connectivity in HuRI than in
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degree-controlled randomized networks (P < 0.001, one-sided empirical test; Extended Data
Fig. 6c), enabling prediction of EV recruiters using the number of EV interaction partners.
Seven of the top 21 most connected proteins in this EV network have established roles in EV
biogenesis or cargo recruitment36. SDCBP (syntenin-1) functions in ESCRT-dependent EV
generation and its knockout shows reduced EV production37. SDCBP has 48 PPIs with other
EV proteins and is frequently detected in EVs, suggesting that it regulates recruitment of
interacting proteins into EVs. To test this hypothesis (Fig. 4a), we knocked out SDCBP in

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the U373vIII cell line (Extended Data Fig. 6d). Of six SDCBP partners detected in the
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U373vIII EV proteome, three (CALM1, CEP55, HPRT1) displayed significantly reduced

protein levels in EVs in the SDCBP knockout line (P < 0.05, one-sided empirical test,
depletion < 0.66; Fig. 4b). In contrast, only 15% of the non-interaction partners of SDCBP
were reduced (P = 0.042, one-sided empirical test; Extended Data Fig. 6e). Thus, SDCBP
may play a role in the recruitment of proteins into EVs, highlighting the potential value of
HuRI in studying protein function within specific subcellular contexts.

Proteins often change subcellular localization, e.g. as part of their maturation process or in
response to external or internal signaling events. These dynamics are difficult to
comprehensively capture at proteome-scale, yet existing efforts have already revealed
numerous proteins that localize to multiple subcellular compartments with some pairs of
compartments exhibiting significantly more crosstalk than others5. Despite a tendency for
interactions in HuRI to link proteins localized to the same compartment (P < 0.01, one-sided
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empirical test; Fig. 2a), a considerable number of interactions were identified between
proteins never reported to co-localize. To explore whether lack of co-localization of
interacting proteins in HuRI might originate from incomplete localization data, we assessed
whether these non-co-localized interacting proteins tend to reside in compartments with
significant crosstalk and observed a significant positive correlation (P < 0.001, one-sided
empirical test; Extended Data Fig. 6f–h). This suggests that HuRI could prove useful in
predicting protein localization dynamics.

Principles of tissue-specific function

Despite recent advances in systematic genome-wide identification of tissue-preferentially
expressed (TiP) genes4 (Extended Data Fig. 7, Supplementary Table 22), we lack a concrete
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understanding of how the surprisingly small set of TiP genes operate together and coordinate
their activity with the core ‘housekeeping’ machinery to mediate tissue-specific functions.
Insights can be obtained from investigation of the tissue-specific network context of TiP
proteins, inferred from integrating protein interactome data with tissue transcriptomes
(Supplementary Table 23). However, we should not expect uniform coverage of TiP proteins
with PPIs using experimental methods that demand expression of both partners within a
single immortalized cell line or result from screening an incomplete ORFeome. Indeed,
contrary to protein complex11–13 and literature-curated interactome maps38 as well as our
previously published binary PPI datasets2,14, we find that TiP proteins are well-represented
in HuRI (Fig. 5a, Extended Data Fig. 8a).

Restricting HuRI to PPIs between proteins expressed in the same tissue, we observe that TiP
proteins engage in as many PPIs and are as central as more uniformly expressed proteins
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(Extended Data Fig. 8b), contrary to previous observations using literature-curated PPI
networks39,40. This result, paired with the fact that PPIs mediated by a TiP protein are
effectively also tissue-specific, leads to the finding that the fraction of tissue-specific PPIs in
the protein interactome as characterized by HuRI is higher than that of tissue-specific genes
in the expressed genome, indicating that substantial information on tissue-specific functions
can only be obtained from the interactome. The opposite is observed for Lit-BM, likely
owing to its bias against TiP genes (Extended Data Fig. 8c).

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To investigate the local network neighborhoods of TiP proteins within their respective tissue
contexts, we used HuRI to derive protein interactome maps for 35 tissues4,41, each of which
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contains about 25,000 PPIs (Supplementary Table 24, Extended Data Fig. 8d). Within each
tissue PPI network, we focused on the interactions involving at least one TiP protein (Fig.
5b). The TiP PPI networks show extensive interactions between TiP and non-TiP proteins,
but with few TiP-TiP PPIs. Despite significant enrichments for HuRI to link proteins that
work in the same biological process, TiP-TiP PPIs, as highlighted for brain in Fig. 5c, are
not enriched, nor is the average shortest path among TiP proteins shorter than in degree-
controlled randomized networks (P > 0.05, empirical test). Using either metric, TiP proteins
were found to be significantly close to each other in only six of 35 tissues. In four of these
six tissues, enrichment for network proximity was driven by clusters of specifically
expressed keratins or late-cornified envelope proteins (Extended Data Fig. 8e). These results
support a model in which tissue-specific functions emerge through interactions between TiP
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proteins and more uniformly expressed members of the basic cellular machinery,
presumably modulating and adapting common cellular processes for cellular context-
specific needs42.

One biological process with both cell-type and developmental stage-specific homeostatic
roles is apoptosis. We used HuRI to identify proteins with interaction partners that were
enriched for known apoptosis regulators (Supplementary Table 25). Five proteins among the
top ten predictions had previously demonstrated roles in apoptosis (Supplementary Note 3).
Among three TiP genes predicted to be implicated in apoptosis (Extended Data Fig. 8f, g),
we further examined OTUD6A. Abundance of OTUD6A negatively correlated with time-of-
death after addition of TRAIL (TNF-related apoptosis-inducing ligand; P = 0.012, two-
sided, empirical test, n = 40 cells), but not after expression of OTUD6A alone (Extended
Data Fig. 8h, Supplementary Table 26), suggesting that OTUD6A participates in the
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apoptosis pathway but is not an inducer of cell death. This and other evidence (Extended
Data Fig. 8f, i, Supplementary Note 3) suggests that OTUD6A exerts an apoptosis
sensitization function via transcriptional activation in a haematopoietic cellular context.

Mechanisms of tissue-specific diseases

Many Mendelian diseases display tissue-specific phenotypes, rarely explained by tissue-
specific expression of genes with disease-associated mutations43 (Fig. 5d, Extended Data
Fig. 9a). Such mutations broadly or specifically affect PPIs involving the mutated protein44.
Perturbations of PPIs between uniformly expressed disease-associated proteins and TiP
proteins in the affected tissues have been suggested to underlie the tissue-specific
phenotypes of those diseases43. In HuRI-derived tissue PPI networks, we find 130 such PPIs
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involving 63 distinct non-TiP disease-causal proteins and 94 TiP proteins. Although we see
no enrichment for PPIs between causal proteins and TiP proteins (Extended Data Fig. 9b,
Supplementary Note 4), this does not rule out the possibility that perturbations of some of
these interactions mediate tissue-specific phenotypes of Mendelian diseases.

To explore this hypothesis, we experimentally tested whether pathogenic variants associated

with Mendelian diseases were able to perturb these PPIs. Of ten causal proteins tested, seven
showed perturbation of PPIs to preferentially expressed interaction partners in the

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corresponding “disease tissues” (Fig 5e, Extended Data Fig. 9c, d, Supplementary Table 27,
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28). One example is PNKP (polynucleotide kinase 3’-phosphatase) whose mutations have
been associated with microcephaly, seizures, and developmental delay. The pathogenic
PNKP mutation Glu326Lys affects neither the DNA kinase nor DNA phosphatase activity of
PNKP, rendering the mechanism of pathogenicity unclear45. We observed that Glu326Lys
perturbed PPIs with two partners preferentially expressed in the brain, SYNGR1 and
TRIM37, whereas a benign control mutation Pro20Ser46 did not affect any PNKP PPIs (Fig.
5f, Extended Data Fig. 9c, e). TRIM37 facilitates DNA repair47, suggesting a potential
mechanism by which perturbation of this interaction could affect the brain-specific DNA
repair function of PNKP. In other examples, HuRI identified CTNNA3 and SUCLA2 to have
respective TiP interaction partners TRIM54 and ARL6IP1 (Extended Data Fig. 10), which
cause similar diseases with overlapping symptoms48,49, supporting the relevance of these
interactions in the physiopathology. Overall, this study yields hypotheses of molecular
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mechanisms for otherwise unexplained tissue-specific phenotypes of seven Mendelian

diseases (Extended Data Fig. 9d) and demonstrates the utility of HuRI as a reference to
study biological mechanisms within specific disease contexts.

Discussion
Here, we present HuRI, a systematically generated human protein interactome map with
more than 50,000 PPIs of high biophysical quality. While HuRI displays highly significant
overlap with known functional relationships, the cellular function of most individual PPIs
remains to be elucidated. We show that follow-up studies on the function of proteins and
PPIs can be guided by integration of HuRI with contextual genome, transcriptome and
proteome data to infer the cellular context in which subnetworks of PPIs operate together to
mediate a function. With advances in single cell transcriptomics8 as well as systematic
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determination of subcellular protein localization, inference of functional PPI subnetworks

that are specific for a given cellular state will further increase in precision. However, a priori
removal of PPIs from HuRI because they are not currently known to function together in a
physiological cellular context could discard data that can help, e.g., to understand the
functional consequences of dysregulated gene and protein expression causing disease.

Despite our extensive screening, many PPIs remained undetected as Y2H, like all
characterized assays15,16,18, has limited sensitivity, detecting no interactions for half of the
tested proteins. This negative result can guide the design of future interactome mapping
efforts to target these proteins. Due to the limitations of Y2H, we expect HuRI to be
depleted for PPIs that depend on post-translational processing of human proteins that the
yeast cell is unable to catalyze or that require additional partners to stabilize the interaction.
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Screening only one isoform per gene also misses interaction partners specific to alternative
spliceforms50. Accurate estimation of the total size of the interactome remains challenging.
PPIs display a continuum of binding strength or stability that, along with other factors, could
underlie a continuum of detectability, as this study suggests. Furthermore, the results
obtained by us and others12 indicate that very stable and functionally conserved PPIs
constitute a minority of the interactome.

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While incomplete, HuRI’s proteome and interactome coverage and uniformity enable its use
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as a reference for the study of most aspects of human cellular function. Efforts to further
complete this reference will require development of new technologies as well as integration
with complementary reference maps of protein complex assemblies13. Although multiple
challenges remain to be solved for a complete and context-specific map of protein functions,
interactions, and higher-level organization, HuRI provides an unbiased genome-scale
scaffold with which to coordinate this information as it emerges.

Extended Data
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Extended Data Fig. 1 |. Y2H assay development and validation of HuRI.

a, Number of protein-coding genes in hORFeome v9.1 and GTEx, FANTOM, and HPA
transcriptome projects. The number of genes in hORFeome v9.1 is on par with the number
of genes found to be expressed in three comprehensive individual transcriptome sequencing
studies and includes 94% of the genes with robust evidence of expression in all three. b,
Overlap between hORFeome v9.1 and intersection of transcriptomes in a. c, Individual and
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combined recovery of PRSv1 and RRSv1 pairs by Y2H assay versions (n = 252, 270). d,
Colored squares showing which protein pairs were detected in PRSv1 (left) and RRSv1
(right) by Y2H assay versions. e, Recovery rates of Lit-BM and PPIs from screens of a 2k-
by-2k gene test space per Y2H assay version in MAPPIT. f, Cumulative PPI count
performing three screens with each Y2H assay version in the test space compared to nine
screens with Y2H assay version 1. g, h, MAPPIT and GPCA recovery of Lit-BM and PPIs
from screens of Space III when split by screen at a RRS rate of 1% (g) or across a range of

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thresholds (h). All error bars, in c, e, g, are 68.3% Bayesian confidence interval, shaded
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error band in h is standard error of proportion and n = between 101 and 395 pairs
successfully tested for each category. i, Number of proteins in HuRI, detected with each
additional screen.
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Extended Data Fig. 2 |. Definition of literature-curated PPI datasets.

a, Categorization of literature-curated PPIs into distinct subsets based on the experimental
methods in which they were detected and the number of pieces of experimental evidence. b-
e, Results of testing the different categories of literature-curated pairs in Y2H (b, d) and
MAPPIT (c, e) where the pairs have been further divided into HT - high throughput and LT -
low throughput subsets (b, c). BM: binary multiple; BS: binary singleton; NB: non-binary.
Between n = 191–471 successfully tested PPIs for each category.
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Extended Data Fig. 3 |. Stericity and interaction strength contribute to PPI detectability.
a, b, Fraction of PPIs with N or C-terminus < 10 Å (a) or 20 Å (b) to PPI interface, for PPIs
with known structure in and not in HuRI (n = 37–1,891 PPIs). Error bars are standard error
of proportion. The structure of UBE2D3 bound to RNF115 illustrates an example of a PPI
found only by Y2H assay version 3 (PDB code: 5ulh). c, MAPPIT recovery rates of HuRI
and Lit-BM PPIs that were also detected in HuRI by the number of screens each pair was
detected in. Error bars are 68.3% Bayesian confidence interval (n = 22–793 PPIs
successfully tested in each category). d, MAPPIT recovery rates of Lit-BM PPIs that were
also detected in HuRI, for increasing number of pieces of experimental evidence per PPI.
Error bars are 68.3% Bayesian confidence interval (n = 24–61 PPIs successfully tested in
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each category). e-f, Distributions of interaction interface area (e) or number of atomic
contacts (f) by the number of HuRI screens in which a PPI is detected, with boxplots
showing median, interquartile range (IQR), and 1.5 × IQR (with outliers), n = 1004 PPIs. g,
Examples of within-complex interactions detected in HuRI (purple) and BioPlex (orange).
Fraction of HuRI PPIs between proteins of protein complexes that link proteins of the same
complex, split by PPIs found in single and multiple screens (dark purple). Error bars are
standard error of proportion, n = 1,042 and 775 PPIs. h, Number of screens each PPI in

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HuRI was detected in, split by Y2H assay version. i, Number of Y2H assay versions each
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PPI in HuRI was detected in. j, Estimates of the size of the total binary protein interactome
and the fraction covered by HuRI, as a function of the minimum number of publications per
gene and the minimum number of evidence for the Lit-BM reference. Error bands are 68.3%
Bayesian confidence interval, n ≥ 170 Lit-BM PPIs.
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Extended Data Fig. 4 |. HuRI provides direct contact information for proteins in complexes.
Intra-complex PPIs are shown for protein complexes from CORUM as found in BioPlex
(orange) or HuRI (purple). HuRI PPIs are further distinguished into PPIs found in single
(light purple) or multiple screens (dark purple).
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Extended Data Fig. 5 |. Topological and functional significance of HuRI.

a, Examples of protein pairs in HuRI with high interaction profile similarity and both high
(left) and low sequence identity (right). b, The number of pairs of proteins in HuRI and 100
random networks at increasing Jaccard similarity cutoffs, with boxplots showing median,
interquartile range (IQR), and 1.5 × IQR (with outliers). PSN: profile similarity network. c,
Enrichment over random networks of the sum of Jaccard similarities of pairs of proteins in
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HuRI above at increasing thresholds of sequence identity. Error bars are 95% confidence
intervals, center is relative to mean of random networks. d, Fraction of PSN edges that are
also PPIs in HuRI, split by the PPIs involving no, one or two self-interacting proteins (SIPs),
at increasing Jaccard similarity cutoffs. Error bars are standard error of proportion. e, f,
Enrichment over random networks of the PPI count (left) or sum of Jaccard similarities
(right) of HuRI PPIs or PSN pairs, respectively, at increasing co-expression (e) and co-
fitness (f) cutoffs. Error bars are 95% confidence interval, center is relative to mean of

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random networks. g, Functional modules in HuRI (top) and its PSN (bottom) with functional
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annotations. h, Heatmaps of PPI counts, ordered by number of publications, for our previous
human interactome maps and Lit-BM i, j, Fraction of genes with at least one PPI for
biomedically interesting genes. Heatmap of HuRI and Lit-BM PPI counts between proteins,
ordered by number of publications, restricted to PPIs involving genes from the
corresponding gene set. k, Schematic of relation between variables: observed PPI degree,
abundance, study bias and lethality. l, Correlation matrices. LoF: Loss-of-Function. PPI
datasets refer to their network degree. m, Degree distribution of various PPI networks,
together. n, Empirical determination of significance of correlation between various network
degrees and gene properties. HuRI-2s = subset of HuRI found in at least two screens, (n =
13,441–53,704 PPIs per network).
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Extended Data Fig. 6 |. Incomplete protein localization annotation likely underlies apparent lack
of co-localization of proteins interacting in HuRI.
a, Odds ratios of proteins in different subcellular compartments and PPI datasets. n = 125–
3,941 proteins per compartment, two-tailed Fisher’s exact test. b, The subnetwork of HuRI
involving extracellular vesicle (EV) proteins. Names of high-degree proteins are shown. c,
Number of PPIs in HuRI between EV proteins compared to the distribution from
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randomized networks (grey). d, Western Blot of SDCBP (left panel) and ACTB (loading
control, right panel) in wild-type (WT) and three knockout (KO) cell lines (#7-#9), repeated
twice in two independent laboratories. Full scanned image was displayed, obtained by
ChemiDoc MP imager (Bio-Rad, Hercules, CA). Cell line #8 was used for EV proteomics.
e, Fraction of proteins whose abundance in EVs was significantly reduced in the SDCBP KO
cell line, split by proteins interacting and not interacting with SDCBP as identified in HuRI.
Error bars are standard error of proportion (n = 6 interactors, 638 non-interactors, *p =
0.042, one-tailed empirical test). f, Schematic illustrating that the number of HuRI PPIs

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between proteins from two different compartments should correlate with the enrichment of
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both compartment pairs to overlap, if co-localization annotation is incomplete. g, Scatter

plot showing, for each pair of subcellular compartments, odds ratios quantifying the
enrichment for proteins located in both compartments versus the enrichment of the density
of PPIs between proteins located to either compartment. Size of points is scaled by the
standard error of the x axis variable. Regression line and 95% confidence interval are shown.
h, The z-score of the regression slope of g compared to those of random networks.
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Extended Data Fig. 7 |. Investigation of tissue-preferential expression data.

a, Examples of genes displaying different levels of tissue-preferential (TiP) expression
across the GTEx tissue panel (left), with boxplots showing median, interquartile range
(IQR), and 1.5 × IQR (with outliers), n = 90–779 samples per tissue. Equation to calculate
tissue-preferential expression for every gene-tissue pair and the maximum TiP value for
every gene (middle). Number of genes showing tissue-preferential expression for increasing
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tissue-preferential expression cutoffs (right). b, Relative number of TiP genes for every
tissue for increasing tissue-preferential expression cutoffs. c-d, Differences in number of TiP
genes upon removal of testis prior to TiP value calculation per tissue (TiP value cutoff = 2)
(c) and in total for increasing tissue-preferential expression cutoffs (d). e, Number of TiP
genes and number of TiP genes that are also exclusively expressed in one tissue (sglTis:
single tissue) for increasing tissue-preferential expression cutoffs.

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Extended Data Fig. 8 |. PPIs between TiP proteins and uniformly expressed proteins likely adapt
basic cellular processes to mediate cellular context-specific functions.
a, TiP protein coverage by CCSB PPI networks for increasing levels of tissue-preferential
expression, (shaded error bands proportional to standard error on proportion, n ≥ 233 genes).
b, Spearman correlation coefficients and 95% confidence intervals for correlations between
degree or betweenness and tissue specificity for HuRI and Lit-BM (n = 6,684 and 4,971
proteins). c, Fraction of HuRI and Lit-BM that involve TiP proteins compared to fraction of
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genome that are TiP genes for increasing levels of tissue-preferential expression. d, Number
of PPIs in HuRI, involving proteins in GTEx, where both proteins are expressed in the same
tissue, and the mean of the tissue-specific subnetworks where error bar is standard deviation.
e, Test for enrichment of TiP-TiP PPIs (left) and significance of average shortest path
between TiP proteins (middle) in each tissue subnetwork, number of TiP proteins in each
subnetwork, interacting with other TiP proteins, being part of Keratin (KRT) or Late-
cornified envelope (LCE) protein family (right). f, g, Transcript expression levels across the
BLUEPRINT hematopoietic cell lineage (f) and GTEx tissue panel (g) for three candidate

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genes predicted to function in apoptosis. EG = esophagus gastroesophageal. h, Histogram of

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number of untransfected cells and their time of death (left) without (top) and with (bottom)
addition of TRAIL. Time of death of cells expressing OTUD6A-GFP fusions versus
OTUD6A expression measured as fluorescence (right) without (top) and with (bottom)
addition of TRAIL. i, Apoptosis-related network context of OTUD6A and C6ORF222 in
HuRI, unfiltered (left) and filtered using colon transverse or mature eosinophil transcript
levels (right).
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Extended Data Fig. 9 |. Potential mechanisms of tissue-specific diseases.

a, Histogram of the number of Mendelian diseases showing symptoms in a number of
tissues. b, Test for enrichment of causal proteins associated with tissue-specific Mendelian
diseases to interact with TiP proteins of affected tissues. c, Network neighborhood of
uniformly expressed causal proteins of tissue-specific diseases found to interact with TiP
proteins in HuRI, indicating PPI perturbation by mutations. d, Causal genes split by
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mutation found to perturb PPI to TiP protein (dashed) or not (solid). e, Expression profile of
PNKP and interactors in brain tissues and PPI perturbation pattern of disease causing
(Glu326Lys) and benign (Pro20Ser) mutation. Yeast growth phenotypes on SC-Leu-Trp
(upper) or SC-Leu-Trp-His+3AT media (lower) are shown; green/grey gene symbols:
preferentially/not expressed.

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Extended Data Fig. 10 |. Mutations in uniformly expressed causal proteins associated with tissue-
specific Mendelian diseases perturb interactions to TiP proteins.
Expression profile and interaction perturbation profile of nine causal proteins and their
interaction partners. Affected tissues were selected for display (top). Control of AD and DB
(Gal 4 DNA binding domain) plasmid presence and cell density by spotting yeast colonies
on SC-Leu-Trp media (upper). Detection of PPIs by spotting yeast on SC-Leu-Trp-His+3AT
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media (lower), where yeast growth indicates PPIs. WT = wild-type, red letters = causal
proteins or alleles, grey gene symbols = interaction partners not expressed in affected
tissues, grey alleles = not pathogenic, green gene symbols = TiP interaction partners in
affected tissues.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

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 Luck et al. Page 25

Authors
Author Manuscript

Katja Luck1,2,3,33, Dae-Kyum Kim1,4,5,6,33, Luke Lambourne1,2,3,33, Kerstin

Spirohn1,2,3,33, Bridget E. Begg1,2,3, Wenting Bian1,2,3, Ruth Brignall1,2,3, Tiziana
Cafarelli1,2,3, Francisco J. Campos-Laborie7,8, Benoit Charloteaux1,2,3, Dongsic
Choi9, Atina G. Coté1,4,5,6, Meaghan Daley1,2,3, Steven Deimling10, Alice
Desbuleux1,2,3,11, Amélie Dricot1,2,3, Marinella Gebbia1,4,5,6, Madeleine F.
Hardy1,2,3, Nishka Kishore1,4,5,6, Jennifer J. Knapp1,4,5,6, István A. Kovács1,12,13,
Irma Lemmens14,15, Miles W. Mee4,5,16, Joseph C. Mellor1,4,5,6,17, Carl Pollis1,2,3,
Carles Pons18, Aaron D. Richardson1,2,3, Sadie Schlabach1,2,3, Bridget Teeking1,2,3,
Anupama Yadav1,2,3, Mariana Babor1,4,5,6, Dawit Balcha1,2,3, Omer Basha19,20,
Christian Bowman-Colin2,3, Suet-Feung Chin21, Soon Gang Choi1,2,3, Claudia
Colabella22,23, Georges Coppin1,2,3,11, Cassandra D’Amata10, David De
Ridder1,2,3, Steffi De Rouck14,15, Miquel Duran-Frigola18, Hanane Ennajdaoui1,4,5,6,
Author Manuscript

Florian Goebels4,5,16, Liana Goehring2,3, Anjali Gopal1,4,5,6, Ghazal Haddad1,4,5,6,

Elodie Hatchi2,3, Mohamed Helmy4,5,16, Yves Jacob24,25, Yoseph Kassa1,2,3,
Serena Landini2,3, Roujia Li1,4,5,6, Natascha van Lieshout1,4,5,6, Andrew
MacWilliams1,2,3, Dylan Markey1,2,3, Joseph N. Paulson26,27,28, Sudharshan
Rangarajan1,2,3, John Rasla1,2,3, Ashyad Rayhan1,4,5,6, Thomas Rolland1,2,3,
Adriana San-Miguel1,2,3, Yun Shen1,2,3, Dayag Sheykhkarimli1,4,5,6, Gloria M.
Sheynkman1,2,3, Eyal Simonovsky19,20, Murat Taşan1,4,5,6,16, Alexander Tejeda1,2,3,
Vincent Tropepe10, Jean-Claude Twizere11, Yang Wang1,2,3, Robert J. Weatheritt4,
Jochen Weile1,4,5,6,16, Yu Xia1,29, Xinping Yang1,2,3, Esti Yeger-Lotem19,20, Quan
Zhong1,2,3,30, Patrick Aloy18,31, Gary D. Bader4,5,16, Javier De Las Rivas7,8,
Suzanne Gaudet1,2,3, Tong Hao1,2,3, Janusz Rak9, Jan Tavernier14,15, David E.
Hill1,2,3,*, Marc Vidal1,2,*, Frederick P. Roth1,4,5,6,16,32,*, Michael A. Calderwood1,2,3,*
Author Manuscript

Affiliations
1Center
for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston,
MA, USA.
2Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA,
USA.
3Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
4The Donnelly Centre, University of Toronto, Toronto, ON, Canada.
5Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
6Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON,
Author Manuscript

Canada.
7Cancer Research Center (CiC-IBMCC, CSIC/USAL), Consejo Superior de
Investigaciones Científicas (CSIC) and University of Salamanca (USAL),
Salamanca, Spain.
8Institute for Biomedical Research of Salamanca (IBSAL), Salamanca, Spain.

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 Luck et al. Page 26

9TheResearch Institute of the McGill University Health Centre, Montreal, QC,

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Canada.
10Department of Cell and Systems Biology, University of Toronto, Toronto, ON,
Canada.
11MolecularBiology of Diseases, Groupe Interdisciplinaire de Génomique Appliquée
(GIGA) and Laboratory of Viral Interactomes, University of Liège, Liège, Belgium.
12Network Science Institute, Northeastern University, Boston, MA, USA.
13WignerResearch Centre for Physics, Institute for Solid State Physics and Optics,
Budapest, Hungary.
14Centerfor Medical Biotechnology, Vlaams Instituut voor Biotechnologie (VIB),
Ghent, Belgium.
Author Manuscript

15Cytokine Receptor Laboratory (CRL), Department of Biomolecular Medicine,

Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium.
16Department of Computer Science, University of Toronto, Toronto, ON, Canada.
17seqWell, Inc. Beverly MA, USA.
18Institute
for Research in Biomedicine (IRB Barcelona), The Barcelona Institute for
Science and Technology, Barcelona, Catalonia, Spain.
19Department of Clinical Biochemistry and Pharmacology, Faculty of Health
Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
20National
Institute for Biotechnology in the Negev, Ben-Gurion University of the
Negev, Beer-Sheva, Israel.
Author Manuscript

21CRUK Cambridge Institute, University of Cambridge, Cambridge, UK.

22Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.
23Istituto
Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati”,
Perugia, Italy.
24Département de Virologie, Unité de Génétique Moléculaire des Virus à ARN
(GMVR), Institut Pasteur, UMR3569, Centre National de la Recherche Scientifique
(CNRS), Paris, France.
25Université Paris Diderot, Paris, France.
26Department of Biostatistics and Computational Biology, Dana-Farber Cancer
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Institute, Boston, MA, USA.

27Department of Biostatistics, Harvard School of Public Health, Boston, MA, USA.
28Department of Biostatistics, Product Development, Genentech Inc., South San
Francisco, CA, USA.
29Department of Bioengineering, McGill University, Montreal, QC, Canada.
30Department of Biological Sciences, Wright State University, Dayton, OH, USA.

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 Luck et al. Page 27

31Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia,

Author Manuscript

Spain.
32Canadian Institute for Advanced Research (CIFAR), Toronto, ON, Canada.
33These authors contributed equally: Katja Luck, Dae-Kyum Kim, Luke Lambourne,
Kerstin Spirohn.

Acknowledgements
This paper is dedicated to the memory of Dr. Deborah Allinger. We thank Pablo Porras Millan and the IntAct team
for their help in disseminating our PPI data via IntAct, pre- and post-publication. We thank Ulrich Braunschweig,
Jonathan Ellis, and Benjamin J. Blencowe for help with data analysis. We also thank Qian Zhu and Olga G.
Troyanskaya as well as Joshua Pan and Cigall Kadoch for sharing co-expression and co-fitness data, respectively.
We thank Katharine S. Tuttle for help with graphics. This work was primarily supported by the National Institute of
Health (NIH) National Human Genome Research Institute (NHGRI) grant U41HG001715 (M.V., F.P.R., D.E.H.,
M.A.C., G.D.B. and J.T.) with additional support from NIH grants P50HG004233 (M.V. and F.P.R.),
Author Manuscript

U01HL098166 (M.V.), U01HG007690 (M.V.), R01GM109199 (M.A.C.), Canadian Institute for Health Research
(CIHR) Foundation Grants (F.P.R. and J.Rak.), the Canada Excellence Research Chairs Program (F.P.R.) and an
American Heart Association grant 15CVGPS23430000 (M.V.). D.-K.K. was supported by a Banting Postdoctoral
Fellowship through the Natural Sciences and Engineering Research Council (NSERC) of Canada and by the Basic
Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of
Education (2017R1A6A3A03004385). C.Pon. was supported by a Ramon Cajal fellowship (RYC-2017-22959).
G.M.S. was supported by NIH Training Grant T32CA009361. M.V. is a Chercheur Qualifié Honoraire from the
Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

Code availability
Analysis code is available at github.com/CCSB-DFCI/HuRI_paper

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Fig. 1 |. Generation of a reference interactome map using a panel of binary assays.

a, Overview of HuRI generation. b, Schematic of the Y2H assay versions. c, Experimental
validation. Lit-BM: literature-curated binary PPIs with multiple evidence; RRS: random
protein pairs. Error bars are 68.3% Bayesian confidence interval, n = 2,281, 383, 475
(MAPPIT) 1,639, 382, 465 (GPCA). d, Number of PPIs and proteins, detected with each
additional screen. e, Fraction of direct contact pairs among five PPI networks. Error bar is
standard error of proportion, n = 121, 410, 1,169, 584, 1,211 PPIs. f, Number of PPIs
identified over time from screening at CCSB and Lit-BM.
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Fig. 2 |. Complementary functional relationships in HuRI between genes.

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a, Enrichment of HuRI and its profile similarity network (PSN) for protein pairs with shared
functional annotation, showing mean and 95% interval of 100 random networks. b,
Functional modules in HuRI and its PSN and in previously published interactome maps
from CCSB.
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Fig. 3 |. Unbiased proteome coverage of HuRI reveals uncharted network neighborhoods of

disease-related genes.
a, Heatmaps of Y2H PPI counts, ordered by number of publications. b, Fraction of HuRI
PPIs in Lit-BM, for increasing values of the minimum number of publications per protein.
Error bar is standard error of proportion, n = 52,569–170 PPIs. c, Fraction of genes with at
least one PPI for biomedically interesting genes. d, As a, but restricted to PPIs involving
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genes from the indicated gene sets. e, Correlation between degree and variables of interest,
before (top) and after (bottom) correcting for the technical confounding factors (n = 13,441–
53,704 PPIs per network, two-tailed permutation test).
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Fig. 4 |. Identification of potential recruiters of proteins into extracellular vesicles.

a, Schematic of experimental design to test EV recruitment function of proteins. MS: Mass
Spectrometry. b, Protein abundance from EVs for each gene in WT (wild-type) and SDCBP
KO (knockout). Mean values of n = 3 biological replicates.
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Fig. 5 |. Tissue-specific functions are largely mediated by interactions between TiP proteins and
uniformly expressed proteins.
a, Tissue-preferentially expressed (TiP) protein coverage by PPI networks for increasing
levels of tissue-preferential expression (shaded error bands proportional to standard error on
proportion, n ≥ 233 genes). b, Tissue-preferential sub-networks. *P < 0.001, 1-sided
empirical test for TiP proteins being close to each other (n = 19,960–30,217 PPIs per
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subnetwork). c, Empirical test of closeness of TiP proteins in the brain sub-network, 1,000
random networks. d, Tissue-specific diseases split by tissue-preferential expression levels of
causal genes. e, Tested tissue-specific diseases split by PPI perturbation result. f, Expression
profile of PNKP and interactors in brain tissues and PPI perturbation pattern of disease
causing (Glu326Lys) and benign (Pro20Ser) mutation. Yeast growth phenotypes on SC-Leu-
Trp (upper) or SC-Leu-Trp-His+3AT (3-Amino-1,2,4-triazole) media (lower). Green gene
symbols: preferentially expressed. Only interactors expressed in brain shown.

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