Human transcription factor protein interaction networks

Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here the authors identify 6703 and 1536 protein-protein interactions for 109 different human TFs through BioID and AP-MS analyses, respectively. Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein interactions for 109 different human TFs through proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS), respectively. The BioID analysis identifies more high-confidence interactions, highlighting the transient and dynamic nature of many of the TF interactions. By performing clustering and correlation analyses, we identify subgroups of TFs associated with specific biological functions, such as RNA splicing or chromatin remodeling. We also observe 202 TF-TF interactions, of which 118 are interactions with nuclear factor 1 (NFI) family members, indicating uncharacterized cross-talk between NFI signaling and other TF signaling pathways. Moreover, TF interactions with basal transcription machinery are mainly observed through TFIID and SAGA complexes. This study provides a rich resource of human TF interactions and also act as a starting point for future studies aimed at understanding TF-mediated transcription.Peer reviewe

CDNF Interacts with ER Chaperones and Requires UPR Sensors to Promote Neuronal Survival

Cerebral dopamine neurotrophic factor (CDNF) is a neurotrophic factor that has beneficial effects on dopamine neurons in both in vitro and in vivo models of Parkinson’s disease (PD). CDNF was recently tested in phase I-II clinical trials for the treatment of PD, but the mechanisms underlying its neuroprotective properties are still poorly understood, although studies have suggested its role in the regulation of endoplasmic reticulum (ER) homeostasis and the unfolded protein response (UPR). The aim of this study was to investigate the mechanism of action of CDNF through analyzing the involvement of UPR signaling in its anti-apoptotic function. We used tunicamycin to induce ER stress in mice in vivo and used cultured primary neurons and found that CDNF expression is regulated by ER stress in vivo and that the involvement of UPR pathways is important for the neuroprotective function of CDNF. Moreover, we used AP-MS and BiFC to perform the first interactome screening for CDNF and report novel binding partners of CDNF. These findings allowed us to hypothesize that CDNF protects neurons from ER-stress-inducing agents by modulating UPR signaling towards cell survival outcomes

Pharmacologically diverse antidepressants facilitate TRKB receptor activation by disrupting its interaction with the endocytic adaptor complex AP-2

Several antidepressant drugs activate tropomyosin-related kinase B (TRKB) receptor, but it remains unclear whether these compounds employ a common mechanism for TRKB activation. Here, using MS, we found that a single intraperitoneal injection of fluoxetine disrupts the interaction of several proteins with TRKB in the hippocampus of mice. These proteins included members of adaptor protein complex-2 (AP-2) involved in vesicular endocytosis. The interaction of TRKB with the cargo-docking ? subunit of the AP-2 complex (AP2M) was confirmed to be disrupted by both acute and repeated fluoxetine treatments. Of note, fluoxetine disrupted the coupling between full-length TRKB and AP2M, but not the interaction between AP2M and the TRKB C-terminal region, indicating that the fluoxetine-binding site in TRKB lies outside the TRKB:AP2M interface. ELISA experiments revealed that in addition to fluoxetine, other chemically diverse antidepressants, such as imipramine, rolipram, phenelzine, ketamine, and its metabolite 2R,6R-hydroxynorketamine, also decreased the interaction between TRKB and AP2M in vitro. Silencing the expression of AP2M in a TRKB-expressing mouse fibroblast cell line (MG87.TRKB) increased cell-surface expression of TRKB and facilitated its activation by brain-derived neurotrophic factor (BDNF), observed as levels of phosphorylated TRKB. Moreover, animals haploinsufficient for the Ap2m1 gene displayed increased levels of active TRKB, along with enhanced cell-surface expression of the receptor in cultured hippocampal neurons. Taken together, our results suggest that disruption of the TRKB:AP2M interaction is a common mechanism underlying TRKB activation by several chemically diverse antidepressants.Peer reviewe

Novel modular chimeric antigen receptor spacer for T cells derived from signal regulatory protein alpha Ig-like domains

Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG’s crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided.Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein α (SIRPα) were designed in silico. An IgG1-based CAR and a CAR lacking both SIRPα and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19+ target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells.Results: T cells carrying the novel SIRPα-based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells. Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon-γ, and production of IL-1β, indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRPα-based CAR T cells with THP-1 cells. Arming T cells with the SIRPα-based CARs favored differentiation towards CD4+ phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRPα- and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRPα-based CARs showed dose dependent leukemia cell control.Conclusion: The novel SIRPα based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy

Background: CCAAT enhancer-binding protein epsilon (C/EBP epsilon) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBP epsilon is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. Objective: The aim of this study was to molecularly characterize the effects of C/EBP epsilon transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. Methods: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. Results: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. Conclusion: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBP epsilon. Mutated C/EBPe acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBP epsilon. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.Peer reviewe

Human Transcription Factor Protein-Protein Interactions in Health and Disease

Transcription factors (TFs) are one of the most important groups of proteins for the development and differentiation of cells. They control the gene expression of all cells in all stages of development. Defects in TF signalling may lead to severely altered development and diseases. However, while TF DNA binding has been widely studied, we are still lacking a systems-level understanding of human TF signalling. TFs’ action in gene expression regulation is highly dependent on their interactions with multiple proteins, such as cofactors, dimerization partners, chromatin modulating proteins, enzymes, inhibitory proteins and general TFs. Therefore, the aim of this study is to shed light on TF protein-protein interactions and, more specifically, to examine the effect of TF mutations found in primary immunodeficiency patients. A comprehensive interactome analysis of 110 TFs revealed over 7,000 TF protein-protein interactions, most of which are nuclear and play a role in transcriptional regulation (I). The large number of TF interactions discovered in this study enabled us to conduct a systems-level analysis that revealed groups of TFs with specific biological functions, such as actin and myosin signalling and RNA splicing. Interestingly, 54 of the TFs studied interacted with the nuclear factor family of TFs. Nuclear factors are known to control a number of genes in development; for instance, they are essential for central nervous system, tooth, brain, skeletal, lung and muscle development. In addition, they are linked to several cancer types. Our data suggest that transcription control by NFIs may be regulated by nuclear factor interactions with other TFs. A219H mutation in the C/EBPε TF was found in a Finnish family with immunodeficiency and autoinflammatory syndrome (II). A data-driven multiomics study of the mutation revealed a novel TF-related disease mechanism; mutation decreased association with transcriptional repressors, increased chromatin binding and widely dysregulated transcription. These changes resulted in disturbed non-canonical inflammasome activation due to the increased expression of NLRP3 and constitutively expressed CASP5. Three different damaging mutations in NFKB1 resulted in diverse immunological phenotypes due to different mechanisms (III): H67R led to decreased nuclear entry, reduced association with RelB and decreased transcriptional activity; I553M led to decreased phosphorylation of S893 and p907 and enhanced p105 subunit degradation upon TNF treatment; and R157X led to an almost total loss of NFKB1 subunits due to proteasome-mediated dominant negative degradation. This study provides valuable information on TF protein-protein interactions at systems level (I). In addition, this study provides examples of how single TF mutation may affect TF signalling on many levels, such as in protein interactions, DNA binding and transcription (II) and how different mutations in the same TF can have different outcomes (III). TFs are downstream players of many signalling cascades and targeting TF protein interactions can offer a high degree of specificity in future therapeutics applications.Transkriptiotekijät eli transkriptiofaktorit (TF:t) ovat tärkeitä proteiineja geenien luennan säätelyssä. Ne vaikuttavat kaikkien solujen toiminnan ylläpidossa ja erilaistumisessa, ja ovat siten välttämättömiä mm. sikiönkehityksessä. Virheet TF:ien signaloinnissa voivat aiheuttaa vakavia kehityshäiriöitä ja sairauksia. Näin ollen TF:ien toiminta soluissa on hyvä ymmärtää mahdollisimman kattavasti, jotta häiriöihin voidaan puuttua esimerkiksi kehittämällä lääkehoitoa. TF:t säätelevät geenien ilmentymistä sitoutumalla perimäainekseen, DNA-juosteeseen, aktivoiden tai estäen kohdegeenien luennan ja valmistuksen aktiivisiksi proteiineiksi. DNA:han sitoutuminen ei kuitenkaan ole tarpeeksi geenien luennan säätelemiseksi, vaan TF:t vuorovaikuttavat useiden muiden proteiinien kanssa halutun vasteen aikaansaamiseksi. Tämän väitöskirjatyön tavoitteena on kartoittaa ihmisen TF:ien proteiinivuorovaikutuksia sekä normaalioloissa että tautitiloissa. Työ koostuu kolmesta osajulkaisusta. Ensimmäisessä osajulkaisussa onnistuimme kartoittamaan solumalleissa yli 7000 proteiinivuorovaikutusta 110 TF:lle. Iso osa näistä vuorovaikutuksista liittyy geenien luennan säätelyyn. Osa TF:stä vuorovaikutti myös erityisten proteiiniryhmien, kuten RNA-silmukointiin liittyvien tai tuma-aktiiniin liittyvien proteiinien, kanssa. Kartoitimme myös TF:ien keskinäisiä vuorovaikutuksia tutkitussa aineistossa ja teimme yllättävän havainnon, että 54 TF:a 110:stä vuorovaikutti Nuclear Factor-perheen (NFI) TF:ien kanssa. Tämä oli mielenkiintoinen löydös, sillä NFI- TF:t ovat välttämättömiä mm. hermoston, hampaiden, aivojen, luuston ja lihasten kehittymisessä sekä ne on yhdistetty usean syövän kehittymiseen. Tulostemme mukaan on mahdollista, että NFI:ien toimintaa säädellään muiden TF:ien kautta. Toisessa osajulkaisussa tutkittiin C/EBPε-TF:n mutaation vaikutuksia soluissa. Mutaatio löydettiin suomalaisesta suvusta, jonka jäsenet kärsivät määrittelemättömästä primääristä immuunipuutoksesta. Solutasolla mutaatio aiheutti laajoja häiriöitä C/EBPε:n toiminassa: virheellinen TF sitoutui enemmän DNA:han, se vuorovaikutti huomattavasti vähemmän TF:ien toimintaa estävien proteiinien kanssa sekä häiritsi yli 460 geenin luentaa. Nämä muutokset johtivat häiriintyneeseen immuunipuolustukseen, mm. yliaktiiviseen non-kanonisen inflammasomin aktitiivisuuteen ja autoimmuunioireisiin. Uusi tauti nimettiin CAIN:ksi (C/EBPε-associated autoinflammation and immune impairment of neutrophils). Kolmannessa osajulkaisussa tutkittiin kolmen erillisen NFKB1 TF-mutaation vaikutuksia primäärissä immuunipuutoksessa kolmessa eri suomalaissuvussa. Mutaatiot eri kohdissa proteiinia vaikuttivat erilaisilla mekanismeilla, mutta jokainen niistä aiheutti virheitä immuunipuolustuksen toimintaan. Kaiken kaikkiaan tämä väitöskirja tarjoaa tärkeän aineiston TF:ien proteiinivuorovaikutuksista, jota voidaan käyttää mm. uusien lääkkeiden ja hoitomuotojen kehittämiseen. Siinä myös kartoitetaan, miten yksittäinen virhe TF:ssa voi aiheuttaa ongelmia monella eri geenien luennan säätelyn tasolla ja miten samassa TF:ssa eri kohdissa olevat mutaatiot voivat aiheuttaa tauteja erilaisilla mekanismeilla

The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naive neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.Peer reviewe

CDNF Interacts with ER Chaperones and Requires UPR Sensors to Promote Neuronal Survival

Cerebral dopamine neurotrophic factor (CDNF) is a neurotrophic factor that has beneficial effects on dopamine neurons in both in vitro and in vivo models of Parkinson's disease (PD). CDNF was recently tested in phase I-II clinical trials for the treatment of PD, but the mechanisms underlying its neuroprotective properties are still poorly understood, although studies have suggested its role in the regulation of endoplasmic reticulum (ER) homeostasis and the unfolded protein response (UPR). The aim of this study was to investigate the mechanism of action of CDNF through analyzing the involvement of UPR signaling in its anti-apoptotic function. We used tunicamycin to induce ER stress in mice in vivo and used cultured primary neurons and found that CDNF expression is regulated by ER stress in vivo and that the involvement of UPR pathways is important for the neuroprotective function of CDNF. Moreover, we used AP-MS and BiFC to perform the first interactome screening for CDNF and report novel binding partners of CDNF. These findings allowed us to hypothesize that CDNF protects neurons from ER-stress-inducing agents by modulating UPR signaling towards cell survival outcomes.Peer reviewe