Domain Fragment Research Articles

Preformed mincle dimers stabilized by an interchain disulfide bond in the neck region.

The sugar-binding receptor mincle stimulates macrophages when it encounters surface glycans on pathogens, such as trehalose dimycolate glycolipid in the outer membrane of mycobacteria. Binding of oligosaccharide ligands to the extracellular C-type carbohydrate-recognition domain (CRD) in mincle initiates intracellular signaling through the common Fc receptor γ (FcRγ) adapter molecule associated with mincle. One potential mechanism for initiation of signaling involves clustering of receptors, so it is important to understand the oligomeric state of mincle. Affinity purification of mincle from transfected mammalian cells has been used to show that mincle exists as a pre-formed, disulfide-linked dimer. Deletion of cysteine residues and chemical crosslinking further demonstrate that the dimers of mincle are stabilized by a disulfide bond between cysteine residues in the neck sequence that links the CRD to the membrane. In contrast, cysteine residues in the transmembrane region of mincle are not required for dimer formation or association with FcRγ. A protocol has been developed for efficient production of a disulfide-linked extracellular domain fragment of mincle in a bacterial expression system by appending synthetic dimerization domains to guide dimer formation in the absence of the membrane anchor.

DNA-Based Molecular Clamp for Probing Protein Interactions and Structure under Force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology, and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA-based device in which the transition between single- and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension, while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Pan-beta-coronavirus subunit vaccine prevents SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV challenge.

Coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, the respective causative agents of coronavirus disease 2019, SARS, and MERS, continually threaten human health. The spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are critical vaccine targets. Nevertheless, the highly mutated RBD of SARS-CoV-2 variants, especially Omicron, significantly reduces the efficacy of current vaccines against SARS-CoV-2 variants. Here a protein-based pan-beta-CoV subunit vaccine is designed by fusing the potent and conserved RBD of MERS-CoV into an RBD-truncated Omicron S protein. The resulting vaccine maintained effective functionality and antigenicity, induced broadly neutralizing antibodies against all of these highly pathogenic human CoVs, and elicited Omicron S-specific cellular immune responses, protecting immunized mice from SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV infections. Taken together, this study rationally designed a pan-beta-CoV subunit vaccine with broad-spectrum efficacy, which has the potential for development as an effective universal vaccine against SARS-CoV-2 variants and other CoVs with pandemic potential.

Phase and defect structures of MnO2‐doped 75BiFeO3–25BaTiO3 ceramics and their effects on electrical properties

AbstractBismuth ferrite (BiFeO3)‐based high‐temperature piezoelectric ceramics have high Curie temperatures and excellent thermal stability; however, their applications are limited by inadequate piezoelectric performance due to large coercive fields and high leakage conduction. This study investigates the impact of MnO2 doping on the phase transition, defect structure, domain morphology, as well as the dielectric, ferroelectric, and electrical conduction behaviors of 75BiFeO3–25BaTiO3 ceramics sintered under an oxygen atmosphere. A structural transformation from distorted rhombohedral to pseudo‐cubic symmetry was observed with increasing MnO2 content, accompanied by domain fragmentation. MnO2 doping at an appropriate level inhibited defects such as and . Regarding electrical properties, the reduction in rhombohedral distortion and enhancement in the pseudo‐cubic phase after MnO2 doping resulted in decreased remanent polarization and electrostrain, which could potentially hinder piezoelectric response. However, appropriate MnO2 doping effectively reduced leakage conduction, significantly enhancing the poling efficiency of the ceramics. The maximum d33 value of 107 pC/N and kp of 0.36 were achieved at an MnO2 doping content of 0.1 wt.%. Excessive MnO2 addition may generate defect dipoles, as indicated by increased coercive field (Ec) and dielectric loss. The observed increase in high‐temperature resistivity with MnO2 content is primarily attributed to reduced grain size and increased concentration of mobile defects. This study provides valuable insights for further research on the application of 75BiFeO3–25BaTiO3 systems in high‐temperature piezoelectric devices.

Helical superstructures between amyloid and collagen in cardiac fibrils from a patient with AL amyloidosis

Systemic light chain (LC) amyloidosis (AL) is a disease where organs are damaged by an overload of a misfolded patient-specific antibody-derived LC, secreted by an abnormal B cell clone. The high LC concentration in the blood leads to amyloid deposition at organ sites. Indeed, cryogenic electron microscopy (cryo-EM) has revealed unique amyloid folds for heart-derived fibrils taken from different patients. Here, we present the cryo-EM structure of heart-derived AL amyloid (AL59) from another patient with severe cardiac involvement. The double-layered structure displays a u-shaped core that is closed by a β-arc lid and extended by a straight tail. Noteworthy, the fibril harbours an extended constant domain fragment, thus ruling out the variable domain as sole amyloid building block. Surprisingly, the fibrils were abundantly concatenated with a proteinaceous polymer, here identified as collagen VI (COLVI) by immuno-electron microscopy (IEM) and mass-spectrometry. Cryogenic electron tomography (cryo-ET) showed how COLVI wraps around the amyloid forming a helical superstructure, likely stabilizing and protecting the fibrils from clearance. Thus, here we report structural evidence of interactions between amyloid and collagen, potentially signifying a distinct pathophysiological mechanism of amyloid deposits.

In vivo CRISPR base editing for treatment of Huntington's disease.

Huntington's disease (HD) is an inherited and ultimately fatal neurodegenerative disorder caused by an expanded polyglutamine-encoding CAG repeat within exon 1 of the huntingtin (HTT) gene, which produces a mutant protein that destroys striatal and cortical neurons. Importantly, a critical event in the pathogenesis of HD is the proteolytic cleavage of the mutant HTT protein by caspase-6, which generates fragments of the N-terminal domain of the protein that form highly toxic aggregates. Given the role that proteolysis of the mutant HTT protein plays in HD, strategies for preventing this process hold potential for treating the disorder. By screening 141 CRISPR base editor variants targeting splice elements in the HTT gene, we identified platforms capable of producing HTT protein isoforms resistant to caspase-6-mediated proteolysis via editing of the splice acceptor sequence for exon 13. When delivered to the striatum of a rodent HD model, these base editors induced efficient exon skipping and decreased the formation of the N-terminal fragments, which in turn reduced HTT protein aggregation and attenuated striatal and cortical atrophy. Collectively, these results illustrate the potential for CRISPR base editing to decrease the toxicity of the mutant HTT protein for HD.

A DNA-based molecular clamp for probing protein interactions and structure under force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Plant-Derived Anti-Human Epidermal Growth Factor Receptor 2 Antibody Suppresses Trastuzumab-Resistant Breast Cancer with Enhanced Nanoscale Binding.

Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitro assays, and in vivo experiments uncover the antibody's nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody's potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

Optimizing recombinant antibody fragment production: A comparison of artificial intelligence and statistical modeling.

Maximizing the recombinant protein yield necessitates optimizing the production medium. This can be done using a variety of methods, including the conventional "one-factor-at-a-time" approach and more recent statistical and mathematical methods such as artificial neural network (ANN), genetic algorithm, etc. Every approach has advantages and disadvantages of its own, yet even when a technique has flaws, it is nevertheless used to get the best results. Here, one categorical variable and four numerical parameters, including post-induction time, inducer concentration, post-induction temperature, and pre-induction cell density, were optimized using the 232 experimental assays of the central composite design. The direct and indirect effects of factors on the yield of anti-epithelial cell adhesion molecule extracellular domain fragment antibody were examined using statistical methods. The analysis of variance results indicate that the response surface methodology (RSM) model is effective in predicting the amount of produced single-chain fragment variable (p-value = 0.0001 and R2 = 0.905). For ANN modeling, the evaluation using normalized root mean square error (NRMSE) and R2 values shows a good fit (R2 = 0.942) and accurate predictions (NRMSE = 0.145). The analysis of error parameters and R2 of a dataset, which contained 30 data points randomly selected from the complete dataset, showed that the ANN model had a higher R2 value (0.968) compared to the RSM model (0.932). Furthermore, the ANN model demonstrated stronger predictive ability with a lower NRMSE (0.048vs. 0.064). Induction at the cell density of 0.7 and an isopropyl β-D-1-thiogalactopyranoside concentration of 0.6mM for 32 h at 30°C in BW25113 was the ideal culture condition leading to the protein yield of 259.51mg/L. Under the optimum conditions, the output values predicted by the ANN model (259.83mg/L) were more in line with the experimental data (259.51mg/L) than the RSM (276.13mg/L) expected value. This outcome demonstrated that the ANN model outperforms the RSM in terms of prediction accuracy.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa’s different domains could potentially be helpful in the rational design of improved toxins.In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

Opinion inertia and coarsening in the persistent voter model.

We consider the persistent voter model (PVM), a variant of the voter model (VM) that includes transient, dynamically induced zealots. Due to peer reinforcement, the internal confidence η_{i} of a normal voter increases in steps of size Δη. Once it surpasses a given threshold, it becomes a zealot. Its opinion remains frozen until enough interactions with the opposing opinion occur, resetting its confidence. No longer a zealot, the regular voter may change opinion once again. This mechanism of opinion inertia, though simplified, is responsible for an effective surface tension, and the PVM exhibits a crossover from a fluctuation-driven dynamics, as in the VM, to a curvature-driven one, akin to the Ising model at low temperature. The average time τ to attain consensus is nonmonotonic with respect to Δη and reaches a minimum at Δη_{min}. In this paper we elucidate the mechanisms that accelerate the system towards consensus close to Δη_{min}. Near the crossover at Δη_{min}, the intermediate region around the domains where the regular voters accumulate (the active region, AR) is large. The surface tension, albeit small, is sufficient to maintain the shape and reduce the domain fragmentation. The large size of the AR in the region of Δη_{min} has two important effects that accelerate the dynamics. First, it dislodges the zealots within the bulk of the domains. Secondly, it maximally suppresses the formation of slowly evolving stripes typical in Ising-like models. This suggests the importance of understanding the role of the AR, where opinion changes are facilitated, and the interplay between regular voters and zealots in disrupting polarized states.

Abstract 1665: Investigating Daple-FLT3 gene fusion in leukemia cells and its response to kinase inhibitors

Abstract Gene fusions are frequently found in leukemia and other cancers. The signaling scaffold protein, Daple (CCDC88C), has been found to undergo gene fusion to the receptor tyrosine kinase, FLT3. Specifically, a fragment of the coiled-coil domain from Daple and tyrosine kinase domain (TKD) of FLT3 are found fused together. How the gene product from this gene fusion functions in rewiring the signaling network in cells and whether this gene product can be targeted though pharmacological inhibitions remain unclear. Cell surface receptors, such as receptor tyrosine kinases (RTKs), activate cell signaling and genetic mutations that lead to constitutive receptor activation can promote unregulated cell proliferation, survival, and migration. Leukemia patients with hyperactive RTKs are treated with tyrosine kinase inhibitors (TKIs), such as Imatinib and Sorafenib. Here we demonstrate how TKIs can directly block the activation of Daple-FLT3 and prevent hyperactivation of its downstream pathways. Our studies unveil a strategy to identify other compounds which may serve as an additive, or alternative, approach to treating patients with Daple gene fusion. Citation Format: Elena Nguyen, Jason Ear. Investigating Daple-FLT3 gene fusion in leukemia cells and its response to kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1665.

Fragmentation of stability domains of dark solitons, dark breathers, and drifting solitons at high pump intensities in normal-dispersion Kerr microresonators

Fragmentation of stability domains of dark solitons, dark breathers, and drifting solitons at high pump intensities in normal-dispersion Kerr microresonators

Evaluation of PDL1 positive cancer cell-specific binding activity of recombinant anti-PDL1 scFv.

Programmed cell death-ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single-chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof-of-concept data for the effect of scFv using PDL1-expressing cells was lacking. In this study, we conducted two kinds of cell-based immunoassays, western blotting and enzyme-linked immunosorbent assay, using anti-PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell-based immunoassays to detect PDL1.

Antiplatelet effects of the CEACAM1-derived peptide QDTT

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) restricts platelet activation via platelet collagen receptor GPVI/FcRγ-chain. In this study, screening against collagen-induced platelet aggregation was performed to identify functional CEACAM1 extracellular domain fragments. CEACAM1 fragments, including Ala-substituted peptides, were synthesized. Platelet assays were conducted on healthy donor samples for aggregation, cytotoxicity, adhesion, spreading, and secretion. Mice were used for tail bleeding and FeCl3-induced thrombosis experiments. Clot retraction was assessed using platelet-rich plasma. Extracellular segments of CEACAM1 and A1 domain-derived peptide QDTT were identified, while N, A2, and B domains showed no involvement. QDTT inhibited platelet aggregation. Ala substitution for essential amino acids (Asp139, Thr141, Tyr142, Trp144, and Trp145) in the QDTT sequence abrogated collagen-induced aggregation inhibition. QDTT also suppressed platelet secretion and “inside-out” GP IIb/IIIa activation by convulxin, along with inhibiting PI3K/Akt pathways. QDTT curtailed FeCl3-induced mesenteric thrombosis without significantly prolonging bleeding time, implying the potential of CEACAM1 A1 domain against platelet activation without raising bleeding risk, thus paving the way for novel antiplatelet drugs.

Mechanisms by which Factor H protects Trypanosoma cruzi from the alternative pathway of complement.

Chagas disease, a chronic disabling disease caused by the protozoan Trypanosoma cruzi, has no standardized treatment or preventative vaccine. The infective trypomastigote form of T. cruzi is highly resistant to killing by the complement immune system. Factor H (FH), a negative regulator of the alternative pathway (AP) of complement on cell surfaces and in blood, contains 20 short consensus repeat domains. The four N-terminal domains of FH inactivate the AP, while the other domains interact with C3b/d and glycan markers on cell surfaces. Various pathogens bind FH to inactivate the AP. T. cruzi uses its trans-sialidase enzyme to transfer host sialic acids to its own surface, which could be one of the approaches it uses to bind FH. Previous studies have shown that FH binds to complement-opsonized T. cruzi and parasite desialylation increases complement-mediated lysis of trypomastigotes. However, the molecular basis of FH binding to T. cruzi remain unknown. Only trypomastigotes, but not epimastigotes (non-infective, complement susceptible) bound FH directly, independent of C3 deposition, in a dose-dependent manner. Domain mapping experiments using 3-5 FH domain fragments showed that domains 5-8 competitively inhibited FH binding to the trypomastigotes by ~35% but did not decrease survival in complement. FH-Fc or mutant FH-Fc fusion proteins (3-11 contiguous FH domains fused to the IgG Fc) also did not kill trypomastigotes. FH-related protein-5, whose domains bear significant sequence identity to all known polyanion-binding FH domains (6-7, 10-14, 19-20), fully inhibited FH binding to trypomastigotes and reduced trypomastigote survival to < 24% in the presence of serum. In conclusion, we have elucidated the role of FH in complement resistance of trypomastigotes.

Genetic analysis of the PAPP-A2 gene and evaluation of free IGF-1, IGFBP-5, and ALS concentrations in a group of 22 patients with idiopathic short stature.

Short stature is one of the main reasons for consultation in outpatient clinics and paediatric endocrinology departments and is defined as height below the 3rd centile or less than -2 standard deviations (SDs). The study's overarching aim was to analyse the PAPP-A2 gene at mutation sites described to date and at exons 3, 4, and 5, which encode the fragment of the catalytic domain with the active site of the pregnancy-associated plasma protein A2 (PAPP-A2) protein. The secondary aims of the study were clinical and auxological analysis of a group of patients with idiopathic short stature and biochemical analysis of growth hormone-insulin-like growth factor-1 (GH-IGF-1) axis parameters not assessed as part of the routine diagnosis of short stature, such as free IGF-1, insulin-like growth factor binding protein 5 (IGFBP-5), and acid-labile subunit (ALS) levels. Molecular analysis of the PAPP-A2 gene was performed using polymerase chain reaction (PCR) and direct sequencing. Biochemical analysis of free IGF-1, IGFBP-5, and ALS was performed by enzyme-linked immunosorbent assay (ELISA). The mean height standard deviation score (HSDS) in the study group was -2.95. None of the patients exhibited previously described mutations in the PAPP-A2 gene or mutations in exons 3, 4, and 5 encoding the fragment of catalytic domain with the active site of the PAPP-A2 protein. In 4 patients, the known, non-pathogenic, heterozygotic polymorphism c.2328C>T(rs10913241) in exon 5 was found. Free IGF-1 levels correlate better with height and HSDS than total IGF-1 levels. The previously described mutations in the PAPP-A2 gene and mutations in exons 3, 4, and 5 encoding the fragment of catalytic domain with the active site of the PAPP-A2 protein were not detected; only the known and non-pathogenic, heterozygotic polymorphism c.2328C>T(rs10913241) in exon 5 of the PAPP-A2 gene was observed.

SLIT Loss or Sequestration Increases Mammary Alveologenesis and Lactogenesis.

SLITs comprise a family of secreted proteins that function as ligands for Roundabout (ROBO) receptors. Previous research showed that ROBO1 promotes the differentiation of milk-producing alveolar cells by inhibiting Notch signaling in mammary luminal cells. Here, we show enhanced alveolar development and increased milk production in Slit2-/-;Slit3-/- knockout mammary gland epithelia. This result can also be achieved by intraperitoneal delivery of recombinant ROBO1 extracellular domain fragment, ROBO1-5Ig-Fc, which sequesters SLITs. Together, our phenotypic studies suggest that SLITs restrict alveologenesis and lactogenesis by inhibiting ROBO1.

Crystal structures of human immune protein FIBCD1 suggest an extended binding site compatible with recognition of pathogen-associated carbohydrate motifs

Fibrinogen C-domain containing-1 (FIBCD1) is an immune protein proposed to be involved in host recognition of chitin on the surface of pathogens. As FIBCD1 readily binds to acetylated molecules, we have determined the high-resolution crystal structures of a recombinant fragment of the FIBCD1 C-terminal domain complexed with small N-acetyl-containing ligands to determine the mode of recognition. All ligands bind at the conserved N-acetyl binding site (S1) with galactose and glucose-derived ligands rotated 180⁰ with respect to each other. One subunit of a native structure derived from protein expressed in mammalian CHO cells binds glycosylation from a neighbouring subunit, in an extended binding site. Across the various structures, the primary S1 binding pocket is occupied by N-acetyl-containing ligands or acetate, with N-acetyl, acetate or a sulfate ion in an adjacent pocket S1(2). Inhibition binding studies of N-acetylglucosamine oligomers, (GlcNAc)n, n=1,2,3,5,11, via ELISA along with Microscale Thermophoresis affinity assays indicate a strong preference of FIBCD1 for longer N-acetylchitooligosaccharides. Binding studies of mutant H396A, located beyond the S1(2) site, showed no significant difference to wildtype but K381L, within the S1(2) pocket, blocked binding to the model ligand acetylated bovine serum albumin suggesting that this pocket may have functional importance in FIBCD1 ligand binding. The binding studies, alongside structural definition of diverse N-acetyl monosaccharide binding in the primary S1 pocket and of additional, adjacent binding pockets, able to accommodate both carbohydrate and sulfate functional groups, suggests a versatility in FIBCD1 to recognise chitin oligomers and other pathogen-associated carbohydrate motifs across an extended surface.

BOD1L Facilitates Chromatin Binding of SETD1A and Promotes Leukemia Cell Growth

The H3K4 methyltransferase SETD1A plays an essential role in leukemia through a non-catalytic activity which regulates transcriptional pause release of RNA polymerase II. SETD1A localizes to the transcriptional start site of numerous actively transcribed genes, but primarily regulates the expression of a restricted set of genes within the DNA damage response as well as heme biosynthesis pathway. However, the roles of upstream regulators or essential components of SETD1A chromatin binding remain unclear. Using the DepMap database and a CRISPR-tiling screen method, we found that BOD1L is the most correlated co-dependency with SETD1A in human cancer cell lines and that the Shg1 domain of BOD1L plays an indispensable role in human MOLM-13 leukemia cell line. BOD1L knockout increases apoptosis and reduces cell cycle. The strong BOD1L dependency in AML cells was also confirmed using a mouse MLL-r leukemia model in vitro and in vivo. SETD1A catalytic domain mutant AML cells still responded to the BOD1L sgRNAs that suggests the role of BOD1L is independent from the SETD1A catalytic function. Importantly, BOD1L knockout mimics the transcriptional profiles of SETD1A knockout cells and reduces the expression of genes in DNA damage response and heme biosynthesis. BOD1L loss immediately reduces SETD1A distribution on chromatin and increased the NELFE and RNA polymerase II signals at transcriptional start sites. These results show that BOD1L is an essential component of the non-catalytic SETD1A function in transcriptional pause release. We also performed an immunoprecipitation assay using tagged constructs to examine the interaction between BOD1L and SETD1A. Our data indicate that SETD1A associates with BOD1L through the FLOS domain, especially in the C-terminal region. In addition, we found that the loop structure in the Shg1 domain of BOD1L is associated with the nonenzymatic domain of SETD1A. A split luciferase complementation assay reveals that the BOD1L Shg1 domain and SETD1A FLOS domain interact. To demonstrate the role of BOD1L chromatin interaction, we enforced localization of the Shg1 domain fragment at a silent gene locus and it successfully induced ectopic SETD1A recruitment to chromatin and formation of the COMPASS complex. However, we observed neither induced H3K4me3 modification nor RNA polymerase II recruitment. Taken together, while the BOD1L-SETD1A complex is not sufficient to act as a pioneer complex for active transcription, our study identified that BOD1L facilitates chromatin binding of SETD1A through the Shg1-FLOS domain interaction and then regulates transcriptional pause release, and that this interaction is a potential therapeutic opportunity for SETD1A-targeting therapy.