Cognate Motifs Research Articles

Construction of Strong Promoters by Assembling Sigma Factor Binding Motifs.

Development of strong promoters is of growing interest in the field of biotechnology and synthetic biology. Here we present a protocol for the construction of strong prokaryotic promoters that can be recognized by designated multiple sigma factors by interlocking their cognate binding motifs on DNA strands. Strong and stress responsive promoters for Escherichia coli and Bacillus subtilis have been created following the presented protocol. Customized promoters could be easily developed for fine-tuning gene expression or overproducing enzymes with prokaryotic cell factories.

FOXO1 Dependent Transcription Network Is a Targetable Vulnerability of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) often has an adverse prognosis and despite aggressive multimodal treatment with conventional and targeted therapies, the median survival of MCL patients remains approximately 4 years. Thus, there is a significant unmet need to find novel targets and rational combination treatments.Targeting lineage vulnerabilities driven by specific transcription factors has been broadly confirmed as an effective intervention in many human cancers. To identify the transcription program dependency of MCL cells, we conducted an unbiased domain-focused CRISPR-Cas9 screening against a library of 8,750 sgRNAs targeting 1,434 transcription factors in MCL cell lines (JEKO1, MAVER1, UPN1, and CCMCL1). We identified Forkhead Box O1 (FOXO1), EBF1, PAX5, and IRF4 as 4 transcription factors that are specifically required for MCL survival and growth. Chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis further revealed that the four transcription factors act together to orchestrate B cell lineage transcriptional program and MCL cell survival.Despite its well-recognized role as a tumor suppressor, FOXO1 has been implicated as a lineage specific transcription factor involved in mature B cell development. Genetic studies revealed a critical role of FOXO1 in germinal center dark zone formation and lymphomagenesis, raising the possibility that FOXO1 acts as a master transcription factor for lineage survival transcription program of MCL. Indeed, hierarchical interaction analysis revealed that FOXO1 functions as a pioneer factor that facilitate the chromatin access of other B cell lineage transcription factors. We demonstrated that interaction of FOXO1 to its cognate motif stabilizes B cell transcription factor complex and supports MCL progression. Along this line, we show that enforced expression of FOXO1 in myeloid leukemia cells induces transdifferentiation and B-cell specific gene expression. Mechanistically, we demonstrate through tiling CRISPR scanning screen that forkhead DNA binding and c-terminal transactivation domains of FOXO1 are specifically required for the viability of MCL cells.Given our finding of FOXO1 as a lineage-specific oncogene in MCL, we next explored the possibility of developing FOXO1-tageted inhibitors. We screened a library of potential small molecule inhibitors of FOXO1 (Forkhead BioTherapeutics) and identified cpd10 as one of the most potent and selective FOXO1 inhibitors (IC 50=76 ×/÷ 1.7. nM) Through the CCMCL1 MCL-NSG preclinical model, we found that cpd10 (100 mg/kg/daily) was well tolerated without overt toxicities. Importantly, prolonged treatment induced a robust cytotoxic response of MCL cells and suppressed MCL progression in vivo. Altogether, our findings identified FOXO1 as a MCL lineage-survival oncogene that can be exploited as a therapeutic target of future drug development. DisclosuresCantley: Petra Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Elemento: Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Janssen: Research Funding; Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy. Belvedere: Forkhead BioTherapeutics: Current Employment. Paik: Forkhead BioTherapeutics: Research Funding.

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris.

The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has become a powerful eukaryotic expression platform for biopharmaceutical and biotechnological applications on both laboratory and industrial scales. Despite the fundamental role that artificial transcription factors (ATFs) play in the orthogonal control of gene expression in synthetic biology, a limited number of ATFs are available for P. pastoris. To establish orthogonal regulators for use in P. pastoris, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant Arabidopsis thaliana, in combination with the activation domains of yeast GAL4 and plant EDLL and a synthetic promoter harboring the cognate cis-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in P. pastoris, ranging from as low as 1- to as high as ∼63-fold induction with only small growth defects. We demonstrated the application of ATF/BSs by generating P. pastoris cells that produce β-carotene. Notably, the productivity of β-carotene in P. pastoris was ∼4.8-fold higher than that in S. cerevisiae, reaching ∼59% of the β-carotene productivity obtained in a S. cerevisiae strain optimized for the production of the β–carotene precursor, farnesyl diphosphate, by rewiring the endogenous metabolic pathways using plant-derived ATF/BSs. Our data suggest that plant-derived regulators have a high degree of transferability from S. cerevisiae to P. pastoris. The plant-derived ATFs, together with their cognate binding sites, powerfully increase the repertoire of transcriptional regulatory modules for the tuning of protein expression levels required in metabolic engineering or synthetic biology in P. pastoris.

Molecular mechanism of methyl-dependent and spatial-specific DNA recognition of c-Jun homodimer.

DNA methylation is important in regulation of gene expression and normal development because it alters the interplay between protein and DNA. Experiments have shown that a single 5-methylcytosine at different CpG sites (mCpG) might have different effects on specific recognition, but the atomistic origin and dynamic details are largely unclear. In this work, we investigated the mechanism of monomethylation at different CpG sites in the cognate motif and the cooperativity of full methylation. By constructing four models of c-Jun/Jun protein binding to the 5[Formula: see text]-XGAGTCA-3[Formula: see text] (X represents C or methylated C) motif, we characterized the dynamics of the contact interface using the all-atom molecular dynamics method. Free energy analysis of MM/GBSA suggests that regardless of whether the C12pG13 site of the bottom strand is methylated, the effects from mC25 of the top strand are dominant and can moderately enhance the binding by [Formula: see text] 31 kcal/mol, whereas mC12 showed a relatively small contribution, in agreement with the experimental data. Remarkably, we found that this spatial-specific influence was induced by different regulatory rules. The influence of the mC25 site is mainly mediated by steric hindrance. The additional methyl group leads to the conformational changes in nearby residues and triggers an obvious structural bending in the protein, which results in the formation of a new T-Asn-C triad that enhances the specific recognition of TCA half-sites. The substitution of the methyl group at the mC12 site of the bottom strand breaks the original H-bonds directly. Such changes in electrostatic interactions also lead to the remote allosteric effects of protein by multifaceted interactions but have negligible contributions to binding. Although these two influence modes are different, they can both fine-tune the local environment, which might produce remote allosteric effects through protein-protein interactions. Further analysis reveals that the discrepancies in these two modes are primarily due to their location. Moreover, when both sites are methylated, the major determinant of binding specificity depends on the context and the location of the methylation site, which is the result of crosstalk and cooperativity.

Hydrogen sulfide-linked persulfidation of ABI4 controls ABA responses through the transactivation of MAPKKK18 in Arabidopsis

Hydrogen sulfide (H2S) is a signaling molecule that regulates plant hormone and stress responses. The phytohormone abscisic acid (ABA) plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1 (DES1) and the production of H2S in guard cells. However, it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals. In this study, we discovered that ABSCISIC ACID INSENSITIVE 4 (ABI4) acts downstream of DES1 in the control of ABA responses in Arabidopsis. ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA, and loss of DES1 function impairs this process. Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination, seedling establishment, and stomatal closure, which are abolished in the ABI4Cys250Ala mutated variant. Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA. Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18 (MAPKKK18), which propagates the MAPK signaling cascade induced by ABA. Furthermore, the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18, and ABI4 can bind the DES1 promoter, forming a regulatory loop. Taken together, these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.

LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate

Master transcription factors reprogram cell fate in multicellular eukaryotes. Pioneer transcription factors have prominent roles in this process because of their ability to contact their cognate binding motifs in closed chromatin. Reprogramming is pervasive in plants, whose development is plastic and tuned by the environment, yet little is known about pioneer transcription factors in this kingdom. Here, we show that the master transcription factor LEAFY (LFY), which promotes floral fate through upregulation of the floral commitment factor APETALA1 (AP1), is a pioneer transcription factor. In vitro, LFY binds to the endogenous AP1 target locus DNA assembled into a nucleosome. In vivo, LFY associates with nucleosome occupied binding sites at the majority of its target loci, including AP1. Upon binding, LFY ‘unlocks’ chromatin locally by displacing the H1 linker histone and by recruiting SWI/SNF chromatin remodelers, but broad changes in chromatin accessibility occur later. Our study provides a mechanistic framework for patterning of inflorescence architecture and uncovers striking similarities between LFY and animal pioneer transcription factor.

A Fluorescence-Based Assay to Determine PDZ-Ligand Binding Thermodynamics.

Postsynaptic density-95, disks-large, and zonula occludens-1 (PDZ) domain interactions with cognate linear binding motifs (i.e., PDZ-binding motifs or PBMs) are important for many biological processes and can be pathological when disrupted. There are hundreds of PDZ-PBM interactions reported but few have been quantitatively determined. Moreover, PDZ-PBM interactions have been identified as potential therapeutic targets. To thoroughly understand PDZ-PBM binding energetics and their specificity, we have developed a sensitive and quantitative equilibrium binding assay. Here, we describe a protocol for determining PDZ-PBM binding energetics using fluorescence anisotropy-based methodology.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species

ABSTRACT The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

Downregulation of the Polycomb-Associated Methyltransferase Ezh2 during Maturation of Hippocampal Neurons Is Mediated by MicroRNAs Let-7 and miR-124.

Ezh2 is a catalytic subunit of the polycomb repressive complex 2 (PRC2) which mediates epigenetic gene silencing through depositing the mark histone H3 lysine 27 trimethylation (H3K27me3) at target genomic sequences. Previous studies have demonstrated that Enhancer of Zeste Homolog 2 (Ezh2) was differentially expressed during maturation of hippocampal neurons; in immature neurons, Ezh2 was abundantly expressed, whereas in mature neurons the expression Ezh2 was significantly reduced. Here, we report that Ezh2 is downregulated by microRNAs (miRs) that are expressed during the hippocampal maturation process. We show that, in mature hippocampal neurons, lethal-7 (let-7) and microRNA-124 (miR-124) are robustly expressed and can target cognate motifs at the 3′-UTR of the Ezh2 gene sequence to downregulate Ezh2 expression. Together, these data demonstrate that the PRC2 repressive activity during hippocampal maturation is controlled through a post-transcriptional mechanism that mediates Ezh2 downregulation in mature neurons.

CTCF confers local nucleosome resiliency after DNA replication and during mitosis.

The access of Transcription Factors (TFs) to their cognate DNA binding motifs requires a precise control over nucleosome positioning. This is especially important following DNA replication and during mitosis, both resulting in profound changes in nucleosome organization over TF binding regions. Using mouse Embryonic Stem (ES) cells, we show that the TF CTCF displaces nucleosomes from its binding site and locally organizes large and phased nucleosomal arrays, not only in interphase steady-state but also immediately after replication and during mitosis. Correlative analyses suggest this is associated with fast gene reactivation following replication and mitosis. While regions bound by other TFs (Oct4/Sox2), display major rearrangement, the post-replication and mitotic nucleosome positioning activity of CTCF is not unique: Esrrb binding regions are also characterized by persistent nucleosome positioning. Therefore, selected TFs such as CTCF and Esrrb act as resilient TFs governing the inheritance of nucleosome positioning at regulatory regions throughout the cell-cycle.

Epigenome editing strategies for the functional annotation of CTCF insulators

The human genome is folded into regulatory units termed ‘topologically-associated domains’ (TADs). Genome-wide studies support a global role for the insulator protein CTCF in mediating chromosomal looping and the topological constraint of TAD boundaries. However, the impact of individual insulators on enhancer-gene interactions and transcription remains poorly understood. Here, we investigate epigenome editing strategies for perturbing individual CTCF insulators and evaluating consequent effects on genome topology and transcription. We show that fusions of catalytically-inactive Cas9 (dCas9) to transcriptional repressors (dCas9-KRAB) and DNA methyltransferases (dCas9-DNMT3A, dCas9-DNMT3A3L) can selectively displace CTCF from specific insulators, but only when precisely targeted to the cognate motif. We further demonstrate that stable, partially-heritable insulator disruption can be achieved through combinatorial hit-and-run epigenome editing. Finally, we apply these strategies to simulate an insulator loss mechanism implicated in brain tumorigenesis. Our study provides strategies for stably modifying genome organization and gene activity without altering the underlying DNA sequence.

The cancer-associated, gain-of-function TP53 variant P152Lp53 activates multiple signaling pathways implicated in tumorigenesis

TP53 is the most frequently mutated tumor suppressor gene in many cancers, yet biochemical characterization of several of its reported mutations with probable biological significance have not been accomplished enough. Specifically, missense mutations in TP53 can contribute to tumorigenesis through gain-of-function of biochemical and biological properties that stimulate tumor growth. Here, we identified a relatively rare mutation leading to a proline to leucine substitution (P152L) in TP53 at the very end of its DNA-binding domain (DBD) in a sample from an Indian oral cancer patient. Although the P152Lp53 DBD alone bound to DNA, the full-length protein completely lacked binding ability at its cognate DNA motifs. Interestingly, P152Lp53 could efficiently tetramerize, and the mutation had only a limited impact on the structure and stability of full-length p53. Significantly, when we expressed this variant in a TP53-null cell line, it induced cell motility, proliferation, and invasion compared with a vector-only control. Also, enhanced tumorigenic potential was observed when P152Lp53-expressing cells were xenografted into nude mice. Investigating the effects of P152Lp53 expression on cellular pathways, we found that it is associated with up-regulation of several pathways, including cell-cell and cell-extracellular matrix signaling, epidermal growth factor receptor signaling, and Rho-GTPase signaling, commonly active in tumorigenesis and metastasis. Taken together, our findings provide a detailed account of the biochemical and cellular alterations associated with the cancer-associated P152Lp53 variant and establish it as a gain-of-function TP53 variant.

Stable enhancers are active in development, and fragile enhancers are associated with evolutionary adaptation

BackgroundDespite continual progress in the identification and characterization of trait- and disease-associated variants that disrupt transcription factor (TF)-DNA binding, little is known about the distribution of TF binding deactivating mutations (deMs) in enhancer sequences. Here, we focus on elucidating the mechanism underlying the different densities of deMs in human enhancers.ResultsWe identify two classes of enhancers based on the density of nucleotides prone to deMs. Firstly, fragile enhancers with abundant deM nucleotides are associated with the immune system and regular cellular maintenance. Secondly, stable enhancers with only a few deM nucleotides are associated with the development and regulation of TFs and are evolutionarily conserved. These two classes of enhancers feature different regulatory programs: the binding sites of pioneer TFs of FOX family are specifically enriched in stable enhancers, while tissue-specific TFs are enriched in fragile enhancers. Moreover, stable enhancers are more tolerant of deMs due to their dominant employment of homotypic TF binding site (TFBS) clusters, as opposed to the larger-extent usage of heterotypic TFBS clusters in fragile enhancers. Notably, the sequence environment and chromatin context of the cognate motif, other than the motif itself, contribute more to the susceptibility to deMs of TF binding.ConclusionsThis dichotomy of enhancer activity is conserved across different tissues, has a specific footprint in epigenetic profiles, and argues for a bimodal evolution of gene regulatory programs in vertebrates. Specifically encoded stable enhancers are evolutionarily conserved and associated with development, while differently encoded fragile enhancers are associated with the adaptation of species.

The oversimplification of ancient Egyptian concepts: Case studies from the Coffin and the Pyramid Texts

The paper presents several case studies on obscure words and passages in the Coffin Texts to demonstrate that this corpus remains significantly understudied and many of its current interpretations suffer from oversimplification. The comparative analysis of cognate passages and motifs throughout the Coffin and the Pyramid Texts highlights some so far unrecognized concepts in the early Egyptian mortuary texts and suggests that these texts are much more complicated than is commonly assumed.

BZLF1 interacts with chromatin remodelers promoting escape from latent infections with EBV.

A hallmark of EBV infections is its latent phase, when all viral lytic genes are repressed. Repression results from a high nucleosome occupancy and epigenetic silencing by cellular factors such as the Polycomb repressive complex 2 (PRC2) and DNA methyltransferases that, respectively, introduce repressive histone marks and DNA methylation. The viral transcription factor BZLF1 acts as a molecular switch to induce transition from the latent to the lytic or productive phase of EBV's life cycle. It is unknown how BZLF1 can bind to the epigenetically silenced viral DNA and whether it directly reactivates the viral genome through chromatin remodeling. We addressed these fundamental questions and found that BZLF1 binds to nucleosomal DNA motifs both in vivo and in vitro. BZLF1 co-precipitates with cellular chromatin remodeler ATPases, and the knock-down of one of them, INO80, impaired lytic reactivation and virus synthesis. In Assay for Transposase-Accessible Chromatin-seq experiments, non-accessible chromatin opens up locally when BZLF1 binds to its cognate sequence motifs in viral DNA. We conclude that BZLF1 reactivates the EBV genome by directly binding to silenced chromatin and recruiting cellular chromatin-remodeling enzymes, which implement a permissive state for lytic viral transcription. BZLF1 shares this mode of action with a limited number of cellular pioneer factors, which are instrumental in transcriptional activation, differentiation, and reprogramming in all eukaryotic cells.

Reverse Immunology Approach to Define a New HIV-gp41-Neutralizing Epitope.

The design of immunogens susceptible to elicit potent and broadly neutralizing antibodies against the human immunodeficiency virus type 1 (HIV-1) remains a veritable challenge in the course of vaccine development. Viral envelope proteins adopt different conformational states during the entry process, allowing the presentation of transient neutralizing epitopes. We focused on the highly conserved 3S motif of gp41, which is exposed to the surface envelope in its trimeric prefusion state. Vaccination with a W614A-modified 3S peptide induces in animals neutralizing anti-HIV-1 antibodies among which we selected clone F8. We used F8 as bait to select for W614A-3S phage-peptide mimics. Binding and molecular docking studies revealed that F8 interacts similarly with W614A-3S and a Mim_F8-1 mimotope, despite their lack of sequence homology, suggesting structural mimicry. Finally, vaccination of mice with the purified Mim_F8-1 phage elicited HIV-1-neutralizing antibodies that bound to the cognate W614A-3S motif. Collectively, our findings provide new insights into the molecular design of immunogens to elicit antibodies with neutralizing properties.

NF-κB and GATA-Binding Factor 6 Repress Transcription of Caveolins in Bladder Smooth Muscle Hypertrophy

Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.

NFAT5 up-regulates expression of the kidney-specific ubiquitin ligase gene Rnf183 under hypertonic conditions in inner-medullary collecting duct cells

We previously reported that among the 37 RING finger protein (RNF) family members, RNF183 mRNA is specifically expressed in the kidney under normal conditions. However, the mechanism supporting its kidney-specific expression pattern remains unclear. In this study, we elucidated the mechanism of the transcriptional activation of murine Rnf183 in inner-medullary collecting duct cells. Experiments with anti-RNF183 antibody revealed that RNF183 is predominantly expressed in the renal medulla. Among the 37 RNF family members, Rnf183 mRNA expression was specifically increased in hypertonic conditions, a hallmark of the renal medulla. RNF183 up-regulation was consistent with the activation of nuclear factor of activated T cells 5 (NFAT5), a transcription factor essential for adaptation to hypertonic conditions. Accordingly, siRNA-mediated knockdown of NFAT5 down-regulated RNF183 expression. Furthermore, the -3,466 to -3,136-bp region upstream of the mouse Rnf183 promoter containing the NFAT5-binding motif is conserved among mammals. A luciferase-based reporter vector containing the NFAT5-binding site was activated in response to hypertonic stress, but was inhibited by a mutation at the NFAT5-binding site. ChIP assays revealed that the binding of NFAT5 to this DNA site is enhanced by hypertonic stress. Of note, siRNA-mediated RNF183 knockdown increased hypertonicity-induced caspase-3 activation and decreased viability of mIMCD-3 cells. These results indicate that (i) RNF183 is predominantly expressed in the normal renal medulla, (ii) NFAT5 stimulates transcriptional activation of Rnf183 by binding to its cognate binding motif in the Rnf183 promoter, and (iii) RNF183 protects renal medullary cells from hypertonicity-induced apoptosis.

Transcription factor Sp2 potentiates binding of the TALE homeoproteins Pbx1:Prep1 and the histone-fold domain protein Nf-y to composite genomic sites

Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.

Characterizing protein-DNA binding event subtypes in ChIP-exo data.

Regulatory proteins associate with the genome either by directly binding cognate DNA motifs or via protein-protein interactions with other regulators. Each recruitment mechanism may be associated with distinct motifs and may also result in distinct characteristic patterns in high-resolution protein-DNA binding assays. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5' → 3' exonuclease digestion. Since different regulatory complexes will result in different protein-DNA crosslinking signatures, analysis of ChIP-exo tag enrichment patterns should enable detection of multiple protein-DNA binding modes for a given regulatory protein. However, current ChIP-exo analysis methods either treat all binding events as being of a uniform type or rely on motifs to cluster binding events into subtypes. To systematically detect multiple protein-DNA interaction modes in a single ChIP-exo experiment, we introduce the ChIP-exo mixture model (ChExMix). ChExMix probabilistically models the genomic locations and subtype memberships of binding events using both ChIP-exo tag distribution patterns and DNA motifs. We demonstrate that ChExMix achieves accurate detection and classification of binding event subtypes using in silico mixed ChIP-exo data. We further demonstrate the unique analysis abilities of ChExMix using a collection of ChIP-exo experiments that profile the binding of key transcription factors in MCF-7 cells. In these data, ChExMix identifies possible recruitment mechanisms of FoxA1 and ERα, thus demonstrating that ChExMix can effectively stratify ChIP-exo binding events into biologically meaningful subtypes. ChExMix is available from https://github.com/seqcode/chexmix. Supplementary data are available at Bioinformatics online.