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1. Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

   https://doi.org/10.1371/journal.pgen.1009039  

   Kuang, Yi; Pyo, Anna; Eafergan, Natanel; Cain, Brittany; Gutzwiller, Lisa M.; Axelrod, Ofri; Gagliani, Ellen K.; Weirauch, Matthew T.; Kopan, Raphael; Kovall, Rhett A.; et al (September 2021, PLOS Genetics)

   Aster, Jon Christopher (Ed.)

   Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the C bf/ S u(H)/ L ag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called S u(H) p aired s ites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo . Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation. 

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2. Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors

   https://doi.org/10.1038/s41467-023-38096-2  

   Li, Miaomiao; Yao, Tao; Lin, Wanru; Hinckley, Will E.; Galli, Mary; Muchero, Wellington; Gallavotti, Andrea; Chen, Jin-Gui; Huang, Shao-shan Carol (May 2023, Nature Communications)

   Abstract Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo-versusheterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers inArabidopsisand show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4cis-elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation. 

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3. Using Restriction Endonuclease, Protection, Selection, and Amplification to Identify Preferred DNA-Binding Sequences of Microbial Transcription Factors

   https://doi.org/10.1128/spectrum.04397-22  

   Barrows, John K.; Van Dyke, Michael W. (February 2023, Microbiology Spectrum)

   Polen, Tino (Ed.)

   ABSTRACT Regulation of gene expression is a vital component of cellular biology. Transcription factor proteins often bind regulatory DNA sequences upstream of transcription start sites to facilitate the activation or repression of RNA polymerase. Research laboratories have devoted many projects to understanding the transcription regulatory networks for transcription factors, as these regulated genes provide critical insight into the biology of the host organism. Various in vivo and in vitro assays have been developed to elucidate transcription regulatory networks. Several assays, including SELEX-seq and ChIP-seq, capture DNA-bound transcription factors to determine the preferred DNA-binding sequences, which can then be mapped to the host organism’s genome to identify candidate regulatory genes. In this protocol, we describe an alternative in vitro , iterative selection approach to ascertaining DNA-binding sequences of a transcription factor of interest using restriction endonuclease, protection, selection, and amplification (REPSA). Contrary to traditional antibody-based capture methods, REPSA selects for transcription factor-bound DNA sequences by challenging binding reactions with a type IIS restriction endonuclease. Cleavage-resistant DNA species are amplified by PCR and then used as inputs for the next round of REPSA. This process is repeated until a protected DNA species is observed by gel electrophoresis, which is an indication of a successful REPSA experiment. Subsequent high-throughput sequencing of REPSA-selected DNAs accompanied by motif discovery and scanning analyses can be used for determining transcription factor consensus binding sequences and potential regulated genes, providing critical first steps in determining organisms’ transcription regulatory networks. IMPORTANCE Transcription regulatory proteins are an essential class of proteins that help maintain cellular homeostasis by adapting the transcriptome based on environmental cues. Dysregulation of transcription factors can lead to diseases such as cancer, and many eukaryotic and prokaryotic transcription factors have become enticing therapeutic targets. Additionally, in many understudied organisms, the transcription regulatory networks for uncharacterized transcription factors remain unknown. As such, the need for experimental techniques to establish transcription regulatory networks is paramount. Here, we describe a step-by-step protocol for REPSA, an inexpensive, iterative selection technique to identify transcription factor-binding sequences without the need for antibody-based capture methods. 

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4. General and Genomic DNA-Binding Specificity for the Thermus thermophilus HB8 Transcription Factor TTHB023

   https://doi.org/10.3390/biom10010094  

   Cox, James Shell; Van Dyke, Michael W. (January 2020, Biomolecules)

   Transcription factors are proteins that recognize specific DNA sequences and affect local transcriptional processes. They are the primary means by which all organisms control specific gene expression. Understanding which DNA sequences a particular transcription factor recognizes provides important clues into the set of genes that they regulate and, through this, their potential biological functions. Insights may be gained through homology searches and genetic means. However, these approaches can be misleading, especially when comparing distantly related organisms or in cases of complicated transcriptional regulation. In this work, we used a biochemistry-based approach to determine the spectrum of DNA sequences specifically bound by the Thermus thermophilus HB8 TetR-family transcription factor TTHB023. The consensus sequence 5′–(a/c)Y(g/t)A(A/C)YGryCR(g/t)T(c/a)R(g/t)–3′ was found to have a nanomolar binding affinity with TTHB023. Analyzing the T. thermophilus HB8 genome, several TTHB023 consensus binding sites were mapped to the promoters of genes involved in fatty acid biosynthesis. Notably, some of these were not identified previously through genetic approaches, ostensibly given their potential co-regulation by the Thermus thermophilus HB8 TetR-family transcriptional repressor TTHA0167. Our investigation provides additional evidence supporting the usefulness of a biochemistry-based approach for characterizing putative transcription factors, especially in the case of cooperative regulation. 

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5. A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer

   https://doi.org/10.1371/journal.pone.0311120  

   Waite, Jack B; Boytz, RuthMabel; Traeger, Alexis R; Lind, Torrey M; Lumbao-Conradson, Koya; Torigoe, Sharon E (September 2024, PLOS ONE)

   Vall-llosera_Camps, Miquel (Ed.)

   Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene,Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the threeKlf4enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site inKlf4enhancer E2 rescued enhancer function andKlf4expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities ofKlf4enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency. 

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