Sequence-specific DNA-binding Protein Research Articles

High efficiency removal of antibiotic resistance gene with designer zinc-finger protein

The use of agricultural biomass-based fertilizers, and the release of feces into the environment leads to last-lasting pollution of antibiotic resistance genes that cannot be removed from waters via traditional methods, resulting in significant health threats. To solve this issue, an antibiotic resistance gene removal method was proposed and tested that used sequence-specific DNA-binding designer zinc finger proteins, which target an 18-bp DNA sequence for specific antibiotic resistance gene binding and removal. Targeting the sulfonamide-resistant sul1 gene, sul1-binding zinc-finger protein was designed, overexpressed, and purified. This protein showed specific binding with sul1 over tetA that do not have the targeted sequence. This protein was further immobilized on agarose-based resins to prepare a sul1-removal column. When loaded with 10 mg protein, this column can remove over 99 % sul1 in water, suggesting high efficiency. This work presents a new method attempting to eliminate environmental and health threats posed by antibiotic resistance genes.

Fusion Expression of Peptides with AflR Binuclear Zinc Finger Motif and Their Enhanced Inhibition of Aspergillus flavus: A Study of Engineered Antimicrobial Peptides.

This study reports a peptide design model for engineering fusion-expressed antimicrobial peptides (AMPs) with the AflR dinuclear zinc finger motif to improve the defense against aflatoxins and Aspergillus flavus. The study identified AflR, a Zn2Cys6-type sequence-specific DNA-binding protein, as a key player in the regulation of aflatoxin biosynthesis. By integrating the AflR motif into AMPs, we demonstrate that these novel fusion peptides significantly lower the minimum inhibitory concentrations (MICs) and reduce aflatoxin B1 and B2 levels, outperforming traditional AMPs. Comprehensive analysis, including bioinformatics and structural determination, elucidates the enhanced structure-function relationship underlying their efficacy. Furthermore, the study reveals the possibility that the fusion peptides have the potential to bind to the DNA binding sites of transcriptional regulators, binding DNA sites of key transcriptional regulators, thereby inhibiting genes critical for aflatoxin production. This research not only deepens our understanding of aflatoxin inhibition mechanisms but also presents a promising avenue for developing advanced antifungal agents, which are essential for global food safety and crop protection.

High prevalence of PRDM9-independent recombination hotspots in placental mammals

In many mammals, recombination events are concentrated in hotspots directed by a sequence-specific DNA-binding protein named PRDM9. Intriguingly, PRDM9 has been lost several times in vertebrates, and notably among mammals, it has been pseudogenized in the ancestor of canids. In the absence of PRDM9, recombination hotspots tend to occur in promoter-like features such as CpG islands. It has thus been proposed that one role of PRDM9 could be to direct recombination away from PRDM9-independent hotspots. However, the ability of PRDM9 to direct recombination hotspots has been assessed in only a handful of species, and a clear picture of how much recombination occurs outside of PRDM9-directed hotspots in mammals is still lacking. In this study, we derived an estimator of past recombination activity based on signatures of GC-biased gene conversion in substitution patterns. We quantified recombination activity in PRDM9-independent hotspots in 52 species of boreoeutherian mammals. We observe a wide range of recombination rates at these loci: several species (such as mice, humans, some felids, or cetaceans) show a deficit of recombination, while a majority of mammals display a clear peak of recombination. Our results demonstrate that PRDM9-directed and PRDM9-independent hotspots can coexist in mammals and that their coexistence appears to be the rule rather than the exception. Additionally, we show that the location of PRDM9-independent hotspots is relatively more stable than that of PRDM9-directed hotspots, but that PRDM9-independent hotspots nevertheless evolve slowly in concert with DNA hypomethylation.

Abstract 1840: MYBBP1A AMPylated by SELO to influence its function

Abstract Introduction: MYBBP1A, named Myb-binding protein 1A, is a protein that mainly locates in nucleolar and shuttles between the nucleus and cytoplasm, nuclear import may be mediated by KPNA2, however export depend partially on XPO1/CRM1. It activates or represses transcription via interactions with sequence specific DNA-binding proteins. Repression may be mediated at least in part by histone deacetylase activity (HDAC activity). Acts as a corepressor and in concert with CRY1, represses the transcription of the core circadian clock component PER2. Preferentially binds to di-methylated histone H3 'Lys-9' (H3K9me2) on the PER2 promoter. MYBBP1A overexpressed in tumor than normal and have a significantly negative correlation with patient overall survival. We aim to explore the role of MYBBP1A in PDAC progression and underlying mechanism. In our research, we find and prove the novel AMPylating enzyme SELO-AMPylate MYBBP1A inhibits PDAC progression and modified site in S1353 and further influence downstream relative RNA, such as 18s rRNA, 28S rRNA. Methods and Materials: 1. The function of MYBBP1A in tumorigenesis at the organismal and cellular levels. MYBBP1A is widely expressed in different tissues. Firstly, we explored the correlation between MYBBP1A expression level and PDAC through TCGA database and Kaplan-Meier plotter. We then established MYBBP1A gene knockout mice mating with PDX/P53K172H/KRASG12C to induced a primary PDAC model to analyze the progression of pancreatic cancer. The effect of MYBBP1A on tumor cell growth and proliferation was explored through cell proliferation analysis and a nude mouse tumor xenograft model. 2. The MYBBP1A AMPylated by SELO influence MYBBP1A function in cell proliferation was investigated at the molecular and cellular levels. AMPylation-specific antibodies were used to confirmed MYBBP1A AMPylated by SELO in both cell lysates and purified proteins through experiments such as CO-IP and WB. Stably expressed MYBBP1A WT/S1353A were constructed to verify the function in cell proliferation or migration by SELO. The signaling molecules were further explored through CO-IP and WB to identify the changed molecular by SELO-AMPylate-MYBBP1A, revealing the entire signaling pathways of AMPylation-mediated regulation of PDAC proliferation. Conclusion: We have demonstrated the function of MYBBP1A in the formation of pancreatic cancer, and further revealed SELO-AMPylate-MYBBP1A importance in the process of reversing MYBBP1A’s role, which has played a certain significance for the treatment of pancreatic cancer and the exploration of related therapeutic targets. Citation Format: Wu Li. MYBBP1A AMPylated by SELO to influence its function [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 1840.

TeaTFactor: a prediction tool for tea plant transcription factors based on BERT.

A transcription factor (TF) is a sequence-specific DNA-binding protein, which plays key roles in cell-fate decision by regulating gene expression. Predicting TFs is key for tea plant research community, as they regulate gene expression, influencing plant growth, development, and stress responses. It is a challenging task through wet lab experimental validation, due to their rarity, as well as the high cost and time requirements. As a result, computational methods are increasingly popular to be chosen. The pre-training strategy has been applied to many tasks in natural language processing (NLP) and has achieved impressive performance. In this paper, we present a novel recognition algorithm named TeaTFactor that utilizes pre-training for the model training of TFs prediction. The model is built upon the BERT architecture, initially pre-trained using protein data from UniProt. Subsequently, the model was fine-tuned using the collected TFs data of tea plants. We evaluated four different word segmentation methods and the existing state-of-the-art prediction tools. According to the comprehensive experimental results and a case study, our model is superior to existing models and achieves the goal of accurate identification. In addition, we have developed a web server at http://teatfactor.tlds.cc, which we believe will facilitate future studies on tea transcription factors and advance the field of crop synthetic biology.

In Silico Functional and Structural Analysis of Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in Human Paired Box 4 Gene.

In human genome, members of Paired box (PAX) transcription factor family are highly sequence-specific DNA-binding proteins. Among PAX gene family members, PAX4 gene has significant role in growth, proliferation, differentiation, and insulin secretion of pancreatic β-cells. Single nucleotide polymorphisms (SNPs) in PAX4 gene progress in the pathogenesis of various human diseases. Hence, the molecular mechanism of how these SNPs in PAX4 gene significantly progress diseases pathogenesis needs to be elucidated. For the reason, a series of bioinformatic analyzes were done to identify the SNPs of PAX4 gene that contribute in diseases pathogenesis. From the analyzes, 4145 SNPs (rsIDs) in PAX4 gene were obtained, where, 362 missense (8.73%), 169 synonymous (4.08%), and 2323 intron variants (56.04%). The rest SNPs were unspecified. Among the 362 missense variants, 118 nsSNPs were found as deleterious in SIFT analysis. Among those, 25 nsSNPs were most probably damaging and 23 were deleterious as observed in PolyPhen-2 and PROVEAN analyzes, respectively. Following all analyzes, 14 nsSNPs (rs149708455, rs115887120, rs147279315, rs35155575, rs370095957, rs373939873, rs145468905, rs121917718, rs2233580, rs3824004, rs372751660, rs369459316, rs375472849, rs372497946) were common and observed as deleterious, probably damaging, affective and diseases associated. Following structural analyzes, 11 nsSNPs guided proteins were found as most unstable and highly conserved. Among these, R20W, R39Q, R45Q, R60H, G65D, and A223D mutated proteins were highly harmful. Hence, the results from above-mentioned integrated comprehensive bioinformatic analyzes guide how different nsSNPs in PAX4 gene alter structural and functional characteristics of the protein that might progress diseases pathogenesis in human including type 2 diabetes.

Long-term follow-up of severe autosomal recessive SP7-related bone disorder

The SP7 gene encodes a zinc finger transcription factor (Osterix), which is a member of the Sp subfamily of sequence-specific DNA-binding proteins, playing an important role in osteoblast differentiation and maturation. SP7 pathogenic variants have been described in association with different allelic disorders. Monoallelic or biallelic SP7 variants cause Osteogenesis imperfecta type XII (OI12), a very rare condition characterized by recurrent fractures, skeletal deformities, undertubulation of long bones, hearing loss, no dentinogenesis imperfecta, and white sclerae. Monoallelic or biallelic SP7 variants may also cause sclerotic skeletal dysplasias (SSD), partially overlapping with Juvenile Paget's disease and craniodiaphyseal dysplasia, characterized by skull hyperostosis, long bones sclerosis, large ribs and clavicles, and possible recurrent fractures. Here, we report the long-term follow-up of an 85-year-old woman presenting with a complex bone disorder including features of either OI12 (bone fragility with multiple fractures, severe deformities and short stature) or SSD (striking skull hyperostosis with optic atrophy, very large ribs and clavicles and long bones sclerosis). Exome sequencing showed previously undescribed biallelic loss of function variants in the SP7 gene: NM_001173467.2(SP7): c.359_362del, p.(Asp120Valfs*11); NM_001173467.2(SP7): c.1163_1174delinsT, p.(Pro388Leufs*33). RT-qPCR confirmed a severely reduced SP7 transcription compared to controls.Our report provides new insights into the clinical and molecular features and long-term outcome of SP7-related bone disorders (SP7-BD), suggesting a continuum phenotypic spectrum characterized by bone fragility, undertubulation of long bones, scoliosis, and very heterogeneous bone mineral density ranging from osteoporosis to osteosclerosis.

The ortholog of human ssDNA-binding protein SSBP3 influences neurodevelopment and autism-like behaviors in Drosophila melanogaster.

1p32.3 microdeletion/duplication is implicated in many neurodevelopmental disorders-like phenotypes such as developmental delay, intellectual disability, autism, macro/microcephaly, and dysmorphic features. The 1p32.3 chromosomal region harbors several genes critical for development; however, their validation and characterization remain inadequate. One such gene is the single-stranded DNA-binding protein 3 (SSBP3) and its Drosophila melanogaster ortholog is called sequence-specific single-stranded DNA-binding protein (Ssdp). Here, we investigated consequences of Ssdp manipulations on neurodevelopment, gene expression, physiological function, and autism-associated behaviors using Drosophila models. We found that SSBP3 and Ssdp are expressed in excitatory neurons in the brain. Ssdp overexpression caused morphological alterations in Drosophila wing, mechanosensory bristles, and head. Ssdp manipulations also affected the neuropil brain volume and glial cell number in larvae and adult flies. Moreover, Ssdp overexpression led to differential changes in synaptic density in specific brain regions. We observed decreased levels of armadillo in the heads of Ssdp overexpressing flies, as well as a decrease in armadillo and wingless expression in the larval wing discs, implicating the involvement of the canonical Wnt signaling pathway in Ssdp functionality. RNA sequencing revealed perturbation of oxidative stress-related pathways in heads of Ssdp overexpressing flies. Furthermore, Ssdp overexpressing brains showed enhanced reactive oxygen species (ROS), altered neuronal mitochondrial morphology, and up-regulated fission and fusion genes. Flies with elevated levels of Ssdp exhibited heightened anxiety-like behavior, altered decisiveness, defective sensory perception and habituation, abnormal social interaction, and feeding defects, which were phenocopied in the pan-neuronal Ssdp knockdown flies, suggesting that Ssdp is dosage sensitive. Partial rescue of behavioral defects was observed upon normalization of Ssdp levels. Notably, Ssdp knockdown exclusively in adult flies did not produce behavioral and functional defects. Finally, we show that optogenetic manipulation of Ssdp-expressing neurons altered autism-associated behaviors. Collectively, our findings provide evidence that Ssdp, a dosage-sensitive gene in the 1p32.3 chromosomal region, is associated with various anatomical, physiological, and behavioral defects, which may be relevant to neurodevelopmental disorders like autism. Our study proposes SSBP3 as a critical gene in the 1p32.3 microdeletion/duplication genomic region and sheds light on the functional role of Ssdp in neurodevelopmental processes in Drosophila.

Overexpression of MMACHC Prevents Craniofacial Phenotypes Caused by Knockdown of znf143b.

ZNF143 is a sequence-specific DNA binding protein that regulates the expression of protein-coding genes and small RNA molecules. In humans, ZNF143 interacts with HCFC1, a transcriptional cofactor, to regulate the expression of downstream target genes, including MMACHC, which encodes an enzyme involved in cobalamin (cbl) metabolism. Mutations in HCFC1 or ZNF143 cause an inborn error of cobalamin metabolism characterized by abnormal cbl metabolism, intellectual disability, seizures, and mild to moderate craniofacial abnormalities. However, the mechanisms by which ZNF143 mutations cause individual phenotypes are not completely understood. Defects in metabolism and craniofacial development are hypothesized to occur because of decreased expression of MMACHC. But recent results have called into question this mechanism as the cause for craniofacial development. Therefore, in the present study, we implemented a loss of function analysis to begin to uncover the function of ZNF143 in craniofacial development using the developing zebrafish. The knockdown of znf143b, one zebrafish ortholog of ZNF143, caused craniofacial phenotypes of varied severity, which included a shortened and cleaved Meckel's cartilage, partial loss of ceratobranchial arches, and a distorted ceratohyal. These phenotypes did not result from a defect in the number of total chondrocytes but were associated with a mild to moderate decrease in mmachc expression. Interestingly, expression of human MMACHC via endogenous transgene prevented the onset of craniofacial phenotypes associated with znf143b knockdown. Collectively, our data establishes that knockdown of znf143b causes craniofacial phenotypes that can be alleviated by increased expression of MMACHC.

Analyzing paramagnetic NMR data on target DNA search by proteins using a discrete-state kinetic model for translocation.

Before reaching their targets, sequence-specific DNA-binding proteins nonspecifically bind to DNA through electrostatic interactions and stochastically change their locations on DNA. Investigations into the dynamics of DNA-scanning by proteins are nontrivial due to the simultaneous presence of multiple translocation mechanisms and many sites for the protein to nonspecifically bind to DNA. Nuclear magnetic resonance (NMR) spectroscopy can provide information about the target DNA search processes at an atomic level. Paramagnetic relaxation enhancement (PRE) is particularly useful to study how the proteins scan DNA in the search process. Previously, relatively simple two-state or three-state exchange models were used to explain PRE data reflecting the target search process. In this work, using more realistic discrete-state stochastic kinetics models embedded into an NMR master equation, we analyzed the PRE data for the HoxD9 homeodomain interacting with DNA. The kinetic models that incorporate sliding, dissociation, association, and intersegment transfer can reproduce the PRE profiles observed at some different ionic strengths. The analysis confirms the previous interpretation of the PRE data and shows that the protein's probability distribution among nonspecific sites is nonuniform during the target DNA search process.

Context-dependent role of Pho binding sites in Polycomb complex recruitment in Drosophila.

Polycomb group (PcG) proteins maintain the silenced state of key developmental genes, but how these proteins are recruited to specific regions of the genome is still not completely understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) comprised of a flexible array of sites for sequence-specific DNA binding proteins, "PcG recruiters," including Pho, Spps, Cg, and GAF. Pho is thought to play a central role in PcG recruitment. Early data showed that mutation of Pho binding sites in PREs in transgenes abrogated the ability of those PREs to repress gene expression. In contrast, genome-wide experiments in pho mutants or by Pho knockdown showed that PcG proteins can bind to PREs in the absence of Pho. Here, we directly addressed the importance of Pho binding sites in 2 engrailed (en) PREs at the endogenous locus and in transgenes. Our results show that Pho binding sites are required for PRE activity in transgenes with a single PRE. In a transgene, 2 PREs together lead to stronger, more stable repression and confer some resistance to the loss of Pho binding sites. Making the same mutation in Pho binding sites has little effect on PcG-protein binding at the endogenous en gene. Overall, our data support the model that Pho is important for PcG binding but emphasize how multiple PREs and chromatin environment increase the ability of PREs to function in the absence of Pho. This supports the view that multiple mechanisms contribute to PcG recruitment in Drosophila.

DNA binding dynamics and target location mechanism of pioneer factor GAF.

The chromatin landscape regulates transcription by localizing and regulating the activity of sequence-specific DNA binding proteins, chromatin remodeling enzymes and transcription machinery. Pioneer transcription factors can target DNA motifs on closed chromatin and trigger local chromatin remodeling to increase chromatin accessibility. Despite this knowledge, how pioneer factors search for their cognate motif on chromatin remains poorly understood. Here, we use multicolor single-molecule fluorescence resonance energy transfer (smFRET) to investigate how a prototypical Drosophila pioneer factor GAF locates its cognate motif on chromatin. To investigate how GAF searches for its cognate motif on DNA and nucleosomes, we strategically placed fluorophores on the DNA binding domain of GAF (GAF-DBD) and DNA motifs, allowing distinction of motif-specific binding and nonspecific DNA binding by FRET. We observed GAF-DBD dwells on a DNA molecule containing one GAF binding motif for 3-4 seconds before dissociation. Interestingly, on a DNA sequence with two GAF binding motifs, GAF-DBD can slide between two motifs while staying bound to the DNA. This suggests GAF monomers use one-dimensional (1D) sliding to search for its motif on nucleosome-free DNA. We next investigated the scenario where one of the two motifs is protected by a nucleosome. If the motif is located at the edge of a 601 nucleosome (SHL7 location), GAF-DBD more frequently lands on the other motif on linker DNA before it slides to the nucleosomal motif. Nucleosomes effectively block 1D sliding when the motif is located more internally on the nucleosome, at SHL5 or SHL3. However, on a nucleosome reconstituted on a native Drosophila DNA sequence, GAF-DBD can directly target internal motifs by three-dimensional diffusion, instead of 1D sliding. Our findings reveal different search modes that can be used by pioneer factors to locate their cognate motifs on chromatin.

Overexpression of Tfap2a in Mouse Oocytes Impaired Spindle and Chromosome Organization.

Transcription factor AP-2-alpha (Tfap2a) is an important sequence-specific DNA-binding protein that can regulate the transcription of multiple genes by collaborating with inducible viral and cellular enhancer elements. In this experiment, the expression, localization, and functions of Tfap2a were investigated in mouse oocytes during maturation. Overexpression via microinjection of Myc-Tfap2a mRNA into the ooplasm, immunofluorescence, and immunoblotting were used to study the role of Tfap2a in mouse oocyte meiosis. According to our results, Tfap2a plays a vital role in mouse oocyte maturation. Levels of Tfap2a in GV oocytes of mice suffering from type 2 diabetes increased considerably. Tfap2a was distributed in both the ooplasm and nucleoplasm, and its level gradually increased as meiosis resumption progressed. The overexpression of Tfap2a loosened the chromatin, accelerated germinal vesicle breakdown (GVBD), and blocked the first polar body extrusion 14 h after maturation in vitro. The width of the metaphase plate at metaphase I stage increased, and the spindle and chromosome organization at metaphase II stage were disrupted in the oocytes by overexpressed Tfap2a. Furthermore, Tfap2a overexpression dramatically boosted the expression of p300 in mouse GV oocytes. Additionally, the levels of pan histone lysine acetylation (Pan Kac), histone H4 lysine 12 acetylation (H4K12ac), and H4 lysine 16 acetylation (H4K16ac), as well as pan histone lysine lactylation (Pan Kla), histone H3 lysine18 lactylation (H3K18la), and H4 lysine12 lactylation (H4K12la), were all increased in GV oocytes after Tfap2a overexpression. Collectively, Tfap2a overexpression upregulated p300, increased the levels of histone acetylation and lactylation, impeded spindle assembly and chromosome alignment, and ultimately hindered mouse oocyte meiosis.

Capture, detection and purification of dsDNA amplicons using a DNA binding protein on magnetic beads

Magnetic separation has been widely exploited for capture and detection of nucleic acids, including amplicons. Streptavidin-magnetic beads (SA-MB) are typically employed for this purpose, as well as in biosensing applications. However, remaining biotinylated primer in the amplification reaction can compete with labeled amplicon for binding to the beads. Also, the harsh conditions needed for elution of bound amplicons restrict their use for purification purposes. Herein we show that a sequence-specific DNA binding protein immobilized on magnetic beads can serve as an alternative to SA-MB for these applications. This is enabled by the high binding affinity of scCro DNA binding protein for its specific sequence and its ability to bind dsDNA but not ssDNA. This specific sequence is easily incorporated in the amplicon during amplification with an extended primer. The scCro-MB exhibited higher amplicon binding capacity and detection sensitivity compared to SA-MB when both synthetic and genomic DNA were used as templates for PCR. This resulted not only from increased protein load on the beads but also from minimized interference of excess labeled primer remaining in the unpurified amplification reactions. Finally, a proof-of-concept was provided for the use of the scCro-MB for PCR amplicon purification under mild elution conditions using salt.

Genetic Diversity of Transcription Factor Genes in Triticum and Mining for Promising Haplotypes for Beneficial Agronomic Traits.

Transcription factor (TF) is a class of the sequence-specific DNA-binding proteins that modulate the transcription of target genes, and thus regulate their expressions. Variations in TF are the crucial determinants for phenotypic traits. Although much progress has been made in the functions of TF genes in wheat, one of the most important staple crops globally, the diversity of TF genes in wheat and its progenitors are not well understood, especially the agronomically promising haplotypes have not yet been characterized. Here, we identified a total of 6,023 TF genes from hexaploid wheat through a genome-search method and classified them into 59 gene families based on the conserved domain. The characteristics and dN/dS values of these genes showed evidently selective effects. Based on re-sequencing data, we found a strong genetic bottleneck among these TF genes on A and D subgenomes while no found in B subgenome during wheat domestication. Combined with selective signals and known QTLs on the whole genome, 21 TF genes were preliminarily found to be associated with yield-related traits. The haplotype frequency of these TF genes was further investigated in bread wheat and its progenitors and 13 major haplotypes were the casual loci related to key traits. Finally, the tissue-specific TF genes were also identified using RNA-seq analysis. This study provided insights into the diversity and evolution of TF genes and the identified TF genes and excellent haplotypes associating with traits will contribute to wheat genetic improvement.

Essential role of Cp190 in physical and regulatory boundary formation.

Boundaries in animal genomes delimit contact domains with enhanced internal contact frequencies and have debated functions in limiting regulatory cross-talk between domains and guiding enhancers to target promoters. Most mammalian boundaries form by stalling of chromosomal loop-extruding cohesin by CTCF, but most Drosophila boundaries form CTCF independently. However, how CTCF-independent boundaries form and function remains largely unexplored. Here, we assess genome folding and developmental gene expression in fly embryos lacking the ubiquitous boundary-associated factor Cp190. We find that sequence-specific DNA binding proteins such as CTCF and Su(Hw) directly interact with and recruit Cp190 to form most promoter-distal boundaries. Cp190 is essential for early development and prevents regulatory cross-talk between specific gene loci that pattern the embryo. Cp190 was, in contrast, dispensable for long-range enhancer-promoter communication at tested loci. Cp190 is thus currently the major player in fly boundary formation and function, revealing that diverse mechanisms evolved to partition genomes into independent regulatory domains.

In Vitro Transcription Factor Binding Site Predictions Using Support Vector Machine Classification

Transcription factors (TFs) are sequence‐specific DNA‐binding proteins essential in regulating gene expression. Determining TF DNA‐binding specificity can help to study gene regulatory networks within cells and how genetic variation can disrupt normal gene expression. One method for characterizing TF specificity is through Support Vector Machines (SVMs) by analyzing chromatin immunoprecipitation followed by DNA‐sequencing (ChIP‐seq) data. However, this can also be achieved using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) data, a method that also aids in determining TF‐DNA preferences. During this project, I implemented a gapped kmer SVM to study TF‐DNA binding preferences by using data from SELEX‐seq. I used a large scale‐gapped kmer, a sequence‐based SVM for analyzing TF specificity. It works by creating a predictive model that is trained with bound and unbound sequences from SELEX data. For purposes of this project, we used the T‐box transcription factor 5 (TBX5). After training the model for TBX5 and testing its performance, it had an AUROC value of 0.8248, indicating a significant degree of reliability. Likewise, the sequences with highest scores contained motifs for the TBX5. Given these results, we concluded that SVM was successfully implemented. In addition, SELEX data had not been previously used to train SVM based predictive models, meaning SELEX data is compatible and useful for developing predictive models.

Centromere Innovations with a Mouse Species

Mammalian centromeres direct faithful genetic inheritance at cell division and are typically found in regions of highly repetitive DNA, such as a‐satellite in humans and minor satellite in many mouse species. In the house mouse M. musculus, the 120 bp DNA monomers are nearly identical (~95%) to each other, and CENP‐A nucleosomes containing the centromeric histone H3 variant, CENP‐A, are positioned with their center position, the nucleosomal dyad, within the DNA recognition element for the sequence‐specific DNA‐binding protein, CENP‐B. We find that CENP‐B‐/‐ mice have precisely the same positioning of CENP‐A nucleosomes as CENP‐B+/+ mice, indicating a potential role for DNA sequence in the positioning of CENP‐A nucleosomes. M. pahari diverged from M. musculus ~ 4 M years ago and has been reported to have a different general DNA sequence organization at centromeres, so we sought to determine the features of centromeric chromatin that were conserved between these species. We found that, on most M. pahari centromeres, CENP‐B is of very low abundance but is extremely enriched on a single chromosome. Using multiple genomic strategies, we identified the most abundant centromere repeat in M. pahari that we term pi‐satellite, which is ~189 bp long and organized into tandem repeat arrays. Chromosomes harboring pi‐satellite lack CENP‐B boxes entirely. Additionally, we employed a more sophisticated strategy to identify a less abundant centromere satellite that contains the CENP‐B box. This variant exhibits greater homogenization, is 188 bp long, and is found exclusively on the single centromere containing extremely high levels of CENP‐B. This centromere also recruits more constitutive heterochromatin, marked by H3K9me3, consistent with a role for CENP‐B in driving pericentromeric heterochromatin. Together, these findings reveal that a mouse species is undergoing dramatic molecular differences directed by evolutionary innovations in centromere DNA sequence.

Gene expression and transcriptional regulation driven by transcription factors involved in congenital heart defects.

Congenital heart disease (CHD) is one of the most important birth defects caused by more than one mutated gene. Mutations in the genes could cause different types of congenital heart defects including atrial septal defect (ASD), tetralogy of Fallot (TOF), and ventricular septal defect (VSD). Cardiac transcription factors are key players for heart development and are actively involved in controlling stress regulation of the heart. Transcription factors are sequence-specific DNA binding proteins that control the process of transcription and work in a synergistic manner. We aim to characterize core cardiac transcription factors including NKX2-5, TBX, SRF, GATA4, and MEF2, which encode homeobox and MADS domain and play a crucial role in heart development. In this study, we have explored the important transcription factors involved in cardiac development and genes controlling the expression and regulation process by using the bioinformatics approach. We have predicted the orthologs and homologs based on their evolutionary history, conserved protein domains, functional sites, and 3D structures for better understanding and presentation of factors responsible for causing CHD. Results showed the importance of these transcription factors for normal heart functioning and development. Understanding the molecular pathways and genetic basis of CHD will help to open a new door for the treatment of patients with cardiac defects.

Effect of the NAMPT Activator P7C3-A20 on γ-Globin Expression in Baboon CD34+ Erythroid Cell Cultures

Increased levels of Fetal Hemoglobin (HbF) reduce the symptoms of sickle cell disease (SCD) and lengthen the life span of patients. New, more effective pharmacological agents that can be safely administered long term to increase HbF levels in SCD patients are highly sought. Expression of the γ-globin gene in adult erythroid cells is normally repressed by the recruitment of multi-protein co-repressor complexes to the γ-globin promoter by sequence-specific DNA binding proteins including BCL11A, LRF1 and TR2/TR4. Enzymes contained within these co-repressor complexes, such as DNMT1, LSD1, G9A, and HDACs, modify the chromatin surrounding the γ-globin promoter by catalyzing repressive epigenetic modifications to both histones and DNA. Small molecule pharmacological inhibitors of these enzymes are potent inducers of HbF in various in cell culture and animal models and in SCD patients, but the use of these drugs in patients has been hindered by their dose-dependent effects on hematopoietic differentiation. An alternative strategy to the use of these pharmacological inhibitors to increase HbF would be to employ pharmacological activators that increase the activity of proteins that positively promote γ-globin expression. Previous studies have shown that pharmacological activators of the Sirtuin 1 protein deacetylase increased γ-globin expression in cultured human CD34+ erythroid progenitor cell cultures (Dai et al; Am J Hematol 92:1177-1186, 2017). Because Sirtuin deacetylase activity is dependent upon nicotinamide adenine dinucleotide (NAD) as a co-factor, we tested the hypothesis that increased concentrations of nicotinamide, an NAD precursor, would also increase γ-globin expression. Baboon bone marrow derived CD34+ erythroid progenitor cells from 4 individual baboons were cultured on AFT024 monolayers for 14 days in the presence and absence of varying concentrations of nicotinamide. Globin chain expression was measured in cell lysates by high performance liquid chromatography (HPLC). Nicotinamide (500μM) appeared to increase γ-globin 2 fold (0.015±0.098 γ/γ+β) compared to untreated controls (0.072±0.04 γ/γ+β; n=4; p<0.08). Because the nicotinamide levels used in this experiments are higher than can be easily achieved by dietary supplementation, additional experiments were performed to test the effect of P7C3-A20, an allosteric activator of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD synthesis, on γ-globin expression. Addition of P7C3-A20 (2.5μM) to CD34+ erythroid progenitor cultures on d1, 4, 7, and 10 increased γ-globin 2.7 fold (0.247±0.10 γ/γ+β) compared to vehicle-treated controls (0.090±0.06 γ/γ+β; n=5; p<0.01). P7C3-A20 treatment did not affect cell viability or growth at concentration< 2.5μM and dose-response experiments showed increased γ-globin in cultures treated with submicromolar concentrations of the drug. Addition of P7C3-A20 to cultures on days 1 and 4 resulted in near maximal stimulation of γ-globin expression with lesser effects when the drug was added on later days (d4 and7 or d7 and 10) strongly suggesting that the drug targets cells at an early stage of differentiation. Additional experiments showed that the effect of P7C3-A20 (2.5μM) in combination with either the DNMT1 inhibitor decitabine (DAC) or the LSD1 inhibitor tranylcypromine (TCP) resulted in a greater than additive effects on γ-globin expression in the absence of cytotoxicity (Figure 1). In conclusion, the NAMPT activator P7C3-A20 increased γ-globin expression in baboon CD34+ erythroid progenitor cells with greater than additive effects in combination with DAC or TCP. P7C3-A20 has potent in vivo effects as a neuroprotective drug in mouse models and non-human primates. Therefore, the potential of this drug for in vivo HbF induction warrants further investigation. [Display omitted] DisclosuresSaunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.