Sci Rep. 2022 Dec 6;12(1):21079. doi: 10.1038/s41598-022-25333-9.

NO ABSTRACT

PMID:36473901 | DOI:10.1038/s41598-022-25333-9

Development. 2022 Oct 24:dev.201016. doi: 10.1242/dev.201016. Online ahead of print.

ABSTRACT

The posterior end of the follicular epithelium is patterned by midline (MID) and its paralog H15, the Drosophila homologs of the mammalian Tbx-20 transcription factor. We previously identified two cis-regulatory modules (CRMs) that recapitulate the endogenous pattern of mid in the follicular epithelium. Here, through CRISPR/Cas9 genome editing, we demonstrate redundant activity of these mid CRMs. While the deletion of either CRM alone generated marginal change in mid expression, the deletion of both CRMs reduced expression by 60%. Unexpectedly, the deletion of the 5' proximal CRM of mid eliminated H15 expression. Interestingly, expression of these paralogs in other tissues remained unaffected in the CRMs deletion backgrounds. These results suggest that the paralogs are regulated by a shared-CRM that coordinates gene expression during posterior fate determination. The consistent overlapping expression of mid and H15 in various tissues may indicate that the paralogs could also be under shared regulation by other CRMs in these tissues.

PMID:36278857 | DOI:10.1242/dev.201016

BMC Genomics. 2022 Oct 19;23(1):714. doi: 10.1186/s12864-022-08933-7.

ABSTRACT

BACKGROUND: Mouse is probably the most important model organism to study mammal biology and human diseases. A better understanding of the mouse genome will help understand the human genome, biology and diseases. However, despite the recent progress, the characterization of the regulatory sequences in the mouse genome is still far from complete, limiting its use to understand the regulatory sequences in the human genome.

RESULTS: Here, by integrating binding peaks in ~ 9,000 transcription factor (TF) ChIP-seq datasets that cover 79.9% of the mouse mappable genome using an efficient pipeline, we were able to partition these binding peak-covered genome regions into a cis-regulatory module (CRM) candidate (CRMC) set and a non-CRMC set. The CRMCs contain 912,197 putative CRMs and 38,554,729 TF binding sites (TFBSs) islands, covering 55.5% and 24.4% of the mappable genome, respectively. The CRMCs tend to be under strong evolutionary constraints, indicating that they are likely cis-regulatory; while the non-CRMCs are largely selectively neutral, indicating that they are unlikely cis-regulatory. Based on evolutionary profiles of the genome positions, we further estimated that 63.8% and 27.4% of the mouse genome might code for CRMs and TFBSs, respectively.

CONCLUSIONS: Validation using experimental data suggests that at least most of the CRMCs are authentic. Thus, this unprecedentedly comprehensive map of CRMs and TFBSs can be a good resource to guide experimental studies of regulatory genomes in mice and humans.

PMID:36261804 | DOI:10.1186/s12864-022-08933-7

BMC Biol. 2022 Oct 5;20(1):221. doi: 10.1186/s12915-022-01426-9.

ABSTRACT

BACKGROUND: Predicting cis-regulatory modules (CRMs) in a genome and their functional states in various cell/tissue types of the organism are two related challenging computational tasks. Most current methods attempt to simultaneously achieve both using data of multiple epigenetic marks in a cell/tissue type. Though conceptually attractive, they suffer high false discovery rates and limited applications. To fill the gaps, we proposed a two-step strategy to first predict a map of CRMs in the genome, and then predict functional states of all the CRMs in various cell/tissue types of the organism. We have recently developed an algorithm for the first step that was able to more accurately and completely predict CRMs in a genome than existing methods by integrating numerous transcription factor ChIP-seq datasets in the organism. Here, we presented machine-learning methods for the second step.

RESULTS: We showed that functional states in a cell/tissue type of all the CRMs in the genome could be accurately predicted using data of only 1~4 epigenetic marks by a variety of machine-learning classifiers. Our predictions are substantially more accurate than the best achieved so far. Interestingly, a model trained on a cell/tissue type in humans can accurately predict functional states of CRMs in different cell/tissue types of humans as well as of mice, and vice versa. Therefore, epigenetic code that defines functional states of CRMs in various cell/tissue types is universal at least in humans and mice. Moreover, we found that from tens to hundreds of thousands of CRMs were active in a human and mouse cell/tissue type, and up to 99.98% of them were reutilized in different cell/tissue types, while as small as 0.02% of them were unique to a cell/tissue type that might define the cell/tissue type.

CONCLUSIONS: Our two-step approach can accurately predict functional states in any cell/tissue type of all the CRMs in the genome using data of only 1~4 epigenetic marks. Our approach is also more cost-effective than existing methods that typically use data of more epigenetic marks. Our results suggest common epigenetic rules for defining functional states of CRMs in various cell/tissue types in humans and mice.

PMID:36199141 | DOI:10.1186/s12915-022-01426-9

Nucleic Acids Res. 2022 Sep 27:gkac795. doi: 10.1093/nar/gkac795. Online ahead of print.

ABSTRACT

Cohesin is a multifunctional protein responsible for transcriptional regulation and chromatin organization. Cohesin binds to chromatin at tens of thousands of distinct sites in a conserved or tissue-specific manner, whereas the function of cohesin varies greatly depending on the epigenetic properties of specific chromatin loci. Cohesin also extensively mediates cis-regulatory modules (CRMs) and chromatin loops. Even though next-generation sequencing technologies have provided a wealth of information on different aspects of cohesin, the integration and exploration of the resultant massive cohesin datasets are not straightforward. Here, we present CohesinDB (https://cohesindb.iqb.u-tokyo.ac.jp), a comprehensive multiomics cohesin database in human cells. CohesinDB includes 2043 epigenomics, transcriptomics and 3D genomics datasets from 530 studies involving 176 cell types. By integrating these large-scale data, CohesinDB summarizes three types of 'cohesin objects': 751 590 cohesin binding sites, 957 868 cohesin-related chromatin loops and 2 229 500 cohesin-related CRMs. Each cohesin object is annotated with locus, cell type, classification, function, 3D genomics and cis-regulatory information. CohesinDB features a user-friendly interface for browsing, searching, analyzing, visualizing and downloading the desired information. CohesinDB contributes a valuable resource for all researchers studying cohesin, epigenomics, transcriptional regulation and chromatin organization.

PMID:36162821 | DOI:10.1093/nar/gkac795

Cancers (Basel). 2022 Sep 8;14(18):4375. doi: 10.3390/cancers14184375.

ABSTRACT

Non-coding segments of the human genome are enriched in cis-regulatory modules that constitute functional elements, such as transcriptional enhancers and Super-enhancers. A hallmark of cancer pathogenesis is the dramatic dysregulation of the "archetype" gene expression profiles of normal human cells. Genomic variations can promote such deficiencies when occurring across enhancers and Super-enhancers, since they affect their mechanistic principles, their functional capacity and specificity, and the epigenomic features of the chromatin microenvironment across which these regulatory elements reside. Here, we comprehensively describe: fundamental mechanisms of gene expression dysregulation in cancers that involve genomic abnormalities within enhancers' and Super-enhancers' (SEs) sequences, which alter the expression of oncogenic transcription factors (TFs); cutting-edge technologies applied for the analysis of variation-enriched hotspots of the cancer genome; and pharmacological approaches for the treatment of Super-enhancers' aberrant function. Finally, we provide an intratumor meta-analysis, which highlights that genomic variations in transcription-factor-driven tumors are accompanied overexpression of genes, a portion of which encodes for additional cancer-related transcription factors.

PMID:36139535 | DOI:10.3390/cancers14184375

Methods Mol Biol. 2022;2573:115-132. doi: 10.1007/978-1-0716-2707-5_9.

ABSTRACT

Cardiac gene therapy has been hampered by off-target expression of gene of interest irrespective of variety of delivery methods. To overcome this issue, cardiac-specific promoters provide target tissue specificity, although expression is often debilitated compared to that of ubiquitous promoters. We have previously shown that sarcolipin promoter with an enhancer calsequestrin cis-regulatory module 4 (CRM4) combination has an improved atrial specificity. Moreover, it showed a minimal extra-atrial expression, which is a significant advantage for AAV9-mediated cardiac gene therapy. Therefore, it can be a useful tool to study and treat atrial-specific diseases such as atrial fibrillation. In this chapter, we introduce practical and simple methodology for atrial-specific gene therapy using sarcolipin promoter with an enhancer CRM4.

PMID:36040590 | DOI:10.1007/978-1-0716-2707-5_9

Int J Mol Sci. 2022 Aug 11;23(16):8981. doi: 10.3390/ijms23168981.

ABSTRACT

(1) Background: The widespread application of ChIP-seq technology requires annotation of cis-regulatory modules through the search of co-occurred motifs. (2) Methods: We present the web server Motifs Co-Occurrence Tool (Web-MCOT) that for a single ChIP-seq dataset detects the composite elements (CEs) or overrepresented homo- and heterotypic pairs of motifs with spacers and overlaps, with any mutual orientations, uncovering various similarities to recognition models within pairs of motifs. The first (Anchor) motif in CEs respects the target transcription factor of the ChIP-seq experiment, while the second one (Partner) can be defined either by a user or a public library of Partner motifs being processed. (3) Results: Web-MCOT computes the significances of CEs without reference to motif conservation and those with more conserved Partner and Anchor motifs. Graphic results show histograms of CE abundance depending on orientations of motifs, overlap and spacer lengths; logos of the most common CE structural types with an overlap of motifs, and heatmaps depicting the abundance of CEs with one motif possessing higher conservation than another. (4) Conclusions: Novel capacities of Web-MCOT allow retrieving from a single ChIP-seq dataset with maximal information on the co-occurrence of motifs and potentiates planning of next ChIP-seq experiments.

PMID:36012247 | DOI:10.3390/ijms23168981

Nat Commun. 2022 Aug 22;13(1):4794. doi: 10.1038/s41467-022-32400-2.

ABSTRACT

Wings have provided an evolutionary advantage to insects and have allowed them to diversify. Here, we have identified in Drosophila a highly robust regulatory mechanism that ensures the specification and growth of the wing not only during normal development but also under stress conditions. We present evidence that a single wing-specific enhancer in the wingless gene is used in two consecutive developmental stages to first drive wing specification and then contribute to mediating the remarkable regenerative capacity of the developing wing upon injury. We identify two evolutionary conserved cis-regulatory modules within this enhancer that are utilized in a redundant manner to mediate these two activities through the use of distinct molecular mechanisms. Whereas Hedgehog and EGFR signalling regulate Wingless expression in early primordia, thus inducing wing specification from body wall precursors, JNK activation in injured tissues induce Wingless expression to promote compensatory proliferation. These results point to evolutionarily linked conservation of wing specification and regeneration to ensure robust development of the wing, perhaps the most relevant evolutionary novelty in insects.

PMID:35995781 | DOI:10.1038/s41467-022-32400-2

Sci Adv. 2022 Aug 12;8(32):eabo6157. doi: 10.1126/sciadv.abo6157. Epub 2022 Aug 10.

ABSTRACT

Gene expression specificity of homeobox transcription factors has remained paradoxical. WUSCHEL activates and represses CLAVATA3 transcription at lower and higher concentrations, respectively. We use computational modeling and experimental analysis to investigate the properties of the cis-regulatory module. We find that intrinsically each cis-element can only activate CLAVATA3 at a higher WUSCHEL concentration. However, together, they repress CLAVATA3 at higher WUSCHEL and activate only at lower WUSCHEL, showing that the concentration-dependent interactions among cis-elements regulate both activation and repression. Biochemical experiments show that two adjacent functional cis-elements bind WUSCHEL with higher affinity and dimerize at relatively lower levels. Moreover, increasing the distance between cis-elements prolongs WUSCHEL monomer binding window, resulting in higher CLAVATA3 activation. Our work showing a constellation of optimally spaced cis-elements of defined affinities determining activation and repression thresholds in regulating CLAVATA3 transcription provides a previously unknown mechanism of cofactor-independent regulation of transcription factor binding in mediating gene expression specificity.

PMID:35947668 | DOI:10.1126/sciadv.abo6157

Funct Plant Biol. 2022 Aug 9. doi: 10.1071/FP21314. Online ahead of print.

ABSTRACT

Synthetic cis-regulatory modules can improve our understanding of gene regulatory networks. We applied an ensemble approach for de novo cis motif discovery among the promoters of 181 drought inducible differentially expressed soybean (Glycine max L.) genes. A total of 43 cis motifs were identified in promoter regions of all gene sets using the binding site estimation suite of tools (BEST). Comparative analysis of these motifs revealed similarities with known cis-elements found in PLACE database and led to the discovery of cis-regulatory motifs that were not yet implicated in drought response. Compiled with the proposed synthetic promoter design rationale, three synthetic assemblies were constructed by concatenating multiple copies of drought-inducible cis motifs in a specific order with inter-motif spacing using random bases and placed upstream of 35s minimal core promoter. Each synthetic module substituted 35S promoter in pBI121 and pCAMBIA3301 to drive glucuronidase expression in soybean hairy roots and Arabidopsis thaliana L. Chimeric soybean seedlings and 3-week-old transgenic Arabidopsis plants were treated with simulated with different levels of osmotic stress. Histochemical staining of transgenic soybean hairy roots and Arabidopsis displayed drought-inducible GUS activity of synthetic promoters. Fluorometric assay and expression analysis revealed that SP2 is the better manual combination of cis-elements for stress-inducible expression. qRT-PCR results further demonstrated that designed synthetic promoters are not tissue-specific and thus active in different parts upon treatment with osmotic stress in Arabidopsis plants. This study provides tools for transcriptional upgradation of valuable crops against drought stress and adds to the current knowledge of synthetic biology.

PMID:35940614 | DOI:10.1071/FP21314

Cells Dev. 2022 Aug 4:203802. doi: 10.1016/j.cdev.2022.203802. Online ahead of print.

ABSTRACT

Segments are repeated anatomical units forming the body of insects. In Drosophila, the specification of the body takes place during the blastoderm through the segmentation cascade. Pair-rule genes such as hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) are of the intermediate level of the cascade and each pair-rule gene is expressed in seven transversal stripes along the antero-posterior axis of the embryo. Stripes are formed by independent cis-regulatory modules (CRMs) under the regulation of transcription factors of maternal source and of gap proteins of the first level of the cascade. The initial blastoderm of Drosophila is a syncytium and it also coincides with the mid-blastula transition when thousands of zygotic genes are transcribed and their products are able to diffuse in the cytoplasm. Thus, we anticipated a complex regulation of the CRMs of the pair-rule stripes. The CRMs of h 1, eve 1, run 1, ftz 1 are able to be activated by bicoid (bcd) throughout the anterior blastoderm and several lines of evidence indicate that they are repressed by the anterior gap genes slp1 (sloppy-paired 1), tll (tailless) and hkb (huckebein). The modest activity of these repressors led to the premise of a combinatorial mechanism regulating the expression of the CRMs of h 1, eve 1, run 1, ftz 1 in more anterior regions of the embryo. We tested this possibility by progressively removing the repression activities of slp1, tll and hkb. In doing so, we were able to expose a mechanism of additive repression limiting the anterior borders of stripes 1. Stripes 1 respond depending on their distance from the anterior end and repressors operating at different levels.

PMID:35934285 | DOI:10.1016/j.cdev.2022.203802

Insects. 2022 Jul 11;13(7):618. doi: 10.3390/insects13070618.

ABSTRACT

We provide here an updated description of the REDfly (Regulatory Element Database for Fly) database of transcriptional regulatory elements, a unique resource that provides regulatory annotation for the genome of Drosophila and other insects. The genomic sequences regulating insect gene expression-transcriptional cis-regulatory modules (CRMs, e.g., "enhancers") and transcription factor binding sites (TFBSs)-are not currently curated by any other major database resources. However, knowledge of such sequences is important, as CRMs play critical roles with respect to disease as well as normal development, phenotypic variation, and evolution. Characterized CRMs also provide useful tools for both basic and applied research, including developing methods for insect control. REDfly, which is the most detailed existing platform for metazoan regulatory-element annotation, includes over 40,000 experimentally verified CRMs and TFBSs along with their DNA sequences, their associated genes, and the expression patterns they direct. Here, we briefly describe REDfly's contents and data model, with an emphasis on the new features implemented since 2020. We then provide an illustrated walk-through of several common REDfly search use cases.

PMID:35886794 | DOI:10.3390/insects13070618

Viruses. 2022 Jun 13;14(6):1284. doi: 10.3390/v14061284.

ABSTRACT

A corticosteroid antagonist impairs Herpes Simplex Virus 1 (HSV-1) productive infection and explant-induced reactivation from latency, suggesting corticosteroids and the glucocorticoid receptor (GR) mediate certain aspects of these complex virus-host interactions. GR-hormone complexes regulate transcription positively and negatively, in part, by binding GR response elements (GREs). Recent studies revealed infected cell protein 0 (ICP0), ICP4, and ICP27 promoter/cis-regulatory modules (CRMs) are cooperatively transactivated by GR and Krüppel-like factor 15 (KLF15), which forms a feed-forward transcription loop. We hypothesized the ICP0 promoter contains independent CRMs that are transactivated by GR, KLF15, and the synthetic corticosteroid dexamethasone (DEX). This hypothesis is based on the finding that the ICP0 promoter contains multiple transcription factor binding sites, and GR and KLF15 cooperatively transactivate the full-length ICP0 promoter. ICP0 promoter sequences spanning -800 to -635 (fragment A) were efficiently transactivated by GR, KLF15, and DEX in monkey kidney cells (Vero), whereas GR and DEX significantly enhanced promoter activity in mouse neuroblastoma cells (Neuro-2A). Furthermore, ICP0 fragment B (-458 to -635) was efficiently transactivated by GR, KLF15, and DEX in Vero cells, but not Neuro-2A cells. Finally, fragment D (-232 to -24) was transactivated significantly in Vero cells by GR, KLF15, and DEX, whereas KLF15 and DEX were sufficient for transactivation in Neuro-2A cells. Collectively, these studies revealed efficient transactivation of three independent CRMs within the ICP0 promoter by GR, KLF15, and/or DEX. Finally, GC-rich sequences containing specificity protein 1 (Sp1) binding sites were essential for transactivation.

PMID:35746756 | DOI:10.3390/v14061284

Plant Cell Rep. 2022 Jun 23. doi: 10.1007/s00299-022-02886-7. Online ahead of print.

ABSTRACT

In SlHDC-A promoter, SlHDC-A core-ES is an essential region for fruit-specific expression and interacts with GATA, HSF and AP1. Triplication of essential region was proposed as a minimal fruit-specific promoter. In plant biotechnology, fruit-specific promoter is an important tool for the improvement and utilization of tomato fruit. To expand our understanding on fruit-specific expression, it is necessary to determine the promoter region involved in fruit-specific transcriptional activity and transcriptional regulations of the promoter. In previous study, we isolated a fruit-specific SlHDC-A core promoter specifically expressed during tomato ripening stages. In this study, we identified SlHDC-A promoter region (SlHDC-A core-ES) that is essential for fruit-specific expression of the SlHDC-A. To understand the molecular mechanisms of fruit-specific expression of the SlHDC-A promoter, we first identified the putative transcription factor binding elements in the SlHDC-A core promoter region and corresponding putative transcription factors which are highly expressed during fruit maturation. Yeast one hybrid analysis confirmed that GATA, HSF, and AP1 interact with the SlHDC-A core-ES promoter region. Further transactivation analysis revealed that expression of the three transcription factors significantly activated expression of a reporter gene driven by SlHDC-A core-ES promoter. These results suggest that GATA, HSF, and AP1 are involved in the fruit-specific expression of SlHDC-A promoter. Furthermore, the synthetic promoter composed of three tandem repeats of SlHDC-A core-ES showed relatively higher activity than the constitutive 35S promoter in the transgenic tomato fruits at the orange stage. Taken together, we propose a new synthetic promoter that is specifically expressed during fruit ripening stage.

PMID:35739429 | DOI:10.1007/s00299-022-02886-7

Proc Natl Acad Sci U S A. 2022 Jun 21;119(25):e2122900119. doi: 10.1073/pnas.2122900119. Epub 2022 Jun 13.

ABSTRACT

Chromatin immunoprecipitation (ChIP) is an important technique for characterizing protein-DNA binding in vivo. One drawback of ChIP-based techniques is the lack of cell type-specificity when profiling complex tissues. To overcome this limitation, we developed SpyChIP to identify cell type-specific transcription factor (TF) binding sites in native physiological contexts without tissue dissociation or nuclei sorting. SpyChIP takes advantage of a specific covalent isopeptide bond that rapidly forms between the 15-amino acid SpyTag and the 17-kDa protein SpyCatcher. In SpyChIP, the target TF is fused with SpyTag by genome engineering, and an epitope tagged SpyCatcher is expressed in cell populations of interest, where it covalently binds to SpyTag-TF. Cell type-specific ChIP is obtained by immunoprecipitating chromatin prepared from whole tissues using antibodies directed against the epitope-tagged SpyCatcher. Using SpyChIP, we identified the genome-wide binding profiles of the Hox protein Ultrabithorax (Ubx) in two distinct cell types of the Drosophila haltere imaginal disc. Our results revealed extensive region-specific Ubx-DNA binding events, highlighting the significance of cell type-specific ChIP and the limitations of whole-tissue ChIP approaches. Analysis of Ubx::SpyChIP results provided insights into the relationship between chromatin accessibility and Ubx-DNA binding, as well as different mechanisms Ubx employs to regulate its downstream cis-regulatory modules. In addition to SpyChIP, we suggest that SpyTag-SpyCatcher technology, as well as other protein pairs that form covalent isopeptide bonds, will facilitate many additional in vivo applications that were previously impractical.

PMID:35696584 | DOI:10.1073/pnas.2122900119

Curr Opin Plant Biol. 2022 Jun 6;68:102232. doi: 10.1016/j.pbi.2022.102232. Online ahead of print.

ABSTRACT

Transcription factors (TFs) play a critical role in determining cell fate decisions by integrating developmental and environmental signals through binding to specific cis-regulatory modules and regulating spatio-temporal specificity of gene expression patterns. Precise identification of functional TF binding sites in time and space not only will revolutionize our understanding of regulatory networks governing cell fate decisions but is also instrumental to uncover how genetic variations cause morphological diversity or disease. In this review, we discuss recent advances in mapping TF binding sites and characterizing the various parameters underlying the complexity of binding site recognition by TFs.

PMID:35679803 | DOI:10.1016/j.pbi.2022.102232

FASEB J. 2022 May;36 Suppl 1. doi: 10.1096/fasebj.2022.36.S1.R3263.

ABSTRACT

The distal tip of the developing vertebrate limb is covered by a rim of thickened ectoderm called the apical ectodermal ridge (AER). The AER secretes fibroblast growth factors (Fgfs) into the underlying mesoderm to control outgrowth and proximal-distal patterning. Patterning during outgrowth proceeds from proximal to distal, with the proximal segment or stylopod being laid down first, then the zeugopod, and the distal segment or autopod forming last. Secreted Fgfs from the AER maintain a sub-AER population of undifferentiated mesoderm, called the progress zone, that provides cells for this progressive differentiation. In mice, Lhx9and its homologue Lhx2are expressed in the sub-AER mesoderm and appear to play redundant roles in maintaining the expression of other patterning molecules, such as Shh. Interestingly in the chicken, the expression of LHX9 is restricted to the anterior aspect of the sub-AER mesoderm, while LHX2 retains the full distal expression seen in mice. Recent clinical reports of an LHX9 missense mutation in a patient with hypoplastic/missing thumbs and great toes suggest that LHX9 can play a critical role in normal limb development. We hypothesized that the chicken cis-regulatory modules (CRMs) regulating Lhx9 might have subtle species-specific sequence variations compared to the murine CRMs that correlate with this differential expression pattern. To test this hypothesis, potential CRMs were identified near the Lhx9 locus by in silicoanalysis, using sequence conservation and regulatory chromatin markers from published ChIP-seq data. The potential CRMs were isolated and inserted into CRM-reporter constructs. The limb-related activity of a potential CRM was then determined by targeted regional electroporation into the distal chicken limb mesoderm. We identified a highly conserved, transcription-factor-rich 623 bp sequence within intron 12 of DENDD1B 132 kbp upstream of the chicken LHX9 transcription start site. The potential CRM was associated with H3k4me2, H3k27ac, p300 and H3k27me3. The associated CRM-reporter construct displayed activity in both anterior and posterior sub-AER limb mesoderm overlapping LHX9 and LHX2 expression. These findings are consistent with a distal sub-AER LHX9enhancer, but do not account for the lack of posterior expression. This suggests that additional CRMs contribute to the final expression pattern of LHX9 in the limb. Twenty-two additional potential CRMs were identified and will be further characterized to determine their role in restricting LHX9expression from the posterior mesoderm in chickens. Characterizing the differential regulation of Lhx9 during limb development may provide insight into the species-specific variation of limb patterning.

PMID:35556850 | DOI:10.1096/fasebj.2022.36.S1.R3263

Biotechnol J. 2022 Apr 28:e2200062. doi: 10.1002/biot.202200062. Online ahead of print.

ABSTRACT

Expression of recombinant genes in HEK293 cells is frequently utilized for production of recombinant proteins and viral vectors. These systems frequently employ the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. However, the mechanistic basis of CMV-mediated transcriptional activation in HEK293 cells is unknown and consequently there are no strategies to engineer CMV for controlled expression of recombinant genes. Extensive bioinformatic analyses of transcription factor regulatory elements (TFREs) within the human CMV sequence and transcription factor mRNAs within the HEK293 transcriptome revealed 80 possible regulatory interactions. Through in vitro functional testing using reporter constructs harboring discrete TFREs or CMV deletion variants we identified key TFRE components and clusters of TFREs (cis-regulatory modules) within the CMV sequence. Our data reveal that CMV activity in HEK293 cells is a function of the promoters various constituent TFREs including AhR:ARNT, CREB, E4F, Sp1, ZBED1, JunB, c-Rel and NF-κB. We also identified critical Sp1-dependent upstream activator elements near the transcriptional start site that were required for efficient transcription and YY1 and RBP-Jκ binding sites that mediate transrepression. Our study shows for the first time that novel, compact CMV-derived promoters can be engineered that exhibit up to 50% higher transcriptional efficiency (activity per unit DNA sequence) or 14% increase in total activity compared to the wild-type counterpart. This article is protected by copyright. All rights reserved.

PMID:35482470 | DOI:10.1002/biot.202200062

Database (Oxford). 2022 Apr 22;2022:baac024. doi: 10.1093/database/baac024.

ABSTRACT

More accurate and more complete predictions of cis-regulatory modules (CRMs) and constituent transcription factor (TF) binding sites (TFBSs) in genomes can facilitate characterizing functions of regulatory sequences. Here, we developed a database predicted cis-regulatory modules (PCRMS) (https://cci-bioinfo.uncc.edu) that stores highly accurate and unprecedentedly complete maps of predicted CRMs and TFBSs in the human and mouse genomes. The web interface allows the user to browse CRMs and TFBSs in an organism, find the closest CRMs to a gene, search CRMs around a gene and find all TFBSs of a TF. PCRMS can be a useful resource for the research community to characterize regulatory genomes. Database URL: https://cci-bioinfo.uncc.edu/.

PMID:35452518 | DOI:10.1093/database/baac024