Principle and application of self-transcribing active regulatory region sequencing in enhancer discovery research.

Self-transcribing active regulatory region sequencing (STARR-seq) is a high-throughput sequencing method capable of simultaneously discovering and validating all enhancers within the genome. In this method, candidate sequences are inserted into plasmid vectors and electroporated into cells. Acting as both enhancers and target genes, the self-transcription of these sequences will also be enhanced by themselves. By sequencing the transcriptome and comparing the results with the non-inserted control, the locations and activity of enhancers can be determined. In traditional enhancer discovery strategies, the chromatin open regions and transcription active regions were sequenced and predicted as enhancers. However, the activity of these putative enhancers could only be validated one by one without a high-throughput method. STARR-seq solved this limitation, allowing simultaneous enhancers discovery and activity validation in a high-throughput manner. Since the introduction of STARR-seq, it has been widely used to discover enhancers and validate enhancer activity in a number of organisms and cells. In this review, we present the traditional enhancer prediction methods and the basic principles, development history, specific applications of STARR-seq, and its future prospects, aiming to provide a reference for researchers in related fields conducting enhancer studies.

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

Background: STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Results: Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~ 50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). Conclusions: HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo.

STARR-seq and other massively-parallel reporter assays are widely used to discover functional enhancers in transfected cell models, which can be confounded by plasmid vector-induced type-I interferon immune responses and lack the multicellular environment and endogenous chromatin state of complex mammalian tissues. Here, we describe HDI-STARR-seq, which combines STARR-seq plasmid library delivery to the liver, by hydrodynamic tail vein injection (HDI), with reporter RNA transcriptional initiation driven by a minimal Albumin promoter, which we show is essential for mouse liver STARR-seq enhancer activity assayed 7 days after HDI. Importantly, little or no vector-induced innate type-I interferon responses were observed. Comparisons of HDI-STARR-seq activity between male and female mouse livers and in livers from males treated with an activating ligand of the transcription factor CAR (Nr1i3) identified many condition-dependent enhancers linked to condition-specific gene expression. Further, thousands of active liver enhancers were identified using a high complexity STARR-seq library comprised of ~50,000 genomic regions released by DNase-I digestion of mouse liver nuclei. When compared to stringently inactive library sequences, the active enhancer sequences identified were highly enriched for liver open chromatin regions with activating histone marks (H3K27ac, H3K4me1, H3K4me3), were significantly closer to gene transcriptional start sites, and were significantly depleted of repressive (H3K27me3, H3K9me3) and transcribed region histone marks (H3K36me3). HDI-STARR-seq offers substantial improvements over current methodologies for large scale, functional profiling of enhancers, including condition-dependent enhancers, in liver tissue in vivo, and can be adapted to characterize enhancer activities in a variety of species and tissues by selecting suitable tissue- and species-specific promoter sequences.

[Treatment of Tile type C pelvic ring fracture using orthopedic robot combined with Starr pelvis reduction frame].

OBJECTIVE: To investigate the clinical effect of orthopedic robot combined with Starr pelvic reduction frame in the treatment of Tile type C pelvic ring fracture. METHODS: From October 2019 to May 2021, 14 patients with type C pelvic ring fracture were treated with robotic combined with Starr pelvic reduction frame, including 9 males and 5 females. The age ranged from 33 to 69 years. All the 14 patients had fresh closed fractures without femur, tibia and fibula fracture. Surgery was completed from 4 to 7 d after hospital admission. During the operation, the X-ray carbon bed was used, the pelvic ring was reduced by Starr pelvis reduction frame, and pelvic ring fracture was treated by orthopedic robot. Operation time, bleeding volume, fluoroscopy times of single screw placement, fracture reduction quality, affected limb function and complications were observed. Radiological reduction was evaluated using Matta scoring standard, and clinical efficacy was evaluated by Majeed pelvic function scoring system at the final follow-up. RESULTS: All of 14 patients successfully completed the operation, the operation time was 84 to 141 min, the bleeding volume was 20 to 50 ml, and the fluoroscopy times of single screw insertion was 4 to 9 times. All of 14 patients were followed up for 12 to 24 months. The healing time was 3 to 7 months. No complications such as fracture of internal fixation, screw loosening, infection and nerve injury were found. According to the evaluation criteria of Matta imaging reduction, 9 cases were excellent, 4 cases were good, and 1 case was fair. At the final follow-up, Majeed pelvic function scoring system was used:10 cases were excellent, 4 cases were good. CONCLUSION: The treatment of type C pelvic ring fracture with robotic combined Starr pelvis reduction frame is simple, time-saving, less trauma, less complications and effective.

KSHV genome harbors both constitutive and lytically induced enhancers.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.

Integrative high-throughput enhancer surveying and functional verification divulges a YY2-condensed regulatory axis conferring risk for osteoporosis.

The precise roles of chromatin organization at osteoporosis risk loci remain largely elusive. Here, we combined chromatin interaction conformation (Hi-C) profiling and self-transcribing active regulatory region sequencing (STARR-seq) to qualify enhancer activities of prioritized osteoporosis-associated single-nucleotide polymorphisms (SNPs). We identified 319 SNPs with biased allelic enhancer activity effect (baaSNPs) that linked to hundreds of candidate target genes through chromatin interactions across 146 loci. Functional characterizations revealed active epigenetic enrichment for baaSNPs and prevailing osteoporosis-relevant regulatory roles for their chromatin interaction genes. Further motif enrichment and network mapping prioritized several putative, key transcription factors (TFs) controlling osteoporosis binding to baaSNPs. Specifically, we selected one top-ranked TF and deciphered that an intronic baaSNP (rs11202530) could allele-preferentially bind to YY2 to augment PAPSS2 expression through chromatin interactions and promote osteoblast differentiation. Our results underline the roles of TF-mediated enhancer-promoter contacts for osteoporosis, which may help to better understand the intricate molecular regulatory mechanisms underlying osteoporosis risk loci.

High-throughput screening of glucocorticoid-induced enhancer activity reveals mechanisms of stress-related psychiatric disorders.

Exposure to stressful life events increases the risk for psychiatric disorders. Mechanistic insight into the genetic factors moderating the impact of stress can increase our understanding of disease processes. Here, we test 3,662 single nucleotide polymorphisms (SNPs) from preselected expression quantitative trait loci in massively parallel reporter assays to identify genetic variants that modulate the activity of regulatory elements sensitive to glucocorticoids, important mediators of the stress response. Of the tested SNP sequences, 547 were located in glucocorticoid-responsive regulatory elements of which 233 showed allele-dependent activity. Transcripts regulated by these functional variants were enriched for those differentially expressed in psychiatric disorders in the postmortem brain. Phenome-wide Mendelian randomization analysis in 4,439 phenotypes revealed potentially causal associations specifically in neurobehavioral traits, including major depression and other psychiatric disorders. Finally, a functional gene score derived from these variants was significantly associated with differences in the physiological stress response, suggesting that these variants may alter disease risk by moderating the individual set point of the stress response.

Prediction accuracy of regulatory elements from sequence varies by functional sequencing technique.

Introduction: Various sequencing based approaches are used to identify and characterize the activities of cis-regulatory elements in a genome-wide fashion. Some of these techniques rely on indirect markers such as histone modifications (ChIP-seq with histone antibodies) or chromatin accessibility (ATAC-seq, DNase-seq, FAIRE-seq), while other techniques use direct measures such as episomal assays measuring the enhancer properties of DNA sequences (STARR-seq) and direct measurement of the binding of transcription factors (ChIP-seq with transcription factor-specific antibodies). The activities of cis-regulatory elements such as enhancers, promoters, and repressors are determined by their sequence and secondary processes such as chromatin accessibility, DNA methylation, and bound histone markers. Methods: Here, machine learning models are employed to evaluate the accuracy with which cis-regulatory elements identified by various commonly used sequencing techniques can be predicted by their underlying sequence alone to distinguish between cis-regulatory activity that is reflective of sequence content versus secondary processes. Results and discussion: Models trained and evaluated on D. melanogaster sequences identified through DNase-seq and STARR-seq are significantly more accurate than models trained on sequences identified by H3K4me1, H3K4me3, and H3K27ac ChIP-seq, FAIRE-seq, and ATAC-seq. These results suggest that the activity detected by DNase-seq and STARR-seq can be largely explained by underlying DNA sequence, independent of secondary processes. Experimentally, a subset of DNase-seq and H3K4me1 ChIP-seq sequences were tested for enhancer activity using luciferase assays and compared with previous tests performed on STARR-seq sequences. The experimental data indicated that STARR-seq sequences are substantially enriched for enhancer-specific activity, while the DNase-seq and H3K4me1 ChIP-seq sequences are not. Taken together, these results indicate that the DNase-seq approach identifies a broad class of regulatory elements of which enhancers are a subset and the associated data are appropriate for training models for detecting regulatory activity from sequence alone, STARR-seq data are best for training enhancer-specific sequence models, and H3K4me1 ChIP-seq data are not well suited for training and evaluating sequence-based models for cis-regulatory element prediction.

High-throughput functional dissection of noncoding SNPs with biased allelic enhancer activity for insulin resistance-relevant phenotypes.

Most of the single-nucleotide polymorphisms (SNPs) associated with insulin resistance (IR)-relevant phenotypes by genome-wide association studies (GWASs) are located in noncoding regions, complicating their functional interpretation. Here, we utilized an adapted STARR-seq to evaluate the regulatory activities of 5,987 noncoding SNPs associated with IR-relevant phenotypes. We identified 876 SNPs with biased allelic enhancer activity effects (baaSNPs) across 133 loci in three IR-relevant cell lines (HepG2, preadipocyte, and A673), which showed pervasive cell specificity and significant enrichment for cell-specific open chromatin regions or enhancer-indicative markers (H3K4me1, H3K27ac). Further functional characterization suggested several transcription factors (TFs) with preferential allelic binding to baaSNPs. We also incorporated multi-omics data to prioritize 102 candidate regulatory target genes for baaSNPs and revealed prevalent long-range regulatory effects and cell-specific IR-relevant biological functional enrichment on them. Specifically, we experimentally verified the distal regulatory mechanism at IRS1 locus, in which rs952227-A reinforces IRS1 expression by long-range chromatin interaction and preferential binding to the transcription factor HOXC6 to augment the enhancer activity. Finally, based on our STARR-seq screening data, we predicted the enhancer activity of 227,343 noncoding SNPs associated with IR-relevant phenotypes (fasting insulin adjusted for BMI, HDL cholesterol, and triglycerides) from the largest available GWAS summary statistics. We further provided an open resource (http://www.bigc.online/fnSNP-IR) for better understanding genetic regulatory mechanisms of IR-relevant phenotypes.

Frontiers and techniques in plant gene regulation.

Understanding plant gene regulation has been a priority for generations of plant scientists. However, due to its complex nature, the regulatory code governing plant gene expression has yet to be deciphered comprehensively. Recently developed methods-often relying on next-generation sequencing technology and state-of-the-art computational approaches-have started to further our understanding of the gene regulatory logic used by plants. In this review, we discuss these methods and the insights into the regulatory code of plants that they can yield.

Genome-wide enhancer identification by massively parallel reporter assay in Arabidopsis.

Enhancers are critical cis-regulatory elements controlling gene expression during cell development and differentiation. However, genome-wide enhancer characterization has been challenging due to the lack of a well-defined relationship between enhancers and genes. Function-based methods are the gold standard for determining the biological function of cis-regulatory elements; however, these methods have not been widely applied to plants. Here, we applied a massively parallel reporter assay on Arabidopsis to measure enhancer activities across the genome. We identified 4327 enhancers with various combinations of epigenetic modifications distinctively different from animal enhancers. Furthermore, we showed that enhancers differ from promoters in their preference for transcription factors. Although some enhancers are not conserved and overlap with transposable elements forming clusters, enhancers are generally conserved across thousand Arabidopsis accessions, suggesting they are selected under evolution pressure and could play critical roles in the regulation of important genes. Moreover, comparison analysis reveals that enhancers identified by different strategies do not overlap, suggesting these methods are complementary in nature. In sum, we systematically investigated the features of enhancers identified by functional assay in A. thaliana, which lays the foundation for further investigation into enhancers' functional mechanisms in plants.

Cis-regulatory atlas of primary human CD4+ T cells.

Cis-regulatory elements (CRE) are critical for coordinating gene expression programs that dictate cell-specific differentiation and homeostasis. Recently developed self-transcribing active regulatory region sequencing (STARR-Seq) has allowed for genome-wide annotation of functional CREs. Despite this, STARR-Seq assays are only employed in cell lines, in part, due to difficulties in delivering reporter constructs. Herein, we implemented and validated a STARR-Seq-based screen in human CD4+ T cells using a non-integrating lentiviral transduction system. Lenti-STARR-Seq is the first example of a genome-wide assay of CRE function in human primary cells, identifying thousands of functional enhancers and negative regulatory elements (NREs) in human CD4+ T cells. We find an unexpected difference in nucleosome organization between enhancers and NRE: enhancers are located between nucleosomes, whereas NRE are occupied by nucleosomes in their endogenous locations. We also describe chromatin modification, eRNA production, and transcription factor binding at both enhancers and NREs. Our findings support the idea of silencer repurposing as enhancers in alternate cell types. Collectively, these data suggest that Lenti-STARR-Seq is a successful approach for CRE screening in primary human cell types, and provides an atlas of functional CREs in human CD4+ T cells.

Using Synthetic DNA Libraries to Investigate Chromatin and Gene Regulation.

Despite the recent explosion in genome-wide studies in chromatin and gene regulation, we are still far from extracting a set of genetic rules that can predict the function of the regulatory genome. One major reason for this deficiency is that gene regulation is a multi-layered process that involves an enormous variable space, which cannot be fully explored using native genomes. This problem can be partially solved by introducing synthetic DNA libraries into cells, a method that can test the regulatory roles of thousands to millions of sequences with limited variables. Here, we review recent applications of this method to study transcription factor (TF) binding, nucleosome positioning, and transcriptional activity. We discuss the design principles, experimental procedures, and major findings from these studies and compare the pros and cons of different approaches.

A Suite of Constitutive Promoters for Tuning Gene Expression in Plants.

The need for convenient tools to express transgenes over a large dynamic range is pervasive throughout plant synthetic biology; however, current efforts are largely limited by the heavy reliance on a small set of strong promoters, precluding more nuanced and refined engineering endeavors in planta. To address this technical gap, we characterize a suite of constitutive promoters that span a wide range of transcriptional levels and develop a GoldenGate-based plasmid toolkit named PCONS, optimized for versatile cloning and rapid testing of transgene expression at varying strengths. We demonstrate how easy access to a stepwise gradient of expression levels can be used for optimizing synthetic transcriptional systems and the production of small molecules in planta. We also systematically investigate the potential of using PCONS as an internal standard in plant biology experimental design, establishing the best practices for signal normalization in experiments. Although our library has primarily been developed for optimizing expression in N. benthamiana, we demonstrate the translatability of our promoters across distantly related species using a multiplexed reporter assay with barcoded transcripts. Our findings showcase the advantages of the PCONS library as an invaluable toolkit for plant synthetic biology.

Computational Analysis of Maize Enhancer Regulatory Elements Using ATAC-STARR-seq.

The blueprints to development, response to the environment, and cellular function are largely the manifestation of distinct gene expression programs controlled by the spatiotemporal activity of cis-regulatory elements. Although biochemical methods for identifying accessible chromatin - a hallmark of active cis-regulatory elements - have been developed, approaches capable of measuring and quantifying cis-regulatory activity are only beginning to be realized. Massively Parallel Reporter Assays coupled to chromatin accessibility profiling present a high-throughput solution for testing the transcription-activating capacity of millions of putatively regulatory DNA sequences in parallel. However, clear computational pipelines for analyzing these high-throughput sequencing-based reporter assays are lacking. In this protocol, I layout and rationalize a computational framework for the processing and analysis of Assay for Transposase Accessible Chromatin profiling followed by Self-Transcribed Active Regulatory Region sequencing (ATAC-STARR-seq) data from a recent study in Zea mays. The approach described herein can be adapted to other sequencing-based reporter assays and is largely agnostic to the model organism with the appropriate input substitutions.

Starr frame-assisted minimally invasive internal fixation for pelvic fractures: Simultaneous anterior and posterior ring stability.

OBJECTIVE: We aimed to investigate the surgical techniques, efficacy, and safety of Starr frame-assisted minimally invasive internal fixation for simultaneous anterior and posterior ring stability in pelvic fractures. METHODS: The clinical data of 22 patients with anterior and posterior pelvic ring injuries who underwent Starr frame-assisted minimally invasive internal fixation were retrospectively collected. The anterior pelvic ring was fixed with an anterior subcutaneous internal fixator (INFIX), and the posterior pelvic ring was fixed with sacroiliac screws. The operative time, intraoperative blood loss, fluoroscopy times, number of assistants, complications, and weight-bearing time were analyzed. The Matta score was used to evaluate the fracture reduction. The Majeed score was used to evaluate clinical efficacy, and Gibbon's classification was used to estimate the occurrence of sacral nerve injury. RESULTS: All 22 patients were treated with sacroiliac screws for posterior ring injuries, including 12 cases with single sacroiliac screws, nine with two sacroiliac screws, and one with three sacroiliac screws. INFIX was used for all anterior ring fixation, including two screws in seven cases, three screws in 13 cases, and four screws in two cases. The fracture reduction quality was excellent in 15 cases, good in four cases, and moderate in three cases by Matta scores. All patients were followed up for 6-20 (12.5 ± 5.7) months. Callus-formation time based on postoperative X-ray was 3-8 (4.3 ± 1.2) weeks. Weight-bearing time was 3-12 (6.3 ± 2.8) weeks after surgery. At the last follow-up, 15 patients were evaluated as excellent, five as good, and two as fair according to the Majeed score. Gibbons' classification showed that one of the three patients with sacral nerve injury recovered completely and two did not recover. Complications occurred in six patients, including one with internal fixation loosening, two with anterolateral thigh paresthesia, one with malunion, one with nonunion, and one with heterotopic ossification. CONCLUSIONS: Starr frame-assisted minimally invasive internal fixation could stabilize the anterior and posterior pelvic rings simultaneously, overcoming the difficulty of fracture reduction. This technique can help patients with early postoperative weight-bearing and improved functional outcomes, providing a novel modality for the minimally invasive treatment of pelvic ring injuries.

Developmental and housekeeping transcriptional programs in Drosophila require distinct chromatin remodelers.

Gene transcription is a highly regulated process in all animals. In Drosophila, two major transcriptional programs, housekeeping and developmental, have promoters with distinct regulatory compatibilities and nucleosome organization. However, it remains unclear how the differences in chromatin structure relate to the distinct regulatory properties and which chromatin remodelers are required for these programs. Using rapid degradation of core remodeler subunits in Drosophila melanogaster S2 cells, we demonstrate that developmental gene transcription requires SWI/SNF-type complexes, primarily to maintain distal enhancer accessibility. In contrast, wild-type-level housekeeping gene transcription requires the Iswi and Ino80 remodelers to maintain nucleosome positioning and phasing at promoters. These differential remodeler dependencies relate to different DNA-sequence-intrinsic nucleosome affinities, which favor a default ON state for housekeeping but a default OFF state for developmental gene transcription. Overall, our results demonstrate how different transcription-regulatory strategies are implemented by DNA sequence, chromatin structure, and remodeler activity.

Genome-wide prediction of activating regulatory elements in rice by combining STARR-seq with FACS.

Self-transcribing active regulatory region sequencing (STARR-seq) is widely used to identify enhancers at the whole-genome level. However, whether STARR-seq works as efficiently in plants as in animal systems remains unclear. Here, we determined that the traditional STARR-seq method can be directly applied to rice (Oryza sativa) protoplasts to identify enhancers, though with limited efficiency. Intriguingly, we identified not only enhancers but also constitutive promoters with this technique. To increase the performance of STARR-seq in plants, we optimized two procedures. We coupled fluorescence activating cell sorting (FACS) with STARR-seq to alleviate the effect of background noise, and we minimized PCR cycles and retained duplicates during prediction, which significantly increased the positive rate for activating regulatory elements (AREs). Using this method, we determined that AREs are associated with AT-rich regions and are enriched for a motif that the AP2/ERF family can recognize. Based on GC content preferences, AREs are clustered into two groups corresponding to promoters and enhancers. Either AT- or GC-rich regions within AREs could boost transcription. Additionally, disruption of AREs resulted in abnormal expression of both proximal and distal genes, which suggests that STARR-seq-revealed elements function as enhancers in vivo. In summary, our work provides a promising method to identify AREs in plants.

Genome-wide identification of functional enhancers and their potential roles in pig breeding.

BACKGROUND: The pig is an economically important livestock species and is a widely applied large animal model in medical research. Enhancers are critical regulatory elements that have fundamental functions in evolution, development and disease. Genome-wide quantification of functional enhancers in the pig is needed. RESULTS: We performed self-transcribing active regulatory region sequencing (STARR-seq) in the porcine kidney epithelial PK15 and testicular ST cell lines, and reliably identified 2576 functional enhancers. Most of these enhancers were located in repetitive sequences and were enriched within silent and lowly expressed genes. Enhancers poorly overlapped with chromatin accessibility regions and were highly enriched in chromatin with the repressive histone modification H3K9me3, which is different from predicted pig enhancers detected using ChIP-seq for H3K27ac or/and H3K4me1 modified histones. This suggests that most pig enhancers identified with STARR-seq are endogenously repressed at the chromatin level and may function during cell type-specific development or at specific developmental stages. Additionally, the PPP3CA gene is associated with the loin muscle area trait and the QKI gene is associated with alkaline phosphatase activity that may be regulated by distal functional enhancers. CONCLUSIONS: In summary, we generated the first functional enhancer map in PK15 and ST cells for the pig genome and highlight its potential roles in pig breeding.

Indications, Feasibility, and Safety of TST STARR Plus Stapler for Degree III Hemorrhoids: A Retrospective Study of 125 Hemorrhoids Patients.

Background: Stapler hemorrhoidopexy (SH) has been widely accepted for hemorrhoids patients because of its low postoperative pain, but it is also associated with a high recurrence rate. The recurrence might be due to failure to completely remove the prolapsed tissue or insufficient removal capacity of the instruments. Removing more prolapsed tissue to reduce the recurrence is believed to benefit more severe prolapsed hemorrhoids patients. Methods: We evaluated the short- and long-term safety and efficacy in 125 hemorrhoids patients who underwent SH in 2013-2015. Eighty patients had prolapsed tissue less than half of the circular anal dilator (CAD) and underwent a procedure for prolapsing hemorrhoids (PPH), while the remaining 45 patients with hemorrhoid prolapse greater than half of the CAD were treated with a tissue selection therapy stapler stapled transanal rectal resection plus (TST STARR+). Results: There were no significant differences between the two groups in terms of operative time, hospitalization time, overall satisfaction or complications. At follow-up of up to 4 years after surgery, there was no significant difference in recurrence rates between TST STARR+ group and PPH group (5.2% vs. 4.7%, p < 0.05). The mean width and volume of the resected tissues were significantly larger in the TST STARR+ group than in the PPH group (4.8 vs. 2.9 cm, 10.2 vs. 4.4 cm3, P < 0.05). Conclusion: The TST STARR+ procedure can remove more hemorrhoidal tissue than PPH and it is better suited for patients with severe annular prolapsed hemorrhoids greater than half of the CAD. It has the advantages of convenient to operate, rapid recovery, fewer complications, and long-term satisfactory results.