PicoGreen assay for nucleic acid quantification - Applications, challenges, and solutions.

Various analytical methods and reagents have been employed for nucleic acid analysis in cells, biological fluids, and formulations. Standard techniques like gel electrophoresis and qRT-PCR are widely used for qualitative and quantitative nucleic acid analysis. However, these methods can be time-consuming and labor-intensive, with limitations such as inapplicability to small RNA at low concentrations and high costs associated with qRT-PCR reagents and instruments. As an alternative, PicoGreen (PG) has emerged as a valuable method for the quantitative analysis of nucleic acids. PG, a fluorescent dye, enables the quantitation of double-stranded DNA (dsDNA) or double-stranded RNA, including miRNA mimic and siRNA, in solution. It is also applicable to DNA and RNA analysis within cells using techniques like FACS and fluorescence microscopy. Despite its advantages, PG's fluorescence intensity is affected by various experimental conditions, such as pH, salts, and chemical reagents. This review explores the recent applications of PG as a rapid, cost-effective, robust, and accurate assay tool for nucleic acid quantification. We also address the limitations of PG and discuss approaches to overcome these challenges, recognizing the expanding range of its applications.

Hematopoietic/erythroid enhancers activate nearby target genes by extending histone H3K27ac and transcribing intergenic RNA.

Enhancers activate gene transcription remotely, which requires tissue specific transcription factors binding to them. GATA1 and TAL1 are hematopoietic/erythroid-specific factors and often bind together to enhancers, activating target genes. Interestingly, we found that some hematopoietic/erythroid genes are transcribed in a GATA1-dependent but TAL1-independnet manner. They appear to have enhancers within a relatively short distance. In this study, we paired highly transcribed hematopoietic/erythroid genes with the nearest GATA1/TAL1-binding enhancers and analyzed these putative enhancer-gene pairs depending on distance between them. Enhancers located at various distances from genes in the pairs, which was not related to transcription level of the genes. However, genes with enhancers at short distances away tended to be transcriptionally unaffected by TAL1 depletion. Histone H3K27ac extended from the enhancers to target genes. The H3K27ac extension was maintained without TAL1, even though it disappeared owing to the loss of GATA1. Intergenic RNA was highly transcribed from the enhancers to nearby target genes, independent of TAL1. Taken together, TAL1-independent transcription of hematopoietic/erythroid genes appears to be promoted by enhancers present in a short distance. These enhancers are likely to activate nearby target genes by tracking the intervening regions.

Risk factors for prediabetes in community-dwelling adults: A generalized estimating equation logistic regression approach with natural language processing insights.

The global prevalence of prediabetes is expected to reach 8.3% (587 million people) by 2045, with 70% of people with prediabetes developing diabetes during their lifetimes. We aimed to classify community-dwelling adults with a high risk for prediabetes based on prediabetes-related symptoms and to identify their characteristics, which might be factors associated with prediabetes. We analyzed homecare nursing records (n = 26,840) of 1628 patients aged over 20 years. Using a natural language processing algorithm, we classified each nursing episode as either low-risk or high-risk for prediabetes based on the detected number and category of prediabetes-symptom words. To identify differences between the risk groups, we employed t-tests, chi-square tests, and data visualization. Risk factors for prediabetes were identified using multiple logistic regression models with generalized estimating equations. A total of 3270 episodes (12.18%) were classified as potentially high-risk for prediabetes. There were significant differences in the personal, social, and clinical factors between groups. Results revealed that female sex, age, cancer coverage as part of homecare insurance coverage, and family caregivers were significantly associated with an increased risk of prediabetes. Although prediabetes is not a life-threatening disease, uncontrolled blood glucose can cause unfavorable outcomes for other major diseases. Thus, medical professionals should consider the associated symptoms and risk factors of prediabetes. Moreover, the proposed algorithm may support the detection of individuals at a high risk for prediabetes. Implementing this approach could facilitate proactive monitoring and early intervention, leading to reduced healthcare expenses and better health outcomes for community-dwelling adults.

Navigating the Landscape of Telemedicine Research: A Topic Modeling Approach for the Present and Future.

Introduction: Telemedicine, which is the provision of remote clinical services via telecommunication technology, has undergone an upsurge since the COVID-19 pandemic. To capture this paradigm, this study surveyed telemedicine literature, including postpandemic publications, to identify dominant research themes and temporal trends and suggest directions for future research. Methods: A corpus of 56,445 telemedicine studies is sourced from PubMed. Latent Dirichlet allocation (LDA) topic modeling performed using the Konstanz Information Miner platform. The textual data for topic modeling were processed by following standard procedures for natural language processing. Moreover, the term frequency-inverse document frequency approach was used to capture the importance of words within the corpus. We assessed perplexity, coherence, and the elbow method to determine the optimal number of topics for modeling. Results: The findings confirm the surge in telemedicine research after 2020, signifying its prominence. LDA topic modeling reveals seven distinct research themes, with the most prominent topic being "patient satisfaction" (21.38%) followed by "perspectives and challenges" (17.95%), and "smartphone apps" (14.32%). Furthermore, the results demonstrate a noticeable shift in topics from screening to therapeutic applications of telemedicine. Conclusions: This study serves as a guide for a broad range of telemedicine research topics. This synthesis of themes reflects the commitment of scholars to address the changing dynamics and health care needs, such as the COVID-19 pandemic, aging in place, smartphone usage, and technological advancement. The analysis also reveals flexible research responses to policy and contextual shifts, highlighting the collective drive to broaden the application of telemedicine in community health care.

Natural Language Processing Application in Nursing Research: A Study Using Text Network Analysis and Topic Modeling.

Although the potential of natural language processing and an increase in its application in nursing research is evident, there is a lack of understanding of the research trends. This study conducts text network analysis and topic modeling to uncover the underlying knowledge structures, research trends, and emergent research themes within nursing literature related to natural language processing. In addition, this study aims to provide a foundation for future scholarly inquiries and enhance the integration of natural language processing in the analysis of nursing research. We analyzed 443 literature abstracts and performed core keyword analysis and topic modeling based on frequency and centrality. The following topics emerged: (1) Term Identification and Communication; (2) Application of Machine Learning; (3) Exploration of Health Outcome Factors; (4) Intervention and Participant Experience; and (5) Disease-Related Algorithms. Nursing meta-paradigm elements were identified within the core keyword analysis, which led to understanding and expanding the meta-paradigm. Although still in its infancy in nursing research with limited topics and research volumes, natural language processing can potentially enhance research efficiency and nursing quality. The findings emphasize the possibility of integrating natural language processing in nursing-related subjects, validating nursing value, and fostering the exploration of essential paradigms in nursing science.

Developing a Classification Algorithm for Prediabetes Risk Detection From Home Care Nursing Notes: Using Natural Language Processing.

This study developed and validated a rule-based classification algorithm for prediabetes risk detection using natural language processing from home care nursing notes. First, we developed prediabetes-related symptomatic terms in English and Korean. Second, we used natural language processing to preprocess the notes. Third, we created a rule-based classification algorithm with 31 484 notes, excluding 315 instances of missing data. The final algorithm was validated by measuring accuracy, precision, recall, and the F1 score against a gold standard testing set (400 notes). The developed terms comprised 11 categories and 1639 words in Korean and 1181 words in English. Using the rule-based classification algorithm, 42.2% of the notes comprised one or more prediabetic symptoms. The algorithm achieved high performance when applied to the gold standard testing set. We proposed a rule-based natural language processing algorithm to optimize the classification of the prediabetes risk group, depending on whether the home care nursing notes contain prediabetes-related symptomatic terms. Tokenization based on white space and the rule-based algorithm were brought into effect to detect the prediabetes symptomatic terms. Applying this algorithm to electronic health records systems will increase the possibility of preventing diabetes onset through early detection of risk groups and provision of tailored intervention.

Development and evaluation of a problem-based learning simulation module for home-visit nursing.

OBJECTIVES: Although home-visit healthcare programs in Korea are expected to expand, providing hands-on experience to nursing students may be limited. This study aimed to develop and evaluate a problem-based learning (PBL) simulation module that reflects home-visit healthcare services provided by public health centers for pre-frail older adults. DESIGN AND SAMPLE: The simulation module, including PBL as prebriefing, was developed by the researchers and revised based on expert reviews. The module was evaluated using a mixed-method embedded one-group post-test-only design with focus group interviews (FGIs). Quantitative data (n = 29) were collected between April and June, 2021. FGIs (n = 10) were conducted twice in June 2021, and qualitative data were analyzed using an inductive content analysis approach. RESULTS: The average score of the Simulation Design Scale was 4.67 ± 0.36. The overall mean score of the Educational Practices Questionnaire was 4.75 ± 0.37. Three themes emerged from the FGIs: immersive learning experience, changes in perspective on nursing, and enhanced nursing competency. CONCLUSION: This PBL-based simulation module was evaluated as a systematic learning process in which nursing students could become self-directed learners, interacting and collaborating with colleagues, instructors, and environments. The module encourages them to practice home visit services.

Multiple CTCF sites cooperate with each other to maintain a TAD for enhancer-promoter interaction in the ß-globin locus.

Insulators are cis-regulatory elements that block enhancer activity and prevent heterochromatin spreading. The binding of CCCTC-binding factor (CTCF) protein is essential for insulators to play the roles in a chromatin context. The ß-globin locus, consisting of multiple genes and enhancers, is flanked by two insulators 3'HS1 and HS5. However, it has been reported that the absence of these insulators did not affect the ß-globin transcription. To explain the unexpected finding, we have deleted a CTCF motif at 3'HS1 or HS5 in the human ß-globin locus and analyzed chromatin interactions around the locus. It was found that a topologically associating domain (TAD) containing the ß-globin locus is maintained by neighboring CTCF sites in the CTCF motif-deleted loci. The additional deletions of neighboring CTCF motifs disrupted the ß-globin TAD, resulting in decrease of the ß-globin transcription. Chromatin interactions of the ß-globin enhancers with gene promoter were weakened in the multiple CTCF motifs-deleted loci, even though the enhancers have still active chromatin features such as histone H3K27ac and histone H3 depletion. Genome-wide analysis using public CTCF ChIA-PET and ChIP-seq data showed that chromatin domains possessing multiple CTCF binding sites tend to contain super-enhancers like the ß-globin enhancers. Taken together, our results show that multiple CTCF sites surrounding the ß-globin locus cooperate with each other to maintain a TAD. The ß-globin TAD appears to provide a compact spatial environment that enables enhancers to interact with promoter.

Histone H3K4me1 and H3K27ac play roles in nucleosome eviction and eRNA transcription, respectively, at enhancers.

Histone H3K4me1 and H3K27ac are enhancer-specific modifications and are required for enhancers to activate transcription of target genes. However, the reciprocal effects of these histone modifications on each other and their roles in enhancers are not clear. Here to comparatively analyze the role of these modifications, we inhibited H3K4me1 and H3K27ac by deleting the SET domains of histone methyltransferases MLL3 and MLL4 and the HAT domain of histone acetyltransferase p300, respectively, in erythroid K562 cells. The loss of H3K4me1 reduced H3K27ac at the ß-globin enhancer LCR HSs, but H3K27ac reduction did not affect H3K4me1. This unequal relationship between two modifications was revealed in putative enhancers by genome-wide analysis using ChIP-seq. Histone H3 eviction at putative enhancers was weakened by the loss of H3K4me1 but not by the loss of H3K27ac. Chromatin remodeling complexes were recruited into the ß-globin LCR HSs in a H3K4me1-dependent manner. In contrast, H3K27ac was required for enhancer RNA (eRNA) transcription, and H3K4me1 was not enough for it. Forced H3K27ac-induced eRNA transcription without affecting H3K4me1 at the ß-globin LCR HSs. These results indicate that H3K4me1 and H3K27ac affect each other in different ways and play more direct roles in nucleosome eviction and eRNA transcription, respectively, at enhancers.

The human ß-globin enhancer LCR HS2 plays a role in forming a TAD by activating chromatin structure at neighboring CTCF sites.

The human ß-globin locus control region (LCR) hypersensitive site 2 (HS2) is one of enhancers for transcription of the ß-like globin genes in erythroid cells. Our previous study showed that the LCR HS2 has active chromatin structure before transcriptional induction of the ß-globin gene, while another enhancer LCR HS3 is activated by the induction. To compare functional difference between them, we deleted each HS (ΔHS2 and ΔHS3) from the human ß-globin locus in hybrid MEL/ch11 cells. Deletion of either HS2 or HS3 dramatically diminished the ß-globin transcription and disrupted locus-wide histone H3K27ac and chromatin interaction between LCR HSs and gene. Surprisingly, ΔHS2 weakened interactions between CTCF sites forming the ß-globin topologically associating domain (TAD), while ΔHS3 did not. CTCF occupancy and chromatin accessibility were reduced at the CTCF sites in the ΔHS2 locus. To further characterize the HS2, we deleted the maf-recognition elements for erythroid activator NF-E2 at HS2. This deletion decreased the ß-globin transcription and enhancer-promoter interaction, but did not affect interactions between CTCF sites for the TAD. In light of these results, we propose that the HS2 has a role in forming a ß-globin TAD by activating neighboring CTCF sites and this role is beyond typical enhancer activity.

Gender differences in the relationship between informal caregiving and subjective health: the mediating role of health promoting behaviors.

BACKGROUND: In most of developed societies, the prevalence of informal care is on the rise due to rapid population ageing. This study investigates longitudinal associations between informal caregiving and health among caregivers and potential gender differences in this relationship. Moreover, drawing on the Health Promotion Model, this study examines the mediating role of health promoting behaviors in the link between informal caregiving and caregiver's health. METHODS: Seven waves of a large-scale (N = 9,608), a nationally representative longitudinal study of middle- and old-aged adults in Korea between 2006 and 2018, were used. To address the possibility of omitted variable bias, this study employed ordinary least squares models with lagged dependent variables (OLS-LDV) as well as fixed effects (FE) models. Univariate Sobel-Goodman mediation tests were used. RESULTS: Findings from OLS-LDV models showed that transition into informal caregiving is negatively associated with health satisfaction and self-rated health. FE results also suggest that our results are robust to controlling for unobserved heterogeneity. In the model where informal caregiving is interacted with gender, we found that these associations were largely driven by women caregivers. Results from Sobel-Goodman tests revealed that a decrease in regular exercise partially explains the observed association between informal caregiving and subjective health outcomes (11% for health satisfaction and 8% for self-rated health). CONCLUSIONS: Although informal caregiving can be a rewarding role, it poses a threat to caregiver's subjective health. Findings of this hold important implications and provide evidence in support of a gender-conscious approach to improve the health and well-being of informal caregivers.

GATA-1-dependent histone H3K27 acetylation mediates erythroid cell-specific chromatin interaction between CTCF sites.

CCCTC-binding factor (CTCF) sites interact with each other in the chromatin environment, establishing chromatin domains. Our previous study showed that interaction between CTCF sites is cell type-specific around the ß-globin locus and is dependent on erythroid-specific activator GATA-1. To find out molecular mechanisms of the cell type-specific interaction, we directly inhibited GATA-1 binding to the ß-globin enhancers by deleting its binding motifs and found that histone H3K27 acetylation (H3K27ac) was decreased at CTCF sites surrounding the ß-globin locus, even though CTCF binding itself was maintained at the sites. Forced H3K27ac by Trichostatin A treatment or CBP/p300 KD affected the interactions between CTCF sites around the ß-globin locus without changes in CTCF binding. Analysis of public ChIA-PET data revealed that H3K27ac is higher at CTCF sites forming short interactions than long interactions. GATA-1 was identified as a representative transcription factor that relates with genes present inside the short interactions in erythroid K562 cells. Depletion of GATA-1-reduced H3K27ac at CTCF sites near erythroid-specific enhancers. These results indicate that H3K27ac at CTCF sites is required for cell type-specific chromatin interactions between them. Tissue-specific activator GATA-1 appears to play a role in H3K27ac at CTCF sites in erythroid cells.

Cholesterol Granuloma of the Maxillary Sinus Misdiagnosed as Nasal Polyp or Mucocele: Four Cases.

ABSTRACT: Cholesterol granuloma is commonly found in the mastoid air cells but is rare in the paranasal sinuses. The most commonly affected sinuses are the frontal sinus, followed by the maxillary sinus. Cholesterol granuloma can be difficult to diagnose because clinical manifestations and radiologic findings are similar to those of other sinonasal disorders. The authors observed 4 cases of cholesterol granuloma that were preoperatively suspected to be nasal polyps or mucoceles. Here, the authors report on their clinical presentation and their successful treatment using the endoscopic sinus approach. These cases expand the current literature on cholesterol granuloma in the paranasal sinuses.

Levels of Interleukin-6 in Saliva, but Not Plasma, Correlate with Clinical Metrics in Huntington's Disease Patients and Healthy Control Subjects.

Growing evidence suggests that inflammatory responses, in both the brain and peripheral tissues, contribute to disease pathology in Huntington's disease (HD), an inherited, progressive neurodegenerative disorder typically affecting adults in their 30-40 s. Hence, studies of inflammation-related markers in peripheral fluids might be useful to better characterize disease features. In this study, we measured levels of C-reactive protein (CRP), Interleukin-6 (IL-6), interleukin 1 beta (IL-1B), and alpha-amylase (AA) in saliva and plasma from n = 125 subjects, including n = 37 manifest HD patients, n = 36 premanifest patients, and n = 52 healthy controls, using immunoassays. We found increases in salivary levels of IL-6, IL-1B and CRP across different disease groups and increased levels of IL-6 in the plasma of HD patients as compared to premanifest patients and controls. The levels of salivary IL-6 were significantly correlated with each of the other salivary markers, as well as with IL-6 levels measured in plasma. Further, salivary IL-6 and IL-1B levels were significantly positively correlated with Total Motor Score (TMS) and chorea scores and negatively correlated with Total Functional Capacity (TFC) in HD patients, whereby in healthy control subjects, IL-6 was significantly negatively correlated with Montreal Cognitive Assessment (MoCA) and the Symbol Digit Modalities test (SDM). Interestingly, the plasma levels of IL-6 did not show similar correlations to any clinical measures in either HD or control subjects. These findings suggest that salivary IL-6 is particularly relevant as a potential non-invasive biomarker for HD symptoms. The advent of an effective, dependable salivary biomarker would meet the urgent need for a less invasive means of identifying and monitoring HD disease progression.

LRF acts as an activator and repressor of the human ß-like globin gene transcription in a developmental stage dependent manner.

Leukemia/lymphoma-related factor (LRF; a hematopoietic transcription factor) has been suggested to repress fetal γ-globin genes in the human adult stage ß-globin locus. Here, to study the role of LRF in the fetal stage ß-globin locus, we knocked out its expression in erythroid K562 cells, in which the γ-globin genes are mainly transcribed. The γ-globin transcription was reduced in LRF knock-out cells, and transcription factor binding to the ß-globin locus control region hypersensitive sites (LCR HSs) and active histone organization in the LCR HSs were disrupted by the depletion of LRF. In contrast, LRF loss in the adult stage ß-globin locus did not affect active chromatin structure in the LCR HSs and induced the fetal γ-globin transcription. These results indicate that LRF may act as an activator and repressor of the human ß-like globin gene transcription in a manner dependent on developmental stage.

Exogenous CLASP2 protein treatment enhances wound healing in vitro and in vivo.

Proliferative and migratory abilities of fibroblasts are essential for wound healing at the skin surface. Cytoplasmic linker-associated protein-2 (CLASP2) was originally found to interact with cytoplasmic linker protein (CLIP)-170. CLASP2 plays an important role in microtubule stabilization and the microtubule-stabilizing activity of CLASP2 depends on its interactions with end binding (EB)-1 and CLIP-170. Although the microtubule-stabilizing role of CLASP2 is well established, the effects of CLASP2 on the migration and proliferation of fibroblasts remain unclear in the context of wound healing. Therefore, we tested the utilization of CLASP2 as a directly applied protein drug to improve wound healing by promoting the migration of effector cells, including skin fibroblasts, to the site of repair or injury using an in vivo excisional wound mouse model and in vitro Hs27 skin fibroblast model. Epidermal growth factor, which is a recognized contributor to cell proliferation and migration, was used as positive control. In vitro and in vivo, CLASP2 treatment significantly enhanced cell migration and accelerated wound closure. Furthermore, in vivo, the CLASP2-treated animal group displayed enhanced epidermal repair and collagen deposition. Next, we studied the mechanism of CLASP2 for wound healing. Increasing the abundance of intracellular free CLASP2 in skin fibroblasts by supplying exogenous CLASP2 seemed to stabilize microtubules through an interaction between CLASP2 and CLIP-170, as well as EB1. Exogenous CLASP2 also showed direct binding with IQGAP1, increasing both cyclic adenosine monophosphate activity and phosphorylation of glycogen synthase kinase 3ß, which in turn reinstated the binding between free CLASP2 and IQGAP1. In summary, exogenous CLASP2 increased Hs27 skin fibroblast migration by interacting with IQGAP1 and other cytoskeletal linker proteins, such as CLIP-170 and EB1. Our results strongly suggest that CLASP2 can be developed in wound healing drugs for skin repair and/or regenerating cosmetic products.

Poloxamer-Based Thermoreversible Gel for Topical Delivery of Emodin: Influence of P407 and P188 on Solubility of Emodin and Its Application in Cellular Activity Screening.

Emodin is a component in a Chinese herb, Rheum officinale Baill, traditionally used for diabetes and anticancer. Its poor solubility is one of the major challenges to pharmaceutical scientists. We previously reported on thermoreversible gel formulations based on poloxamer for the topical delivery of emodin. The present study was to understand the effect of poloxamer type on emodin solubility and its application in cellular activity screening. Various gel formulations composed of poloxamer 407 (P407), poloxamer 188 (P188) and PEG400 were prepared and evaluated. Major evaluation parameters were the gelation temperature (Tgel) and solubility of emodin. The emodin solubility increased with increasing poloxamer concentration and the Tgel was modulated by the proper combination of P407. In particular, this study showed that the amount of P407 in thermoreversible poloxamer gel (PG) was the dominant factor in enhancing solubility and P188 was effective at fixing gelation temperature in the desired range. A thermoreversible emodin PG was selected as the proper composition with the liquid state at room temperature and gel state at body temperature. The gel showed the solubility enhancement of emodin at least 100-fold compared to 10% ethanol or water. The thermoreversible formulation was applied for in vitro cellular activity screening in the human dermal fibroblast cell line and DLD-1 colon cancer cell line after dilution with cell culture media. The thermoreversible gel formulation remained as a clear solution in the microplate, which allowed reliable cellular activity screening. In contrast, emodin solution in ethanol or DMSO showed precipitation at the corresponding emodin concentration, complicating data interpretation. In conclusion, the gel formulation is proposed as a useful prototype topical formulation for testing emodin in vivo as well as in vitro.

Optimization of thermoreversible poloxamer gel system using QbD principle.

The objective of this study was to optimize thermoreversible gel formulations with respect to their gelation temperature and solubilizing capacity, using quality by design (QbD) principles based on design of experiment (DoE). Independent variables, X1, X2 and X3, represent the weight percentages of poloxamer 407 (P407), poloxamer 188 (P188) and the polyethylene glycol 400, respectively (these polymers are either thermoreversible gelling agents or solubilizers). Emodin, a poorly water-soluble compound, was used as a model drug. Fifteen gel formulations were prepared based on the compositions generated by an extreme vertices type of mixture design using a DoE software, in which their gelation temperatures (Y1) and emodin solubility (Y2) were measured. The gelation temperature and emodin solubility were each described adequately using a full cubic model and a quadratic model, respectively. Theoretical and experimental responses showed linearity with R2=0.9943 for the gelation temperature and R2=0.9629 for emodin solubility. The design space was established from the models describing the gelation temperature (test accuracy: 104.5%) and emodin solubility (test accuracy: 96.6%). This study demonstrates successful application of DoE for prediction of gelation temperature and solubility of emodin thermoreversible poloxamer gel (PG). In conclusion, the present DoE approach has led to the establishment of a design space and manufacturing control strategy for gel formulations.

KLF1 stabilizes GATA-1 and TAL1 occupancy in the human ß-globin locus.

KLF1 is an erythroid specific transcription factor that binds to regulatory regions of erythroid genes. Binding sites of KLF1 are often found near binding sites of GATA-1 and TAL1. In the ß-globin locus, KLF1 is required for forming active chromatin structure, although its role is unclear. To explore the role of KLF1 in transcribing the human γ-globin genes, we stably reduced the expression of KLF1 in erythroid K562 cells, compromising its association in the ß-globin locus. The γ-globin transcription was reduced with disappearance of active chromatin structure of the locus in the KLF1 knockdown cells. Interestingly, GATA-1 and TAL1 binding was reduced in the ß-globin locus, even though their expressions were not affected by KLF1 knockdown. The KLF1-dependent GATA-1 and TAL1 binding was observed in the adult locus transcribing the ß-globin gene and in several erythroid genes, where GATA-1 occupancy is independent from TAL1. These results indicate that KLF1 plays a role in facilitating and/or stabilizing GATA-1 and TAL1 occupancy in the erythroid genes, contributing to the generation of active chromatin structure such as histone acetylation and chromatin looping.