Systematic transcriptome profiling of hPSC-derived osteoblasts unveils CORIN's mastery in governing osteogenesis through CEBPD modulation.

The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and intrinsic transcriptional machinery. While rodent osteoblastic differentiation has been extensively studied, research on human osteogenesis has been limited by cell sources and existing models. Here, we systematically dissect human pluripotent stem cell-derived osteoblasts to identify functional membrane proteins and their downstream transcriptional networks involved in human osteogenesis. Our results reveal an enrichment of type II transmembrane serine protease CORIN in humans but not rodent osteoblasts. Functional analyses demonstrated that CORIN depletion significantly impairs osteogenesis. Genome-wide chromatin immunoprecipitation enrichment and mechanistic studies show that p38 MAPK-mediated CCAAT enhancer binding protein delta (CEBPD) upregulation is required for CORIN-modulated osteogenesis. Contrastingly, the type I transmembrane heparan sulfate proteoglycan SDC1 enriched in mesenchymal stem cells exerts a negative regulatory effect on osteogenesis through a similar mechanism. Chromatin immunoprecipitation-seq, bulk and single-cell transcriptomes, and functional validations indicated that CEBPD plays a critical role in controlling osteogenesis. In summary, our findings uncover previously unrecognized CORIN-mediated CEBPD transcriptomic networks in driving human osteoblast lineage commitment.

Decreased expression of KLF6 in ectopic endometrial stromal cells contributes to endometriosis progression by targeting CTNNB1.

Despite decades of research, endometriosis remains a mysterious gynecological disease with unknown etiology and pathogenesis. Krüppel-like Factor 6 (KLF6), a transcription factor, has a wide expression profile and regulates a variety of biological processes. Here, we investigated the expression and function of KLF6 and its possible regulatory mechanisms in endometriosis. To determine the function of KLF6, knockdown and overexpression experiments were performed in eutopic endometrial stromal cells (EU-ESCs) and ectopic endometrial stromal cells (EC-ESCs), respectively. Cell viability, apoptosis, migration, invasion, and angiogenesis assays were conducted in ESCs. ChIP-sequencing and mRNA-sequencing were performed to investigate the functional mechanism of KLF6 in regulating ESCs. We found that KLF6 was highly expressed in eutopic endometrium of endometriosis patients, compared with ectopic endometrium. Similarly, the same was true in EU-ESCs, which was compared with EC-ESCs. Overexpression of KLF6 significantly suppressed EC-ESC proliferation, migration and invasion and induced cell apoptosis, while knockdown of KLF6 resulted in the opposite effects on EU-ESCs. Overexpression of KLF6 significantly inhibited EC-ESC angiogenesis. Mechanistically, the results of ChIP sequencing and mRNA sequencing revealed that CTNNB1 may be a transcriptional target regulated by KLF6. Reintroduction of KLF6 reversed the effects of KLF6 knockdown on EU-ESCs. KLF6 inhibited the proliferation, migration and angiogenesis of EC-ESCs by inhibiting the expression of CTNNB1. Our findings provided a new perspective on the role of KLF6 in endometriosis progression and inspire potential targeted therapeutic strategies.

AP-1/C-FOS and AP-1/FRA2 differentially regulate early and late adipogenic differentiation of mesenchymal stem cells.

Obesity is defined as an abnormal accumulation of adipose tissue in the body and is a major global health problem due to increased morbidity and mortality. Adipose tissue is made up of adipocytes, which are fat-storing cells, and the differentiation of these fat cells is known as adipogenesis. Several transcription factors (TFs) such as CEBPß, CEBPα, PPARγ, GATA, and KLF have been reported to play a key role in adipogenesis. In this study, we report one more TF AP-1, which is found to be involved in adipogenesis. Human mesenchymal stem cells  were differentiated into adipocytes, and the expression pattern of different subunits of AP-1 was examined during adipogenesis. It was observed that C-FOS was predominantly expressed at an early stage (Day 2), whereas FRA2 expression peaked at later stages (Days 6 and 8) of adipogenesis. Chromatin immunoprecipitation-sequencing analysis revealed that C-FOS binds mainly to the promoters of WNT1, miR-30a, and ANAPC7 and regulates their expression during mitotic clonal expansion. In contrast, FRA2 binds to the promoters of CIDEA, NOTCH1, ARAF, and MYLK, regulating their expression and lipid metabolism. Data obtained clearly indicate that the differential expression of C-FOS and FRA2 is crucial for different stages of adipogenesis. This also raises the possibility of considering AP-1 as a therapeutic target for treating obesity and related disorders.

How diverse a monocentric chromosome can be? Repeatome and centromeric organization of Juncus effusus (Juncaceae).

Juncus is the largest genus of Juncaceae and was considered holocentric for a long time. Recent findings, however, indicated that 11 species from different clades of the genus have monocentric chromosomes. Thus, the Juncus centromere organization and evolution need to be reassessed. We aimed to investigate the major repetitive DNA sequences of two accessions of Juncus effusus and its centromeric structure by employing whole-genome analyses, fluorescent in situ hybridization, CENH3 immunodetection, and chromatin immunoprecipitation sequencing. We showed that the repetitive fraction of the small J. effusus genome (~270 Mbp/1C) is mainly composed of Class I and Class II transposable elements (TEs) and satellite DNAs. Three identified satellite DNA families were mainly (peri)centromeric, with two being associated with the centromeric protein CENH3, but not strictly centromeric. Two types of centromere organization were discerned in J. effusus: type 1 was characterized by a single CENH3 domain enriched with JefSAT1-155 or JefSAT2-180, whereas type 2 showed multiple CENH3 domains interrupted by other satellites, TEs or genes. Furthermore, while type 1 centromeres showed a higher degree of satellite identity along the array, type 2 centromeres had less homogenized arrays along the multiple CENH3 domains per chromosome. Although the analyses confirmed the monocentric organization of J. effusus chromosomes, our data indicate a more dynamic arrangement of J. effusus centromeres than observed for other plant species, suggesting it may constitute a transient state between mono- and holocentricity.

Loss of NSD2 causes dysregulation of synaptic genes and altered H3K36 dimethylation in mice.

Background: Epigenetic disruptions have been implicated in neurodevelopmental disorders. NSD2 is associated with developmental delay/intellectual disability; however, its role in brain development and function remains unclear. Methods: We performed transcriptomic and epigenetic analyses using Nsd2 knockout mice to better understand the role of NSD2 in the brain. Results and discussion: Transcriptomic analysis revealed that the loss of NSD2 caused dysregulation of genes related to synaptic transmission and formation. By analyzing changes in H3 lysine 36 dimethylation (H3K36me2), NSD2-mediated H3K36me2 mainly marked quiescent state regions and the redistribution of H3K36me2 occurred at transcribed genes and enhancers. By integrating transcriptomic and epigenetic data, we observed that H3K36me2 changes in a subset of dysregulated genes related to synaptic transmission and formation. These results suggest that NSD2 is involved in the regulation of genes important for neural function through H3K36me2. Our findings provide insights into the role of NSD2 and improve our understanding of epigenetic regulation in the brain.

The molecular circadian rhythms regulating the cell cycle.

The circadian clock controls the expression of a large proportion of protein-coding genes in mammals and can modulate a wide range of physiological processes. Recent studies have demonstrated that disruption or dysregulation of the circadian clock is involved in the development and progression of several diseases, including cancer. The cell cycle is considered to be the fundamental process related to cancer. Accumulating evidence suggests that the circadian clock can control the expression of a large number of genes related to the cell cycle. This article reviews the mechanism of cell cycle-related genes whose chromatin regulatory elements are rhythmically occupied by core circadian clock transcription factors, while their RNAs are rhythmically expressed. This article further reviews the identified oscillatory cell cycle-related genes in higher organisms such as baboons and humans. The potential functions of these identified genes in regulating cell cycle progression are also discussed. Understanding how the molecular clock controls the expression of cell cycle genes will be beneficial for combating and treating cancer.

The mitotic checkpoint kinase BUB1 is a direct and actionable target of MYB in adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a head and neck cancer that frequently originates in salivary glands, but can also strike other exocrine glands such as the breast. A key molecular alteration found in the majority of ACC cases is MYB gene rearrangements, leading to activation of the oncogenic transcription factor MYB. In this study, we used immortalised breast epithelial cells and an inducible MYB transgene as a model of ACC. Molecular profiling confirmed that MYB-driven gene expression causes a transition into an ACC-like state. Using this new cell model, we identified BUB1 as a targetable kinase directly controlled by MYB, whose pharmacological inhibition caused MYB-dependent synthetic lethality, growth arrest and apoptosis of patient-derived cells and organoids.

Resveratrol and 3,3'-Diindolylmethane Differentially Regulate Aryl Hydrocarbon Receptor and Estrogen Receptor Alpha Activity through Multiple Transcriptomic Targets in MCF-7 Human Breast Cancer Cells.

Inhibitory crosstalk between estrogen receptor alpha (ERα) and aryl hydrocarbon receptor (AHR) regulates 17ß-estradiol (E2)-dependent breast cancer cell signaling. ERα and AHR are transcription factors activated by E2 and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Dietary ligands resveratrol (RES) and 3,3'diindolylmethane (DIM) also activate ERα while only DIM activates AHR and RES represses it. DIM and RES are reported to have anti-cancer and anti-inflammatory properties. Studies with genome-wide targets and AHR- and ERα-regulated genes after DIM and RES are unknown. We used chromatin immunoprecipitation with high-throughput sequencing and transcriptomics to study ERα as well as AHR coregulation in MCF-7 human breast cancer cells treated with DIM, RES, E2, or TCDD alone or E2+TCDD for 1 and 6 h, respectively. ERα bound sites after being DIM enriched for the AHR motif but not after E2 or RES while AHR bound sites after being DIM and E2+TCDD enriched for the ERE motif but not after TCDD. More than 90% of the differentially expressed genes closest to an AHR binding site after DIM or E2+TCDD also had an ERα site, and 60% of the coregulated genes between DIM and E2+TCDD were common. Collectively, our data show that RES and DIM differentially regulate multiple transcriptomic targets via ERα and ERα/AHR coactivity, respectively, which need to be considered to properly interpret their cellular and biological responses. These novel data also suggest that, when both receptors are activated, ERα dominates with preferential recruitment of AHR to ERα target genes.

High-throughput methods for the analysis of transcription factors and chromatin modifications: Low input, single cell and spatial genomic technologies.

Genome-wide analysis of transcription factors and epigenomic features is instrumental to shed light on DNA-templated regulatory processes such as transcription, cellular differentiation or to monitor cellular responses to environmental cues. Two decades of technological developments have led to a rich set of approaches progressively pushing the limits of epigenetic profiling towards single cells. More recently, disruptive technologies using innovative biochemistry came into play. Assays such as CUT&RUN, CUT&Tag and variations thereof show considerable potential to survey multiple TFs or histone modifications in parallel from a single experiment and in native conditions. These are in the path to become the dominant assays for genome-wide analysis of TFs and chromatin modifications in bulk, single-cell, and spatial genomic applications. The principles together with pros and cons are discussed.

Multi-omics data of gastric cancer cell lines.

OBJECTIVES: Gastric cancer (GC) is the fourth most common cancer worldwide, with the highest incidence and mortality regardless of sex. Despite technological advances in diagnosing and treating gastric cancer, GC still has high incidence and mortality rates. Therefore, continuous research is needed to overcome GC. In various studies, cell lines are used to find and verify the cause of specific diseases. Large-scale genomic studies such as ENCODE and Roadmap epigenomic projects provide multiomics data from various organisms and samples. However, few multi-omics data for gastric tissues and cell lines have been generated. Therefore, we performed RNA-seq, Exome-seq, and ChIP-seq with several gastric cell lines to generate a multi-omics data set in gastric cancer. DATA DESCRIPTION: Multiomic data, such as RNA-seq, Exome-seq, and ChIP-seq, were produced in gastric cancer and normal cell lines. RNA-seq data were generated from nine GC and one normal gastric cell line, mapped to a human reference genome (hg38) using the STAR alignment tool, and quantified with HTseq. Exome sequence data were produced in nine GC and two normal gastric lines. Sequenced reads were mapped and processed using BWA-MEM and GATK, variants were called by stralka2, and annotation was performed using ANNOVAR. Finally, for the ChIP-seq, nine GC cell lines and four GC cell lines were used in two experimental sets; chip-seq was performed to confirm changes in H3K4me3 and H3K27me3. Data was mapped to human reference hg38 with BWA-MEM, and peak calling and annotation were performed using the Homer tool. Since these data provide multi-omics data for GC cell lines, it will be useful for researchers who use the GC cell lines to study.

In vivo RNA-seq and ChIP-seq analyses show an obligatory role for the C terminus of p53 in conferring tissue-specific radiation sensitivity.

Thymus and spleen, in contrast to liver, are radiosensitive tissues in which p53-dependent apoptosis is triggered after whole-body radiation in vivo. Combined RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analyses of radiation-treated mouse organs identifies both shared and tissue-specific p53 transcriptional responses. As expected, the p53 targets shared among thymus and spleen are enriched in apoptotic targets. The inability to upregulate these genes in the liver is not due to reduced gene occupancy. Use of an engineered mouse model shows that deletion of the C terminus of p53 can confer radiation-induced expression of p53 apoptotic targets in the liver with concomitant increased cell death. Global RNA-seq analysis reveals that an additional role of the C terminus is also needed for transcriptional activation of liver-specific p53 targets. It is hypothesized that both suppression of apoptotic gene expression combined with enhanced activation of liver-specific targets confers tissue-specific radio-resistance.

Cell Type-Specific Whole-Genome Landscape of ΔFOSB Binding in the Nucleus Accumbens After Chronic Cocaine Exposure.

BACKGROUND: The ability of neurons to respond to external stimuli involves adaptations of gene expression. Induction of the transcription factor ΔFOSB in the nucleus accumbens, a key brain reward region, is important for the development of drug addiction. However, a comprehensive map of ΔFOSB's gene targets has not yet been generated. METHODS: We used CUT&RUN (cleavage under targets and release using nuclease) to map the genome-wide changes in ΔFOSB binding in the 2 main types of nucleus accumbens neurons-D1 or D2 medium spiny neurons-after chronic cocaine exposure. To annotate genomic regions of ΔFOSB binding sites, we also examined the distributions of several histone modifications. Resulting datasets were leveraged for multiple bioinformatic analyses. RESULTS: The majority of ΔFOSB peaks occur outside promoter regions, including intergenic regions, and are surrounded by epigenetic marks indicative of active enhancers. BRG1, the core subunit of the SWI/SNF chromatin remodeling complex, overlaps with ΔFOSB peaks, a finding consistent with earlier studies of ΔFOSB's interacting proteins. Chronic cocaine use induces broad changes in ΔFOSB binding in both D1 and D2 nucleus accumbens medium spiny neurons of male and female mice. In addition, in silico analyses predict that ΔFOSB cooperatively regulates gene expression with homeobox and T-box transcription factors. CONCLUSIONS: These novel findings uncover key elements of ΔFOSB's molecular mechanisms in transcriptional regulation at baseline and in response to chronic cocaine exposure. Further characterization of ΔFOSB's collaborative transcriptional and chromatin partners specifically in D1 and D2 medium spiny neurons will reveal a broader picture of the function of ΔFOSB and the molecular basis of drug addiction.

Genome-wide Analysis of Histone H3 Lysine 27 Trimethylation Profiles in Sciatic Nerve of Chronic Constriction Injury Rats.

The histone H3 lysine 27 trimethylation (H3K27me3) is one of the most important chromatin modifications, which is associated with injury-activated gene expression in Schwann cells (SCs). However, the alteration of genome-wide H3K27me3 enrichments in the development of neuropathic pain is still unknown. Here, we applied the chromatin immunoprecipitation sequencing (ChIP-seq) approach to identify the alteration of differential enrichments of H3K27me3 in chronic constriction injury (CCI) sciatic nerve of rats and potential molecular mechanisms underlying the development of neuropathic pain. Our results indicated that CCI increased the numbers of SCs displaying H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) and H3K27me3 in the sciatic nerve. ChIP-seq data showed that CCI significantly changed H3K27me3 enrichments on gene promoters in the sciatic nerve. Bioinformatics analyses exhibited that genes gaining H3K27me3 were mostly associated with regulation of cell proliferation, response to stress and oxidation-reduction process. Genes losing this mark were enriched in neuronal generation, and MAPK, cAMP as well as ERBB signaling pathways. Importantly, IL1A, CCL2, NOS2, S100A8, BDNF, GDNF, ERBB3 and C3 were identified as key genes in neuropathic pain. CCI led to significant upregulation of key genes in the sciatic nerve. EZH2 inhibitor reversed CCI-induced increases of H3K27me3 and key gene protein levels, which were accompanied by relieved mechanical allodynia and thermal hyperalgesia in CCI rats. These results indicate that genes with differential enrichments of H3K27me3 in SCs function in various cellular processes and pathways, and many are linked to neuropathic pain after peripheral nerve injury.

Upregulation of PD-L1 by SARS-CoV-2 promotes immune evasion.

Patients with severe COVID-19 often suffer from lymphopenia, which is linked to T-cell sequestration, cytokine storm, and mortality. However, it remains largely unknown how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces lymphopenia. Here, we studied the transcriptomic profile and epigenomic alterations involved in cytokine production by SARS-CoV-2-infected cells. We adopted a reverse time-order gene coexpression network approach to analyze time-series RNA-sequencing data, revealing epigenetic modifications at the late stage of viral egress. Furthermore, we identified SARS-CoV-2-activated nuclear factor-κB (NF-κB) and interferon regulatory factor 1 (IRF1) pathways contributing to viral infection and COVID-19 severity through epigenetic analysis of H3K4me3 chromatin immunoprecipitation sequencing. Cross-referencing our transcriptomic and epigenomic data sets revealed that coupling NF-κB and IRF1 pathways mediate programmed death ligand-1 (PD-L1) immunosuppressive programs. Interestingly, we observed higher PD-L1 expression in Omicron-infected cells than SARS-CoV-2 infected cells. Blocking PD-L1 at an early stage of virally-infected AAV-hACE2 mice significantly recovered lymphocyte counts and lowered inflammatory cytokine levels. Our findings indicate that targeting the SARS-CoV-2-mediated NF-κB and IRF1-PD-L1 axis may represent an alternative strategy to reduce COVID-19 severity.

Timing of Blood Sample Processing Affects the Transcriptomic and Epigenomic Profiles in CD4+ T-cells of Atopic Subjects.

Optimal pre-analytical conditions for blood sample processing and isolation of selected cell populations for subsequent transcriptomic and epigenomic studies are required to obtain robust and reproducible results. This pilot study was conducted to investigate the potential effects of timing of CD4+ T-cell processing from peripheral blood of atopic and non-atopic adults on their transcriptomic and epigenetic profiles. Two heparinized blood samples were drawn from each of three atopic and three healthy individuals. For each individual, CD4+ T-cells were isolated from the first blood sample within 2 h (immediate) or from the second blood sample after 24 h storage (delayed). RNA sequencing (RNA-Seq) and histone H3K27 acetylation chromatin immunoprecipitation sequencing (ChIP-Seq) analyses were performed. A multiplicity of genes was shown to be differentially expressed in immediately processed CD4+ T-cells from atopic versus healthy subjects. These differences disappeared when comparing delayed processed cells due to a drastic change in expression levels of atopy-related genes in delayed processed CD4+ T-cells from atopic donors. This finding was further validated on the epigenomic level by examining H3K27 acetylation profiles. In contrast, transcriptomic and epigenomic profiles of blood CD4+ T-cells of healthy donors remained rather unaffected. Taken together, for successful transcriptomics and epigenomics studies, detailed standard operation procedures developed on the basis of samples from both healthy and disease conditions are implicitly recommended.

Global Gene Expression Regulation Mediated by TGFß Through H3K9me3 Mark.

Background: Epigenetic alterations play an important part in carcinogenesis. Different biological responses, including cell proliferation, migration, apoptosis, invasion, and senescence, are affected by epigenetic alterations in cancer. In addition, growth factors, such as transforming growth factor beta (TGFß) are important regulators of tumorigenesis. Our understanding of the interplay between the epigenetic bases of tumorigenesis and growth factor signaling in tumorigenesis is rudimentary. Some studies suggest a link between TGFß signaling and the heterochromatinizing histone mark H3K9me3. There is evidence for signal-dependent interactions between R-Smads and histone methyltransferases. However, the effects of TGFß signaling on genome wide H3K9me3 landscape remains unknown. Our research examines TGFß -induced genome-wide H3K9me3 in prostate cancer. Method: Chromatin-Immunoprecipitation followed by sequencing was performed to analyze genome-wide association of H3K9me3 epigenetic mark. DAVID Functional annotation tool was utilized to understand the involvement of different Biological Processes and Molecular Function. MEME-ChIP tool was also used to analyze known and novel DNA-binding motifs. Results: H3K9me3 occupancy appears to increase at intronic regions after short-term (6 hours) TGFß stimulation and at distal intergenic regions during long-term stimulation (24 hours). We also found evidence for a possible association of SLC transporters with H3K9me3 mark in presence of TGFß during tumorigenesis. No direct correlation was found between the occupancy of H3K9me3 mark and the expression of various genes. The epigenetic mechanisms-mediated regulation of gene expression by TGFß was concentrated at promoters rich in SRY and FOXJ3 binding sites. Conclusion: Our results point toward a positive association of oncogenic function of TGFß and the H3K9me3 mark and provide a context to the role of H3K9me3 in TGFß-induced cell migration and cell adhesion. Interestingly, these functions of TGFß through H3K9me3 mark regulation seem to depend on transcriptional activation in contrast to the conventionally known repressive nature of H3K9me3.

Interspecies Single-Cell RNA-Seq Analysis Reveals the Novel Trajectory of Osteoclast Differentiation and Therapeutic Targets.

Bone turnover is finely tuned by cells in the bone milieu, including osteoblasts, osteoclasts, and osteocytes. Osteoclasts are multinucleated giant cells with a bone-resorbing function that play a critical role in regulating skeletal homeostasis. Osteoclast differentiation is characterized by dramatic changes in morphology and gene expression following receptor activator of nuclear factor-kappa-Β ligand (RANKL) stimulation. We performed single-cell RNA-sequencing analyses of human and murine osteoclast-lineage cells (OLCs) and found that OLCs in the mitotic phase do not differentiate into mature osteoclasts. We also identified a guanosine triphosphatase (GTPase) family member, RAB38, as a highly expressed molecule in both human and murine osteoclast clusters; RAB38 gene expression is associated with dynamic changes in histone modification and transcriptional regulation. Silencing Rab38 expression by using short hairpin RNA (shRNA) inhibited osteoclast differentiation and maturation. In summary, we established an integrated fate map of human and murine osteoclastogenesis; this will help identify therapeutic targets in bone diseases. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

An optimized ChIP-Seq framework for profiling histone modifications in Chromochloris zofingiensis.

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high-quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein cross-linking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

Genome-Wide ChIP-seq and RNA-seq Analyses of STAT3 Target Genes in TLRs Activated Human Peripheral Blood B Cells.

Toll like receptors (TLRs) induced response plays a vital role in B-cell development and activation, in which TLR7-mediated and TLR9-mediated response interact together and play antagonistic or cooperative roles at different situations. Previous studies showed that the transcription factor signal transducer and activator of transcription (STAT) 3 was one of the key transcriptional factors (TFs) needed for both TLR7 and TLR9 signaling in B cell, and patients with autosomal dominant hyper IgE syndromes (AD-HIES) due to STAT3 mutations having defective TLRs response in B cells. However, how STAT3 affects its target genes and the downstream signaling pathways in B cell upon TLRs stimulation remains unclarified on a genome-wide level. ChIP-seq and RNA-seq was used in this study to identify the STAT3 targets in response to TLRs stimulation in human B cell. STAT3 ChIP-seq results showed a total of 611 and 2,289 differential STAT3-binding sites in human B cell after TLR7 and TLR9 agonists stimulation, respectively. RNA-seq results showed 1,186 and 1,775 differentially expressed genes after TLR7 and TLR9 activation, respectively. We identified 47 primary STAT3 target genes after TLR7 activation and 189 target genes after TLR9 activation in B cell by integration of STAT3 ChIP-seq and RNA-seq data. Among these STAT3 primary targets, we identified 7 TFs and 18 TFs for TLR7 and TLR9 response, respectively. Besides, we showed that STAT3 might regulate TLR9, but not TLR7 response in B cells through directly regulating integrin signaling pathway, which might further affect the antagonism between TLR7 and TLR9 signaling in B cell. Our study provides insights into the molecular mechanism of human TLRs response in B cell and how it can be regulated, which helps to better understand and modulate TLR-mediated pathogenic immune responses in B cell.

SEAseq: a portable and cloud-based chromatin occupancy analysis suite.

BACKGROUND: Genome-wide protein-DNA binding is popularly assessed using specific antibody pulldown in Chromatin Immunoprecipitation Sequencing (ChIP-Seq) or Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing experiments. These technologies generate high-throughput sequencing data that necessitate the use of multiple sophisticated, computationally intensive genomic tools to make discoveries, but these genomic tools often have a high barrier to use because of computational resource constraints. RESULTS: We present a comprehensive, infrastructure-independent, computational pipeline called SEAseq, which leverages field-standard, open-source tools for processing and analyzing ChIP-Seq/CUT&RUN data. SEAseq performs extensive analyses from the raw output of the experiment, including alignment, peak calling, motif analysis, promoters and metagene coverage profiling, peak annotation distribution, clustered/stitched peaks (e.g. super-enhancer) identification, and multiple relevant quality assessment metrics, as well as automatic interfacing with data in GEO/SRA. SEAseq enables rapid and cost-effective resource for analysis of both new and publicly available datasets as demonstrated in our comparative case studies. CONCLUSIONS: The easy-to-use and versatile design of SEAseq makes it a reliable and efficient resource for ensuring high quality analysis. Its cloud implementation enables a broad suite of analyses in environments with constrained computational resources. SEAseq is platform-independent and is aimed to be usable by everyone with or without programming skills. It is available on the cloud at https://platform.stjude.cloud/workflows/seaseq and can be locally installed from the repository at https://github.com/stjude/seaseq .