Cell Type-specific Transcription Factors Research Articles

Single-cell insights: pioneering an integrated atlas of chromatin accessibility and transcriptomic landscapes in diabetic cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) poses a growing health threat, elevating heart failure risk in diabetic individuals. Understanding DCM is crucial, with fibroblasts and endothelial cells playing pivotal roles in driving myocardial fibrosis and contributing to cardiac dysfunction. Advances in Multimodal single-cell profiling, such as scRNA-seq and scATAC-seq, provide deeper insights into DCM’s unique cell states and molecular landscape for targeted therapeutic interventions.MethodsSingle-cell RNA and ATAC data from 10x Multiome libraries were processed using Cell Ranger ARC v2.0.1. Gene expression and ATAC data underwent Seurat and Signac filtration. Differential gene expression and accessible chromatin regions were identified. Transcription factor activity was estimated with chromVAR, and Cis-coaccessibility networks were calculated using Cicero. Coaccessibility connections were compared to the GeneHancer database. Gene Ontology analysis, biological process scoring, cell-cell communication analysis, and gene-motif correlation was performed to reveal intricate molecular changes. Immunofluorescent staining utilized various antibodies on paraffin-embedded tissues to verify the findings.ResultsThis study integrated scRNA-seq and scATAC-seq data obtained from hearts of WT and DCM mice, elucidating molecular changes at the single-cell level throughout the diabetic cardiomyopathy progression. Robust and accurate clustering analysis of the integrated data revealed altered cell proportions, showcasing decreased endothelial cells and macrophages, coupled with increased fibroblasts and myocardial cells in the DCM group, indicating enhanced fibrosis and endothelial damage. Chromatin accessibility analysis unveiled unique patterns in cell types, with heightened transcriptional activity in myocardial cells. Subpopulation analysis highlighted distinct changes in cardiomyocytes and fibroblasts, emphasizing pathways related to fatty acid metabolism and cardiac contraction. Fibroblast-centered communication analysis identified interactions with endothelial cells, implicating VEGF receptors. Endothelial cell subpopulations exhibited altered gene expressions, emphasizing contraction and growth-related pathways. Candidate regulators, including Tcf21, Arnt, Stat5a, and Stat5b, were identified, suggesting their pivotal roles in DCM development. Immunofluorescence staining validated marker genes of cell subpopulations, confirming PDK4, PPARγ and Tpm1 as markers for metabolic pattern-altered cardiomyocytes, activated fibroblasts and endothelial cells with compromised proliferation.ConclusionOur integrated scRNA-seq and scATAC-seq analysis unveils intricate cell states and molecular alterations in diabetic cardiomyopathy. Identified cell type-specific changes, transcription factors, and marker genes offer valuable insights. The study sheds light on potential therapeutic targets for DCM.

Evolution of the enhancer-rich regulatory region of the gene for the cell-type specific transcription factor POU1F1

Precise spatio-temporal expression of genes in organogenesis is regulated by the coordinated interplay of DNA elements such as promoter and enhancers present in the regulatory region of a given locus. POU1F1 transcription factor plays a crucial role in the development of somatotrophs, lactotrophs and thyrotrophs in the anterior pituitary gland, and in maintaining high expression of growth hormone, prolactin and TSH. In mouse, expression of POU1F1 is controlled by a region fenced by two CTCF sites, containing 5 upstream enhancer elements, designated E-A (5′ to 3′). Elements C, B and A correspond to elements shown previously to play a role in pituitary development and hormonal expression; functional roles for elements E and D have not been reported. We performed comparative sequence analysis of this regulatory region and discovered that three elements, B, C and E, are present in all vertebrate groups except Agnatha. One very long (>2 kb) element (A) is unique to mammals suggesting a specific change in regulation of the gene in this group. Using DNA accessibility assay (ATAC-seq) we showed that conserved elements in anterior pituitary of four non-mammals are open, suggesting functionality as regulatory elements. We showed that, in many non-mammalian vertebrates, an additional upstream exon closely follows element E, leading to alternatively spliced transcripts. Here, element E functions as an alternative promoter, but in mammals this feature is lost, suggesting conversion of alternative promoter to enhancer. Our work shows that regulation of POU1F1 changed markedly during the course of vertebrate evolution, use of a low number of enhancer elements combined with alternative promoters in non-mammalian vertebrates being replaced by use of a unique combination of regulatory units in mammals. Most importantly, our work suggests that evolutionary conversion of alternate promoter to enhancer could be one of the evolutionary mechanisms of enhancer birth.

Abstract 1686: Dissecting the 3D enhancer-promoter hubs in prostate tumorigenesis

Abstract The transcription process is controlled by regulatory elements that are often located in non-coding regions. Among regulatory elements, activities of enhancers that are bound by cell-type specific transcription factors (TFs) are tightly linked to cell identity. Enhancers loop to interact with the promoters of target genes at a distance to drive different cell fates. Evidently, transcriptional dysregulation linked to enhancers that are differentially activated in cancer is an underlying factor in tumorigenesis. To identify enhancers linked to prostate tumorigenesis, we analyzed hundreds of in-house and public H3K27ac ChIP-seq data generated from normal prostate and prostate cancer cell lines and tissues. We identified thousands of prostate cancer-specific enhancers (PSEs) that were activated exclusively in cancer conditions. To further identify the target genes of PSEs, we performed 3C-derived assays, such as Hi-C and Micro-C, and mapped genome-wide interactions. However, they were limited by the resolution to detect enhancer-promoter interactions. The recent development of Region Capture Micro-C (RCMC), a hybridization-based capture Micro-C method, allowed us to deeply map the highly nested interactions between regulatory elements. By utilizing RCMC, we generated high-resolution, focal contact maps that revealed comprehensive enhancer-promoter interactions near PSEs in prostate cancer cells. Interestingly, we found that PSEs loop to other PSEs clustered in the proximal genomic region, in addition to promoters of significantly upregulated genes in cancer compared to normal cells. We termed these regions as multi-connected "3D enhancer-promoter hubs”. To mechanistically dissect these hubs, we deleted 5+ PSEs one by one using the CRISPR/Cas9 system and performed multi-omics analyses, including RNA-seq and ChIP-seq. We found that single deletions of a subset of PSEs led to the downregulation of multiple genes looped to the deleted PSE as well as to other PSEs. Moreover, we found that deletion reduced the activity of other connected PSEs. When we further evaluated the chromatin contacts of regulatory elements in the PSE-deleted cells, we observed that many chromatin contacts remained unaffected. Finally, we identified TFs that bind to PSEs, and we are currently in the process of investigating enhancer-promoter hub connectivity by performing knockdown experiments on these TFs. Furthermore, we are using molecular assays to identify PSEs that could exert a phenotypic shift in cancer cell identity to drive prostate tumorigenesis. Overall, our study not only provides mechanistic insights into the spatial regulation of 3D enhancer-promoter hubs but also identifies new non-coding drivers in prostate tumorigenesis. Citation Format: Huan Cao, Zexun Wu, Leonardo Gonzalez-Smith, Seolyn Yang, Suhn K. Rhie. Dissecting the 3D enhancer-promoter hubs in prostate tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1686.

Deciphering the spatiotemporal transcriptional and chromatin accessibility of human retinal organoid development at the single-cell level

Molecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids (ROs) provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-seq and spatial transcriptomics we present for the first-time a single cell spatiotemporal transcriptome of RO development. Our data demonstrate that ROs recapitulate key events of retinogenesis including optic vesicle/cup formation, presence of a putative ciliary margin zone, emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-seq data, we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development, and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal neurons.

Exploring intrinsic variability between cultured nasal and bronchial epithelia in cystic fibrosis

The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have uncovered that nasal and bronchial tissues exhibit intrinsic variability, including differences in mucociliary cell composition and expression of unique transcriptional regulatory proteins which relate to germ layer origin. In the present study, we explored whether intrinsic differences between nasal and bronchial epithelial cells persist in cell cultures and affect epithelial cell functioning in CF. Comparison of air–liquid interface (ALI) differentiated epithelial cells from subjects with CF revealed distinct mucociliary differentiation states of nasal and bronchial cultures. Moreover, using RNA sequencing we identified cell type-specific signature transcription factors in differentiated nasal and bronchial epithelial cells, some of which were already poised for expression in basal progenitor cells as evidenced by ATAC sequencing. Analysis of differentiated nasal and bronchial epithelial 3D organoids revealed distinct capacities for fluid secretion, which was linked to differences in ciliated cell differentiation. In conclusion, we show that unique phenotypical and functional features of nasal and bronchial epithelial cells persist in cell culture models, which can be further used to investigate the effects of tissue-specific features on upper and lower respiratory disease development in CF.

Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

Analysis of potential biomarkers for diabetic kidney disease based on single-cell RNA-sequencing integrated with a single-cell sequencing assay for transposase-accessible chromatin.

Diabetic kidney disease (DKD) is a renal microvascular disease caused by hyperglycemia that involves metabolic remodeling, oxidative stress, inflammation, and other factors. The mechanism is complex and not fully unraveled. We performed an integrated single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) and single-cell RNA-sequencing (scRNA-seq) analyses of kidneys from db/db and db/m mice to identify differential open chromatin regions and gene expression, particularly in genes related to proximal tubular reabsorption and secretion. We identified 9,776 differentially expressed genes (DEGs) and 884 cell type-specific transcription factors (TFs) across 15 cell types. Glucose and lipid transporters, and TFs related to the circadian rhythm in the proximal tubules had significantly higher expression in db/db mice than in db/m mice (P<0.01). Crosstalk between podocytes and tubular cells in the proximal tubules was enhanced, and renal inflammation, oxidative stress, and fibrosis pathways were activated in db/db mice. Western blotting and immunohistochemical staining results showed that Wfdc2 expression in the urine and kidneys of DKD patients was higher than that in non-diabetic kidney disease (NDKD) controls. The revealed landscape of chromatin accessibility and transcriptional profiles in db/db mice provide insights into the pathological mechanism of DKD.

Single-cell ATAC sequencing illuminates the cis-regulatory differentiation of taxol biosynthesis between leaf mesophyll and leaf epidermal cells in Taxus mairei

Taxol is a widely used anticancer drug; however, its accumulation pattern can vary largely among leaf tissues. Taxol biosynthesis is driven by a series of transcription factors (TFs), but the extent of chromatin changes that functionally regulate gene expression associated with taxol biosynthesis remain poorly understood. To assess the effects of tissue heterogeneity on taxol biosynthesis, we applied single-cell transposase-accessible chromatin sequencing to Taxus mairei leaves and identified differentially accessible regions. We captured 9488 independent cells with 42,192 fragment reads per cell. In total, 66.64% were confidently mapped onto the T. mairei genome. Projection analysis divided all the cells into nine clusters, which suggests a high degree of cell heterogeneity in T. mairei leaves. Based on the cell type markers, Clusters 2 and 7 were annotated as leaf mesophyll cells, and Clusters 5 and 6 were annotated as leaf epidermal cells. A differential accessibility analysis identified 9600 and 8538 leaf mesophyll and epidermal, respectively, cell-specific open chromatin regions. We predicted the genomic features of the genes involved in different stages of taxol biosynthesis, and there was a close correlation between chromatin open peaks and the expression levels of several taxol biosynthesis-related genes. We further identified a number of cell type-specific TF motifs and reference TFs that appeared to be involved in taxol biosynthesis. The study of T. mairei leaves at a single-cell resolution provides a valuable resource for determining the basic principles of the cell type-specific regulation of taxol biosynthesis.

Smarca4 deficiency induces Pttg1 oncogene upregulation and hyperproliferation of tubular and interstitial cells during kidney development.

Kidney formation and nephrogenesis are controlled by precise spatiotemporal gene expression programs, which are coordinately regulated by cell-cycle, cell type-specific transcription factors and epigenetic/chromatin regulators. However, the roles of epigenetic/chromatin regulators in kidney development and disease remain poorly understood. In this study, we investigated the impact of deleting the chromatin remodeling factor Smarca4 (Brg1), a human Wilms tumor-associated gene, in Wnt4-expressing cells. Smarca4 deficiency led to severe tubular defects and a shortened medulla. Through unbiased single-cell RNA sequencing analyses, we identified multiple types of Wnt4 Cre-labeled interstitial cells, along with nephron-related cells. Smarca4 deficiency increased interstitial cells but markedly reduced tubular cells, resulting in cells with mixed identity and elevated expression of cell-cycle regulators and genes associated with extracellular matrix and epithelial-to-mesenchymal transition/fibrosis. We found that Smarca4 loss induced a significant upregulation of the oncogene Pttg1 and hyperproliferation of Wnt4 Cre-labeled cells. These changes in the cellular state could hinder the cellular transition into characteristic tubular structures, eventually leading to fibrosis. In conclusion, our findings shed light on novel cell types and genes associated with Wnt4 Cre-labeled cells and highlight the critical role of Smarca4 in regulating tubular cell differentiation and the expression of the cancer-causing gene Pttg1 in the kidney. These findings may provide valuable insights into potential therapeutic strategies for renal cell carcinoma resulting from SMARCA4 deficiency.

Refined read-out: The hUHRF1 Tandem-Tudor domain prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3.

UHRF1 is an essential chromatin protein required for DNA methylation maintenance, mammalian development, and gene regulation. We investigated the Tandem-Tudor domain (TTD) of human UHRF1 that is known to bind H3K9me2/3 histones and is a major driver of UHRF1 localization in cells. We verified binding to H3K9me2/3 but unexpectedly discovered stronger binding to H3 peptides and mononucleosomes containing K9me2/3 with additional K4me1. We investigated the combined binding of TTD to H3K4me1-K9me2/3 versus H3K9me2/3 alone, engineered mutants with specific and differential changes of binding, and discovered a novel read-out mechanism for H3K4me1 in an H3K9me2/3 context that is based on the interaction of R207 with the H3K4me1 methyl group and on counting the H-bond capacity of H3K4. Individual TTD mutants showed up to a 10,000-fold preference for the double-modified peptides, suggesting that after a conformational change, WT TTD could exhibit similar effects. The frequent appearance of H3K4me1-K9me2 regions in human chromatin demonstrated in our TTD chromatin pull-down and ChIP-western blot data suggests that it has specific biological roles. Chromatin pull-down of TTD from HepG2 cells and full-length murine UHRF1 ChIP-seq data correlate with H3K4me1 profiles indicating that the H3K4me1-K9me2/3 interaction of TTD influences chromatin binding of full-length UHRF1. We demonstrate the H3K4me1-K9me2/3 specific binding of UHRF1-TTD to enhancers and promoters of cell-type-specific genes at the flanks of cell-type-specific transcription factor binding sites, and provided evidence supporting an H3K4me1-K9me2/3 dependent and TTD mediated downregulation of these genes by UHRF1. All these findings illustrate the important physiological function of UHRF1-TTD binding to H3K4me1-K9me2/3 double marks in a cellular context.

METB-03. THE DISTINCT GENOMICS OF HIGH-GRADE INFANT GLIOMAS AMONG PEDIATRIC GLIOMAS HARBORING RECEPTOR TYROSINE KINASE FUSIONS

Abstract Gliomas account for about 60% of pediatric brain tumors, about half of which are classified as high-grade. Recent studies have shown that infants with high-grade gliomas (HGG) harboring receptor tyrosine kinase (RTK) fusions have a more favorable prognosis than pediatric HGG patients. The molecular mechanisms for this favorable, but from the histology divergent clinical outcome, are yet unknown. We analyzed n=18 brain tumor samples (n=7 infant, n=11 pediatric) of patients with low-grade glioma (LGGs) or HGG presenting with RTK fusions using single-cell RNA sequencing. Considering the different fusion genes (ALK n=6, NTRK n=8, and MET n=4), age groups, and glioma grades, we evaluated the metaprograms of tumor cells and their proportions using non-negative matrix factorization. Preliminary results show similar metaprograms comparing infant and pediatric LGG. Cells detected as MES-like did not occur in LGG but were found in several HGG samples. However, in HGG, the MES-like cells show different expression patterns for infant and pediatric samples. In addition, compared with pediatric HGG, infant HGG has a higher proportion of programs that are predominantly present in LGG, such as AC-like and NPC-like. These initial findings serve as a starting point for cell type-specific transcription factor regulatory networks and are an important step toward understanding the underlying biology of this distinct pediatric brain tumor entity.

Single-Cell RNA Sequencing of Sox17-Expressing Lineages Reveals Distinct Gene Regulatory Networks and Dynamic Developmental Trajectories.

During early embryogenesis, the transcription factor SOX17 contributes to hepato-pancreato-biliary system formation and vascular-hematopoietic emergence. To better understand Sox17 function in the developing endoderm and endothelium, we developed a dual-color temporal lineage-tracing strategy in mice combined with single-cell RNA sequencing to analyze 6934 cells from Sox17-expressing lineages at embryonic days 9.0-9.5. Our analyses showed 19 distinct cellular clusters combined from all 3 germ layers. Differential gene expression, trajectory and RNA-velocity analyses of endothelial cells revealed a heterogenous population of uncommitted and specialized endothelial subtypes, including 2 hemogenic populations that arise from different origins. Similarly, analyses of posterior foregut endoderm revealed subsets of hepatic, pancreatic, and biliary progenitors with overlapping developmental potency. Calculated gene-regulatory networks predict gene regulons that are dominated by cell type-specific transcription factors unique to each lineage. Vastly different Sox17 regulons found in endoderm versus endothelial cells support the differential interactions of SOX17 with other regulatory factors thereby enabling lineage-specific regulatory actions.

Super-enhancer-associated transcription factors collaboratively regulate trophoblast-active gene expression programs in human trophoblast stem cells.

The placenta is an essential organ that supports the growth and development of the fetus during pregnancy. However, cell type-specific enhancers and transcription factors (TFs), and the mechanisms underlying the maintenance and differentiation of trophoblast stem cell (TSC) populations in the human placenta remain elusive. Here, using human TSCs as a model system, we identify 31,362 enhancers that are enriched with the motifs of previously reported TSC-pivotal TFs, including TEAD4, GATA2/3 and TFAP2C. Subsequently, we identify 580 super-enhancers (SEs) and 549 SE-associated genes. These genes are robustly expressed in the human placenta and include numerous TFs, implying that SE-associated TFs (SE-TFs) may play crucial roles in placental development. Additionally, we identify the global binding sites of five TSC-pivotal SE-TFs (FOS, GATA2, MAFK, TEAD4 and TFAP2C), revealing that they preferentially co-occupy enhancers, regulate each otherand form a trophoblast-active gene regulatory network. Loss-of-function studies unveil that the five TFs promote self-renewal of TSCs by activating proliferation-associated genes while repressing developmental genes. We further reveal that the five TFs exert conserved and unique functions on placental development between humans and mice. Our study provides important insights into the roles of human TSC-pivotal TFs in regulating placenta-specific gene expression programs.

How Deepbics Quantifies Intensities of Transcription Factor-DNA Binding and Facilitates Prediction of Single Nucleotide Variant Pathogenicity With a Deep Learning Model Trained On ChIP-Seq Data Sets.

The binding of DNA sequences to cell type-specific transcription factors is essential for regulating gene expression in all organisms. Many variants occurring in these binding regions play crucial roles in human disease by disrupting the cis-regulation of gene expression. We first implemented a sequence-based deep learning model called deepBICS to quantify the intensity of transcription factors-DNA binding. The experimental results not only showed the superiority of deepBICS on ChIP-seq data sets but also suggested deepBICS as a language model could help the classification of disease-related and neutral variants. We then built a language model-based method called deepBICS4SNV to predict the pathogenicity of single nucleotide variants. The good performance of deepBICS4SNV on 2 tests related to Mendelian disorders and viral diseases shows the sequence contextual information derived from language models can improve prediction accuracy and generalization capability.

Integrative single-cell RNA-seq and ATAC-seq analysis of myogenic differentiation in pig

BackgroundSkeletal muscle development is a multistep process whose understanding is central in a broad range of fields and applications, from the potential medical value to human society, to its economic value associated with improvement of agricultural animals. Skeletal muscle initiates in the somites, with muscle precursor cells generated in the dermomyotome and dermomyotome-derived myotome before muscle differentiation ensues, a developmentally regulated process that is well characterized in model organisms. However, the regulation of skeletal muscle ontogeny during embryonic development remains poorly defined in farm animals, for instance in pig. Here, we profiled gene expression and chromatin accessibility in developing pig somites and myotomes at single-cell resolution.ResultsWe identified myogenic cells and other cell types and constructed a differentiation trajectory of pig skeletal muscle ontogeny. Along this trajectory, the dynamic changes in gene expression and chromatin accessibility coincided with the activities of distinct cell type-specific transcription factors. Some novel genes upregulated along the differentiation trajectory showed higher expression levels in muscular dystrophy mice than that in healthy mice, suggesting their involvement in myogenesis. Integrative analysis of chromatin accessibility, gene expression data, and in vitro experiments identified EGR1 and RHOB as critical regulators of pig embryonic myogenesis.ConclusionsCollectively, our results enhance our understanding of the molecular and cellular dynamics in pig embryonic myogenesis and offer a high-quality resource for the further study of pig skeletal muscle development and human muscle disease.

Deep learning approaches for noncoding variant prioritization in neurodegenerative diseases.

Determining how noncoding genetic variants contribute to neurodegenerative dementias is fundamental to understanding disease pathogenesis, improving patient prognostication, and developing new clinical treatments. Next generation sequencing technologies have produced vast amounts of genomic data on cell type-specific transcription factor binding, gene expression, and three-dimensional chromatin interactions, with the promise of providing key insights into the biological mechanisms underlying disease. However, this data is highly complex, making it challenging for researchers to interpret, assimilate, and dissect. To this end, deep learning has emerged as a powerful tool for genome analysis that can capture the intricate patterns and dependencies within these large datasets. In this review, we organize and discuss the many unique model architectures, development philosophies, and interpretation methods that have emerged in the last few years with a focus on using deep learning to predict the impact of genetic variants on disease pathogenesis. We highlight both broadly-applicable genomic deep learning methods that can be fine-tuned to disease-specific contexts as well as existing neurodegenerative disease research, with an emphasis on Alzheimer's-specific literature. We conclude with an overview of the future of the field at the intersection of neurodegeneration, genomics, and deep learning.

SEMplMe: a tool for integrating DNA methylation effects in transcription factor binding affinity predictions

MotivationAberrant DNA methylation in transcription factor binding sites has been shown to lead to anomalous gene regulation that is strongly associated with human disease. However, the majority of methylation-sensitive positions within transcription factor binding sites remain unknown. Here we introduce SEMplMe, a computational tool to generate predictions of the effect of methylation on transcription factor binding strength in every position within a transcription factor’s motif.ResultsSEMplMe uses ChIP-seq and whole genome bisulfite sequencing to predict effects of methylation within binding sites. SEMplMe validates known methylation sensitive and insensitive positions within a binding motif, identifies cell type specific transcription factor binding driven by methylation, and outperforms SELEX-based predictions for CTCF. These predictions can be used to identify aberrant sites of DNA methylation contributing to human disease.Availability and ImplementationSEMplMe is available from https://github.com/Boyle-Lab/SEMplMe.

Toward a base-resolution panorama of the in vivo impact of cytosine methylation on transcription factor binding

BackgroundWhile methylation of CpG dinucleotides is traditionally considered antagonistic to the DNA-binding activity of most transcription factors (TFs), recent in vitro studies have revealed a more complex picture, suggesting that over a third of TFs may preferentially bind to methylated sequences. Expanding these in vitro observations to in vivo TF binding preferences is challenging since the effect of methylation of individual CpG sites cannot be easily isolated from the confounding effects of DNA accessibility and regional DNA methylation. Thus, in vivo methylation preferences of most TFs remain uncharacterized.ResultsWe introduce joint accessibility-methylation-sequence (JAMS) models, which connect the strength of the binding signal observed in ChIP-seq to the DNA accessibility of the binding site, regional methylation level, DNA sequence, and base-resolution cytosine methylation. We show that JAMS models quantitatively explain TF occupancy, recapitulate cell type-specific TF binding, and have high positive predictive value for identification of TFs affected by intra-motif methylation. Analysis of 2209 ChIP-seq experiments results in high-confidence JAMS models for 260 TFs, revealing a negative association between in vivo TF occupancy and intra-motif methylation for 45% of studied TFs, as well as 16 TFs that are predicted to bind to methylated sites, including 11 novel methyl-binding TFs mostly from the multi-zinc finger family.ConclusionsOur study substantially expands the repertoire of in vivo methyl-binding TFs, but also suggests that most TFs that prefer methylated CpGs in vitro present themselves as methylation agnostic in vivo, potentially due to the balancing effect of competition with other methyl-binding proteins.

SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues

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

From genetic variants to diseases: the role of LACC1 in T cells

Abstract Expression quantitative trait loci (eQTLs) studies provide associations of genetic variants with the expression of particular genes. But how these eQTLs directly affect gene expression and in what cell types of the immune system they act remain largely unknown. One hypothesis is that functional eQTLs in the cis-regulatory elements may affect cell type-specific transcription factor binding, thus reducing gene expression and even contributing to the cause of autoimmune and inflammatory diseases. Previously, we identified T cell-specific eQTLs of a gene known as laccase domain containing 1 (LACC1), which are GWAS hits for Crohn’s disease. Four of them are located in the LACC1 promoter region in the same haploblock. Direct association of disease-risk variants with lower LACC1 expression was confirmed by comparing pre-mRNA quantity of the different alleles in LACC1 heterozygous human CD4 T cells. We further validated the functional role of the eQTLs using CRISPR Cas9-mediated base-pair editing. LACC1 function has been analyzed primarily in macrophages and shown to affect their function, but the GWAS hits do not affect expression in macrophages. Moreover, in our in vitro studies, human CD4 T cells with LACC1 knockdown showed altered metabolic programming and proliferation, which may contribute to inflammatory diseases. Overall, our study provides an excellent model for analyzing and explaining how genetic variants contribute to diseases by influencing gene expression in specific cell types and changing cell functions.