Chromatin Mapping Research Articles

Abstract 4728: Biochemical and genomic approaches for high throughput drug discovery in chromatin remodeling research

Abstract Chromatin remodeling is mediated by ATP-dependent enzymes that play key roles regulating gene expression and genome replication/repair. Aberrant nucleosome organization from dysregulated chromatin remodeling can severely alter chromatin accessibility and disrupt these important processes, thereby driving various cancers. Remarkably, nearly 20% of all human cancers contain mutations in subunits from the SWI/SNF family of chromatin remodeling complexes, making them of great interest to basic research and therapeutic intervention. In vitro studies on the remodeling enzymes (and their multi-subunit complexes) are challenging, partially due to the strong preference for nucleosome-based substrates (the physiological target of these enzymes). We have created the EpiDyne® nucleosome portfolio to examine chromatin remodeler activity in biochemical assays, and here present the development of novel readouts (-PicoGreenTM and -TR-FRET). These nonradioactive plate-based assays are automation adaptable, ready for high-throughput inhibitor screening, and can be customized for various remodeling enzymes that exhibit preferences in nucleosome composition (e.g. histone type or DNA linker length).For parallel in vivo studies we note that genome-wide remodeler localization and open chromatin mapping are fundamental for understanding the function/activity of these enzymes in cancer development and inhibitor response. However, traditional genomic approaches have significant issues: e.g. ChIP-seq is unable to effectively map ATPases without heavily modified high-noise protocols; while ATAC-seq to map open regions cannot deal with cross-linking that could stabilize transient states on interest. To this end, we have optimized the CUTANA™ CUT&RUN approach to efficiently capture the localization of all major classes of chromatin remodelers with high signal to background. We have also adopted NicE-seq for chromatin accessibility profiling in cross-linked material. As complementary tools to the EpiDyne platform, CUT&RUN and NicE-seq facilitate epigenomic research on chromatin remodelers in cancer therapeutic intervention. Keywords: Chromatin Remodeling, EpiDyne, Nucleosome repositioning, high-throughput screening (HTS), CUTANA™ CUT&RUN, NicE-seq Citation Format: Lu Sun, Hannah Willis, Matthew R. Marunde, Vishnu U. Kumary, Matthew J. Meiners, Saarang Gopinath, Jonathan M. Burg, Bryan J. Venters, Allison Hickman, Zu-Wen Sun, Martis W. Cowles, Pierre Esteve, Hang Gyeong Chin, Chaithanya Ponnaluri, Sriharsa Pradhan, Michael-Christopher Keogh. Biochemical and genomic approaches for high throughput drug discovery in chromatin remodeling research. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4728.

Plasmodium falciparum gametocytes display global chromatin remodelling during sexual differentiation

BackgroundThe protozoan malaria parasite Plasmodium falciparum has a complex life cycle during which it needs to differentiate into multiple morphologically distinct life forms. A key process for transmission of the disease is the development of male and female gametocytes in the human blood, yet the mechanisms determining sexual dimorphism in these haploid, genetically identical sexual precursor cells remain largely unknown. To understand the epigenetic program underlying the differentiation of male and female gametocytes, we separated the two sexual forms by flow cytometry and performed RNAseq as well as comprehensive ChIPseq profiling of several histone variants and modifications.ResultsWe show that in female gametocytes the chromatin landscape is globally remodelled with respect to genome-wide patterns and combinatorial usage of histone variants and histone modifications. We identified sex specific differences in heterochromatin distribution, implicating exported proteins and ncRNAs in sex determination. Specifically in female gametocytes, the histone variants H2A.Z/H2B.Z were highly enriched in H3K9me3-associated heterochromatin. H3K27ac occupancy correlated with stage-specific gene expression, but in contrast to asexual parasites this was unlinked to H3K4me3 co-occupancy at promoters in female gametocytes.ConclusionsCollectively, we defined novel combinatorial chromatin states differentially organising the genome in gametocytes and asexual parasites and unravelled fundamental, sex-specific differences in the epigenetic code. Our chromatin maps represent an important resource for future understanding of the mechanisms driving sexual differentiation in P. falciparum.

Abstract 1953: Cell penetration and nucleus delivery of DNA methyltransferases for in vivo chromatin mapping

Abstract 1953: Cell penetration and nucleus delivery of DNA methyltransferases for in vivo chromatin mapping

ScATAC-Seq reveals heterogeneity associated with spermatogonial differentiation in cultured male germline stem cells

Spermatogonial stem cells are the most primitive spermatogonia in testis, which can self-renew to maintain the stem cell pool or differentiate to give rise to germ cells including haploid spermatids. All-trans-retinoic acid (RA), a bioactive metabolite of vitamin A, plays a fundamental role in initiating spermatogonial differentiation. In this study, single-cell ATAC-seq (scATAC-seq) was used to obtain genome-wide chromatin maps of cultured germline stem cells (GSCs) that were in control and RA-induced differentiation states. We showed that different subsets of GSCs can be distinguished based on chromatin accessibility of self-renewal and differentiation signature genes. Importantly, both progenitors and a subset of stem cells are able to respond to RA and give rise to differentiating cell subsets with distinct chromatin accessibility profiles. In this study, we identified regulatory regions that undergo chromatin remodeling and are associated with the retinoic signaling pathway. Moreover, we reconstructed the differentiation trajectory and identified novel transcription factor candidates enriched in different spermatogonia subsets. Collectively, our work provides a valuable resource for understanding the heterogeneity associated with differentiation and RA response in GSCs.

High quality mapping of chromatin at or near the nuclear lamina from small numbers of cells reveals cell cycle and developmental changes of chromatin at the nuclear periphery.

The chromatin associated with the nuclear lamina (NL) is referred to as lamina-associated domains (LADs). Here, we present an adaptation of the tyramide-signal amplification sequencing (TSA-seq) protocol, which we call chromatin pull down-based TSA-seq (cTSA-seq), that can be used to map chromatin regions at or near the NL from as little as 50 000 cells. The cTSA-seq mapped regions are composed of previously defined LADs and smaller chromatin regions that fall within the Hi-C defined B-compartment containing nuclear peripheral heterochromatin. We used cTSA-seq to map chromatin at or near the assembling NL in cultured cells progressing through early G1. cTSA-seq revealed that the distal ends of chromosomes are near or at the reassembling NL during early G1, a feature similar to those found in senescent cells. We expand the use of cTSA-seq to the mapping of chromatin at or near the NL from fixed-frozen mouse cerebellar tissue sections. This mapping reveals a general conservation of NL-associated chromatin and identifies global and local changes during cerebellar development. The cTSA-seq method reported here is useful for analyzing chromatin at or near the NL from small numbers of cells derived from both in vitro and in vivo sources.

3D chromatin maps of the human pancreas reveal lineage-specific regulatory architecture of T2D risk

Three-dimensional (3D) chromatin organization maps help dissect cell-type-specific gene regulatory programs. Furthermore, 3D chromatin maps contribute to elucidating the pathogenesis of complex genetic diseases by connecting distal regulatory regions and genetic risk variants to their respective target genes. To understand the cell-type-specific regulatory architecture of diabetes risk, we generated transcriptomic and 3D epigenomic profiles of human pancreatic acinar, alpha, and beta cells using single-cell RNA-seq, single-cell ATAC-seq, and high-resolution Hi-C of sorted cells. Comparisons of these profiles revealed differential A/B (open/closed) chromatin compartmentalization, chromatin looping, and transcriptional factor-mediated control of cell-type-specific gene regulatory programs. We identified a total of 4,750 putative causal-variant-to-target-gene pairs at 194 type 2 diabetes GWAS signals using pancreatic 3D chromatin maps. We found that the connections between candidate causal variants and their putative target effector genes are cell-type stratified and emphasize previously underappreciated roles for alpha and acinar cells in diabetes pathogenesis.

LB971 HiCuT: An efficient and low input method to identify protein-centric chromatin interactions

Protein-DNA interactions regulate gene expression, and some interactions occur over large distances, such that they are nearby in 3-D space but are separated by many nucleotides in the linear genome. These long-range chromatin loops are essential for gene regulation but remain difficult to interrogate. Methods to capture these chromatin interactions mediated by a specific protein factor include Hi-C sequencing coupled with ChIP-seq, ChIA-PET, PLAC-seq, and HiChIP. These methods all require high amounts of starting material (0.5M – 100M cells) and sequencing at high depth (a minimum of 150M reads per sample), which limits their general use. Here, we describe Hi-C Coupled chromatin cleavage and Tagmentation (HiCuT), an enzyme-based tagmentation strategy that provides efficient and high-resolution protein-centric chromatin mapping from as few as 100,000 cells and 12M sequencing reads per sample. Activated transposase generates fragment libraries with extremely low background signal that are easily interpreted with minimal computational processing. This permits cost-effective protein-centric 3D genome profiling in systems previously unmeasurable, including primary cells and human tissue samples. We validated HiCuT method for two different proteins, CTCF and RNA polymerase 2 in GM12878 cells. Next, we performed HiCuT on primary skin cells with an anti H3K27ac antibody and identified previously validated long-range chromatin interactions. Importantly, the high resolution generated by HiCuT permitted annotation of previously identified single nucleotide polymorphisms (SNPs) in skin disease to potential target genes. Thus, HiCuT will permit protein-centric 3-D genome binding assessment in rare cell populations that were not feasible previously, including primary cells, human tissue samples, and personalized epigenomics.

Studying Epigenetics of Cardiovascular Diseases on Chip Guide

Epigenetics is defined as the study of inheritable changes in the gene expressions and phenotypes that occurs without altering the normal DNA sequence. These changes are mainly due to an alteration in chromatin or its packaging, which changes the DNA accessibility. DNA methylation, histone modification, and noncoding or microRNAs can best explain the mechanism of epigenetics. There are various DNA methylated enzymes, histone-modifying enzymes, and microRNAs involved in the cause of various CVDs (cardiovascular diseases) such as cardiac hypertrophy, heart failure, and hypertension. Moreover, various CVD risk factors such as diabetes mellitus, hypoxia, aging, dyslipidemia, and their epigenetics are also discussed together with CVDs such as CHD (coronary heart disease) and PAH (pulmonary arterial hypertension). Furthermore, different techniques involved in epigenetic chromatin mapping are explained. Among these techniques, the ChIP-on-chip guide is explained with regard to its role in cardiac hypertrophy, a final form of heart failure. This review focuses on different epigenetic factors that are involved in causing cardiovascular diseases.

Abstract 3749: Mapping the DNA accessibility landscape of BCP-ALL patients revealed principles of tumor evolution

Abstract Pediatric B-Acute Lymphoblastic Leukemia (B-ALL) is the primary cause of death from hematological disease in children. Despite the enormous improvement of treatments based on innovative immunotherapy, applications of new approaches constitute a significant emergency in clinical practice. We have built the most extensive map of the accessibility landscape of 35 B-Cell Precursor ALL (BCP-ALL) to date. We integrated this map with a plethora of transcriptomic and epigenomics pan-cancer profiles to define the key determinant of BCP-ALL post-therapy relapse. We show that chromatin accessibility dynamics play a major regulatory role in BCP-ALL heterogeneity and sustain disease evolution. We have analyzed a large cohort of tissues from healthy donors (N=6) and BCP-ALL patients at time of Primary onset (N=11), Remission (N=7),or Relapse (N=9. We generated genome-wide accessibility profiles using the Assay for Transposase- Accessible Chromatin (ATAC-seq) at the coverage of 50-60 million reads of each sample. We observed that regulatory elements are strongly associated with epigenetic heterogeneity, but dominant elements have a higher chance of being shared across patients. By differential analysis of chromatin layout between healthy profiles and primary tissues, we identified 688 regulatory regions being deactivated and 758 activated during cancer early stages. Interestingly, novel activated sites recapitulate B-ALL characteristics interrogating the Disease Ontology database and gene ontology (Pval< 10-5). Relapsed patients are dominated by regulatory elements (N=~6000)originally represented at diagnosis that shrinks undertreatments and subsequently re-expand, driving the relapse. HiC maps, H3K27ac, and H3K4me1 ChIP-seq experiments integrated with chromatin maps obtained from ENCODE, GTEX, TCGA, and transcriptomic data generated by the TARGET project confirm that a large percentage of these elements are high fidelity active enhancers. The activity of these enhancers increases the risk of relapse significantly. Particularly, DCTD, MYB, and BCL2 genes are actively regulated by a set of plastic enhancer elements characterized with experimental and computational approaches. We gained insights into the role ofthe DCTD gene regulation using CRISPR-Cas9-mediated deletion of the DCTD-enhancer, leading to decreased level of DCTD gene mRNA of 40% accompanied by acell proliferation reduction of 30%. Next, we characterized the cistrome landscape of our patient cohort at accessible sites relative to the disease status. Foot printing analysis suggested ERG and EBF transcription factors (TFs) as a fundamental player in sustaining the activity of the most dynamical enhancers. In conclusion, chromatin accessibility shifts can be used to trace phenotypic changes throughout the journey of BCP-ALL patients and provide novel insights in targeting cancer phenotypical modulators. Citation Format: Giacomo Corleone, Cristina Sorino, Matteo Caforio, Stefano Di Giovenale, Francesca De Nicola, Valentina Bertaina, Angela Pitisci, Franco Locatelli, Maurizio Fanciulli, Valentina Folgiero. Mapping the DNA accessibility landscape of BCP-ALL patients revealed principles of tumor evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3749.

Chromatin accessibility analysis reveals regulatory dynamics and therapeutic relevance of Vogt-Koyanagi-Harada disease

The barrier to curing Vogt–Koyanagi–Harada disease (VKH) is thought to reside in a lack of understanding in the roles and regulations of peripheral inflammatory immune cells. Here we perform a single-cell multi-omic study of 166,149 cells in peripheral blood mononuclear cells from patients with VKH, profile the chromatin accessibility and gene expression in the same blood samples, and uncover prominent cellular heterogeneity. Immune cells in VKH blood are highly activated and pro-inflammatory. Notably, we describe an enrichment of transcription targets for nuclear factor kappa B in conventional dendritic cells (cDCs) that governed inflammation. Integrative analysis of transcriptomic and chromatin maps shows that the RELA in cDCs is related to disease complications and poor prognosis. Ligand-receptor interaction pairs also identify cDC as an important predictor that regulated multiple immune subsets. Our results reveal epigenetic and transcriptional dynamics in auto-inflammation, especially the cDC subtype that might lead to therapeutic strategies in VKH.

Distant‐acting Enhancers in Development and Disease

Multiple converging lines of evidence from experimental and human genetic studies indicate that both common and rare sequence variants involving enhancers play major roles in Mendelian and complex human disease phenotypes. We use a combination of sequence‐based molecular approaches including chromatin mapping and synthesis‐enabled modification of enhancer sequences, large‐scale transgenic mouse studies (http://enhancer.lbl.gov), and CRISPR genome editing in the mouse model to study the in vivofunction of enhancers in developmental, evolutionary, and disease‐related processes. Using examples from our ongoing work, I will illustrate how these methods provide insight into the function and evolution of distant‐acting regulatory sequences and offer a starting point for understanding their role in human disease. I will discuss how improved in vivo mouse assays can be used to study candidate pathogenic human mutations in enhancers at scale, revealing the extent to which human genetic studies alone can be insufficient to identify truly causal mutations in enhancers. I will also show how such assays can be used to explore the sensitivity of enhancers that are extremely conserved (“ultraconserved”) in evolution to mutations, revealing a remarkable functional robustness to sequence changes that contrasts with apparent evolutionary constraints.

Extensive sequence divergence between the reference genomes of two zebrafish strains, Tuebingen and AB.

The zebrafish (Danio rerio) is one of the most widely used model organisms for studying vertebrate gene function and human disease, given the 70% conserved protein-coding genes between zebrafish and human. Two of the most common laboratory zebrafish strains are Tuebingen and AB. Despite the fact that the zebrafish reference genome is derived from the Tuebingen strain, the AB strain is still widely used although a high-quality genome comparable to Tuebingen is lacking. Here, we report a 1.40-Gb representative de novo genome assembly of the AB strain (DrAB1), with contig N50length of 21Mb, by integrating Illumina short-read sequencing, Nanopore long-read sequencing and HiC-based chromatin mapping. Compared with the published zebrafish Zv11 reference genome (GRCz11), this genome assembly shows considerable improvements in both contiguity and completeness. In addition, substantial structural differences and extensive sequence divergence of unprecedented resolution have been uncovered, especially with respect to 9,029,929single nucleotide polymorphisms, 2,376,812 InDels, 32,623 insertions, 22,089 deletions and 220 inversions, which constitute ~2.6% of the DrAB1genome. Many of these variants may have potential functional effects on phenotype, which should be considered in further experimental designs. Consequently, our study provides additional genomic resources and a high-resolution structural variation map based on whole-genome alignment for the zebrafish community, which could also be an indispensable reference genome from a model species in future research on fish phylogenetic genomics, comparative genomics and adaptive evolution.

Capturing 3D Chromatin Maps of Human Primary Monocytes: Insights From High-Resolution Hi-C.

Although the variation in chromatin architecture during adaptive immune responses has been thoroughly investigated, the 3D landscape of innate immunity is still unknown. Herein, chromatin regulation and heterogeneity among human primary monocytes were investigated. Peripheral blood was collected from two healthy persons and two patients with systemic lupus erythematosus (SLE), and CD14+ monocytes were selected to perform Hi-C, RNA-seq, ATAC-seq and ChIP-seq analyses. Raw data from the THP1 cell line Hi-C library were used for comparison. For each sample, we constructed three Hi-C libraries and obtained approximately 3 billion paired-end reads in total. Resolution analysis showed that more than 80% of bins presented depths greater than 1000 at a 5 kb resolution. The constructed high-resolution chromatin interaction maps presented similar landscapes in the four individuals, which showed significant divergence from the THP1 cell line chromatin structure. The variability in chromatin interactions around HLA-D genes in the HLA complex region was notable within individuals. We further found that the CD16-encoding gene (FCGR3A) is located at a variable topologically associating domain (TAD) boundary and that chromatin loop dynamics might modulate CD16 expression. Our results indicate both the stability and variability of high-resolution chromatin interaction maps among human primary monocytes. This work sheds light on the potential mechanisms by which the complex interplay of epigenetics and spatial 3D architecture regulates chromatin in innate immunity.

An effector index to predict target genes at GWAS loci.

Drug development and biological discovery require effective strategies to map existing genetic associations to causal genes. To approach this problem, we selected 12 common diseases and quantitative traits for which highly powered genome-wide association studies (GWAS) were available. For each disease or trait, we systematically curated positive control gene sets from Mendelian forms of the disease and from targets of medicines used for disease treatment. We found that these positive control genes were highly enriched in proximity of GWAS-associated single-nucleotide variants (SNVs). We then performed quantitative assessment of the contribution of commonly used genomic features, including open chromatin maps, expression quantitative trait loci (eQTL), and chromatin conformation data. Using these features, we trained and validated an Effector Index (Ei), to map target genes for these 12 common diseases and traits. Ei demonstrated high predictive performance, both with cross-validation on the training set, and an independently derived set for type 2 diabetes. Key predictive features included coding or transcript-altering SNVs, distance to gene, and open chromatin-based metrics. This work outlines a simple, understandable approach to prioritize genes at GWAS loci for functional follow-up and drug development, and provides a systematic strategy for prioritization of GWAS target genes.

TAF4b transcription networks regulating early oocyte differentiation.

Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.

Single-Molecule Multikilobase-Scale Profiling of Chromatin Accessibility Using m6A-SMAC-Seq and m6A-CpG-GpC-SMAC-Seq.

A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq , NOMeseq, and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m6A-SMAC-seq and m6A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets.

The landscape of accessible chromatin in quiescent cardiac fibroblasts and cardiac fibroblasts activated after myocardial infarction

ABSTRACT After myocardial infarction, the massive death of cardiomyocytes leads to cardiac fibroblast proliferation and myofibroblast differentiation, which contributes to the extracellular matrix remodelling of the infarcted myocardium. We recently found that myofibroblasts further differentiate into matrifibrocytes, a newly identified cardiac fibroblast differentiation state. Cardiac fibroblasts of different states have distinct gene expression profiles closely related to their functions. However, the mechanism responsible for the gene expression changes during these activation and differentiation events is still not clear. In this study, the gene expression profiling and genome-wide accessible chromatin mapping of mouse cardiac fibroblasts isolated from the uninjured myocardium and the infarct at multiple time points corresponding to different differentiation states were performed by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), respectively. ATAC-seq peaks were highly enriched in the promoter area and the distal area where the enhancers are located. A positive correlation was identified between the expression and promoter accessibility for many dynamically expressed genes, even though evidence showed that mechanisms independent of chromatin accessibility may also contribute to the gene expression changes in cardiac fibroblasts after MI. Moreover, motif enrichment analysis and gene regulatory network construction identified transcription factors that possibly contributed to the differential gene expression between cardiac fibroblasts of different states.

Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation

Cell type specification during pancreatic development is tightly controlled by a transcriptional and epigenetic network. The precise role of most transcription factors, however, has been only described in mice. To convey such concepts to human pancreatic development, alternative model systems such as pancreatic in vitro differentiation of human pluripotent stem cells can be employed. Here, we analyzed stage-specific RNA-, ChIP-, and ATAC-sequencing data to dissect transcriptional and regulatory mechanisms during pancreatic development. Transcriptome and open chromatin maps of pancreatic differentiation from human pluripotent stem cells provide a stage-specific pattern of known pancreatic transcription factors and indicate ONECUT1 as a crucial fate regulator in pancreas progenitors. Moreover, our data suggest that ONECUT1 is also involved in preparing pancreatic progenitors for later endocrine specification. The dissection of the transcriptional and regulatory circuitry revealed an important role for ONECUT1 within such network and will serve as resource to study human development and disease.

Mapping the landscape of chromatin dynamics during na\xefve CD4+\u2009T-cell activation

T-cell activation induces context-specific gene expression programs that promote energy generation and biosynthesis, progression through the cell cycle and ultimately cell differentiation. The aim of this study was to apply the omni ATAC-seq method to characterize the landscape of chromatin changes induced by T-cell activation in mature naïve CD4+ T-cells. Using a well-established ex vivo protocol of canonical T-cell receptor signaling, we generated genome-wide chromatin maps of naïve T-cells from pediatric donors in quiescent or recently activated states. We identified thousands of individual chromatin accessibility peaks that are associated with T-cell activation, the majority of which were annotated intronic and intergenic enhancer regions. A core set of 3268 gene promoters underwent chromatin remodeling and concomitant changes in gene expression in response to activation, and were enriched in multiple pathways controlling cell cycle regulation, metabolism, inflammatory response genes and cell survival. Leukemia inhibitory factor (LIF) was among those factors that gained the highest accessibility and expression, in addition to IL2-STAT5 dependent chromatin remodeling in the T-cell activation response. Using publicly available data we found the chromatin response was far more dynamic at 24-h compared with 72-h post-activation. In total 546 associations were reproduced at both time-points with similar strength of evidence and directionality of effect. At the pathways level, the IL2-STAT5, KRAS signalling and UV response pathways were replicable at both time-points, although differentially modulated from 24 to 72 h post-activation.

Genome of the butterfly hillstream loach provides insights into adaptations to torrential mountain stream life

Butterfly hillstream loach (Beaufortia kweichowensis), a benthic fish in the torrential mountain streams, possesses a totally flat ventrum, flattened craniofacial and body skeletons, and enlarged paired fins covered by substantially small keratinous structures. However, little is known about the genetic basis of these specialized morphological adaptations. Here we present a 448.52-Mb genome assembly with contig N50 length of 5.53Mb by integrating Illumina short-read sequencing, Nanopore long-read sequencing and HiC-based chromatin map. Demographic history reconstruction of the butterfly hillstream loach reveals that the population dynamics is correlated with the different stages of uplifting of the Tibetan Plateau. Comparative genomic analysis finds evidence of six keratin genes in butterfly hillstream loach evolving under positive selection. Within these genes, two keratin genes exhibit species-specific and divergent amino acid changes, suggesting a role in the formation of the unculi. Additionally, a series of positively selected genes, rapid evolving genes, specific variant genes and expanded gene families are found, including genes related to Hedgehog, Notch and BMP pathways, which may be involved in craniofacial development. These findings may have important implications for understanding the genetic basis of phenotypic adaptation to torrential mountain stream life.