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Start from Scratch: Precisely Identify Massive Active Enhancers by Sequencing

ABSTRACT: Enhancer loci identified by ChIP-Seq or other experimental methods occupy hundreds of base pairs on the genome. It is the paradox comparing with the motif analysis, which usually contains only a few or tens nucleotides, achieved by bioinformatics analysis. To address this issue, we develop an experimental method, termed as massive active enhancer sequencing (MAE-Seq), to designate active enhancer sequences from arbitrary sources of 25bp random DNA libraries. These sequences are constructed in a mini-promoter vector with fluorescent reporter. After transfection, positive cells are sorted out and for sequencing. With the results, we successfully identify hundreds known accurate active enhancer sequences as expected. Besides that, large amounts of unmarked regulatory elements (UREs) that might control gene expression without epigenetic features are also been spotted. In conclusion, MAE-Seq would be useful to refine enhancer sequences and precisely annotate the genome in eukaryotes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE129453 | GEO | 2023/03/29

REPOSITORIES: GEO

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Refine regulatory elements across the genome

Project description:We develop an experimental method termed as MAE-seq, to identify active enhancer across the genome. Enhancer loci are mapped precisely to 25 bp in different types of cells. In mouse embryonic stem cells, 19,514 active enhancers are identified including 49.25% known and 50.75% de novo ones. In human HEK293T cell , 3,0359 active enhancers are identified.

2023-04-20 | GSE149028 | GEO

Whole RNA-seq on clones of GM12878 lymphoblastoid clones DF1 and DF2

Project description:Analysis of Allelic bias in clonal lymphoblastoid cells. Abstract: In mammals, numerous autosomal genes are subject to mitotically stable monoallelic expression (MAE), including genes that play critical roles in a variety of human diseases. Due to challenges posed by the clonal nature of MAE, very little is known about its regulation; in particular, no molecular features have been specifically linked to MAE. Here we report an approach that distinguishes MAE genes in human cells with great accuracy: a chromatin signature consisting of chromatin marks associated with active transcription (H3K36me3) and silencing (H3K27me3) simultaneously occurring in the gene body. The MAE signature is present in ~20% of ubiquitously expressed genes and over 30% of tissue-specific genes across cell types. Notably, it is enriched among key developmental genes that have bivalent chromatin structure in pluripotent cells. Our results open a new approach to the study of MAE that is independent of polymorphisms, and suggest that MAE is linked to cell differentiation.

2013-12-31 | GSE52090 | GEO

Whole RNA-seq on clones of GM12878 lymphoblastoid clones DF1 and DF2

2013-12-31 | E-GEOD-52090 | biostudies-arrayexpress

MAE-seq refines regulatory elements across the genome

Project description:Proper cell fate determination relies on precise spatial and temporal genome-wide cooperativities among regulatory elements (REs) and their targeted genes. However, the lengths of REs defined by different methods are varied, which indicates sequence redundancy contained and the context of the genome will be unintelligible. To figure this out, we developed a method termed as MAE-seq (Massive Active Enhancers by Sequencing) to experimentally identify functional REs at a 25-bp scale. In this study, more than 626,879 active enhancers are identified in mouse embryonic stem cells. Comparative analysis indicates most of histone modification datasets could be annotated by MAE-Seq loci. Additionally, 35.03% (219,623) are identified as de novo ones without any epigenetic modification. Validation results show that these 25-bp sequences could act as a functional unit, which shows identical or similar expression patterns as the previously defined larger elements. Additionally, we examined the functions of some novel elements that can up-regulate gene activities. By integrating with Hi-C data, over 55.64% novel elements may have distal association with different targeted genes. As an example, we find Cdh1 gene is interacting with one novel and two known REs in mESCs. And their biological effects are investigated via CRISPR-Cas9. It indicates these distal associations can coordinate Cdh1 gene expression and mESCs proliferation. Therefore, our study provides an experimental approach to refine the REs at 25-bp resolution and it will be a great improvement to unscramble the underlying genome context.

2023-01-06 | GSE193494 | GEO

A UTX–MLL4–p300 Transcriptional Regulatory Network Coordinately Shapes Active Enhancer Landscapes for Eliciting Transcription (RNA-Seq)

Project description:Enhancer activation is a critical step for gene activation. Here we report a novel epigenetic crosstalk at enhancers between the UTX (H3K27 demethylase)-MLL4 (H3K4 methyltransferase) complex and the histone acetyltransferase p300. We demonstrate that UTX, in a demethylase activity-independent manner, facilitates conversion of naïve (unmarked) enhancers in embryonic stem cells to an active (H3K4me1+/H3K27ac+) state by recruiting and coupling the enzymatic functions of MLL4 and p300. Loss of UTX leads to attenuated enhancer activity, characterized by reduced levels of H3K4me1 and H3K27ac as well as impaired transcription. The UTX-MLL4 complex enhances p300-dependent H3K27 acetylation through UTX-dependent stimulation of p300 recruitment while MLL4-mediated H3K4 monomethylation, reciprocally, requires p300 function. Importantly, MLL4-generated H3K4me1 further enhances p300-dependent transcription. This work reveals a previously unrecognized cooperativity among enhancer-associated chromatin modulators, including a unique function for UTX, in establishing an “active enhancer landscape” and defines a mechanism for the joint deposition of H3K4me1 and H3K27ac.

2017-07-06 | GSE97702 | GEO

A UTX–MLL4–p300 Transcriptional Regulatory Network Coordinately Shapes Active Enhancer Landscapes for Eliciting Transcription (ChIP-Seq)

2017-07-06 | GSE97701 | GEO

Chromatin signature identifies monoallelic gene expression across mammalian cell types (ChIP-seq)

Project description:Monoallelic expression of autosomal genes (MAE) is a widespread epigenetic phenomenon which is poorly understood, due in part to current limitations of genome-wide approaches for assessing it. Recently, we reported that a specific histone modification signature is strongly associated with MAE, and demonstrated that it can serve as a proxy of MAE in human lymphoblastoid cells (Nag et al. Elife. 2013 Dec 31;2:e01256). Here, we use murine cells to establish that this chromatin signature is conserved between mouse and human, and is associated with MAE in every tested cell type. Our analyses reveal extensive conservation in the identity of MAE genes between the two species. By applying MAE chromatin signature analysis to a large number of cell and tissue types, we show that the MAE state remains consistent during terminal cell differentiation and is predominant among cell-type specific genes, suggesting a link between MAE and specification of cell identity.

2015-06-17 | GSE67380 | GEO

Chromatin signature identifies monoallelic gene expression across mammalian cell types (RNA-seq)

2015-06-17 | GSE67379 | GEO

Octamer sequences with HEXplorer score amplitude of -10.35, –0.15 or +10.32

Project description:Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5’ss and 3’ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We performed pulldown analysis to identify RNA-binding proteins, that bind either sequence segments with a HEXplorer score amplitude of +10.32, –0.15 or -10.35.

2022-10-13 | PXD030139 | Pride

Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features

Project description:Gene expression is determined by genomic elements called enhancers, which contain short motifs bound by different transcription factors (TFs). However, how enhancer sequences and TF motifs relate to enhancer activity is unknown and general sequence requirements for enhancers or comprehensive sets of important enhancer sequence elements have remained elusive. Here, we computationally dissect thousands of functional enhancer sequences from three different Drosophila cell lines. We find that the enhancers display distinct cis-regulatory sequence signatures, which are predictive of the enhancersM-bM-^@M-^Y cell type-specific or broad activities. These signatures contain transcription factor motifs and a novel class of enhancer sequence elements, dinucleotide repeat motifs (DRMs). DRMs are highly enriched in enhancers, particularly in enhancers that are broadly active across different cell types. We experimentally validate the importance of the identified TF motifs and DRMs for enhancer function and show that they can be sufficient to create an active enhancer de novo from non-functional sequence. The function of DRMs as a novel class of general enhancer features that are also enriched in human regulatory regions might explain their implication in several diseases and provides important insights into gene regulation. STARR-seq was performed in BG3 cells with paired-end sequencing in two replicates and respective inputs.

2014-04-02 | E-GEOD-49809 | biostudies-arrayexpress

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