scholarly journals De novo identification of in vivo binding sites from ChIP-chip data

2008 ◽  
Vol 9 (S7) ◽  
Author(s):  
Victor Jin ◽  
Alina Rabinovich ◽  
Henny O'Geen ◽  
Sushma Iyengar ◽  
Peggy Farnham
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Chip Data ◽  
In Vivo Binding ◽  
Chip Chip

scholarly journals Telomere Formation by Rap1p Binding Site Arrays Reveals End-Specific Length Regulation Requirements and Active Telomeric Recombination

2001 ◽  
Vol 21 (23) ◽  
pp. 8117-8128 ◽  
Author(s):  
Simona Grossi ◽  
Alessandro Bianchi ◽  
Pascal Damay ◽  
David Shore
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Repeat Sequence ◽  
Independent Manner ◽  
Telomere Repeat ◽  
Length Regulation ◽  
End Capping ◽  
Original Array

ABSTRACT Rap1p, the major telomere repeat binding protein in yeast, has been implicated in both de novo telomere formation and telomere length regulation. To characterize the role of Rap1p in these processes in more detail, we studied the generation of telomeres in vivo from linear DNA substrates containing defined arrays of Rap1p binding sites. Consistent with previous work, our results indicate that synthetic Rap1p binding sites within the internal half of a telomeric array are recognized as an integral part of the telomere complex in an orientation-independent manner that is largely insensitive to the precise spacing between adjacent sites. By extending the lengths of these constructs, we found that several different Rap1p site arrays could never be found at the very distal end of a telomere, even when correctly oriented. Instead, these synthetic arrays were always followed by a short (≈100-bp) “cap” of genuine TG repeat sequence, indicating a remarkably strict sequence requirement for an end-specific function(s) of the telomere. Despite this fact, even misoriented Rap1p site arrays promote telomere formation when they are placed at the distal end of a telomere-healing substrate, provided that at least a single correctly oriented site is present within the array. Surprisingly, these heterogeneous arrays of Rap1p binding sites generate telomeres through a RAD52-dependent fusion resolution reaction that results in an inversion of the original array. Our results provide new insights into the nature of telomere end capping and reveal one way by which recombination can resolve a defect in this process.

scholarly journals E2F in vivo binding specificity: Comparison of consensus versus nonconsensus binding sites

2008 ◽  
Vol 18 (11) ◽  
pp. 1763-1777 ◽  
Author(s):  
A. Rabinovich ◽  
V. X. Jin ◽  
R. Rabinovich ◽  
X. Xu ◽  
P. J. Farnham
Keyword(s):  
Binding Sites ◽  
Binding Specificity ◽  
In Vivo Binding

C/ebpα and Pu.1 Are Members of a Critical Regulatory Network Required for Hoxa9-Mediated Immortalization

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 650-650
Author(s):  
Cailin Collins ◽  
Jingya Wang ◽  
Joel Bronstein ◽  
Jay L. Hess
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Target Genes ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modifications ◽  
Conditional Knockout ◽  
Motif Analysis ◽  
Myeloid Leukemias

Abstract Abstract 650 HOXA9 is a homeodomain-containing transcription factor that plays important roles in both development and hematopoiesis. Deregulation of HOXA9 occurs in a variety of acute lymphoid and myeloid leukemias and plays a key role in their pathogenesis. More than 50% of acute myeloid leukemia (AML) cases show up-regulation of HOXA9, which correlates strongly with poor prognosis. Nearly all cases of AML with mixed lineage leukemia (MLL) translocations have increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. Despite the crucial role that HOXA9 plays in development, hematopoiesis and leukemia, its transcriptional targets and mechanisms of action are poorly understood. Previously we identified Hoxa9 and Meis1 binding sites in myeloblastic cells, profiled their epigenetic modifications, and identified the target genes regulated by Hoxa9. Hoxa9 and Meis1 co-bind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/p300 binding characteristic of enhancers. Hoxa9 association at these sites correlates strongly with increases in histone H3K27 acetylation and activation of downstream target genes, including many proleukemic gene loci. De novo motif analysis of Hoxa9 binding sites shows a marked enrichment of motifs for the transcription factors in the C/EBP and ETS families, and C/ebpα and the ETS transcription factor Pu.1 were found to cobind at Hoxa9-regulated enhancers. Both C/ebpα and Pu.1 are known to play critical roles in the establishment of functional enhancers during normal myeloid development and are mutated or otherwise deregulated in various myeloid leukemias. To determine the importance of co-association of Hoxa9, C/ebpα and Pu.1 at myeloid enhancers, we generated cell lines from C/ebpα and Pu.1 conditional knockout mice (kindly provided by Dr. Daniel Tenen, Harvard University) by immortalization with Hoxa9 and Meis1. In addition we transformed bone marrow with a tamoxifen-regulated form of Hoxa9. Strikingly, loss of C/ebpα or Pu.1, or inactivation of Hoxa9, blocks proliferation and leads to myeloid differentiation. ChIP experiments show that both C/ebpα and Pu.1 remain bound to Hoxa9 binding sites in the absence of Hoxa9. After the loss of Pu.1, both Hoxa9 and C/ebpα dissociate from Hoxa9 binding sites with a corresponding decrease in target gene expression. In contrast, loss of C/ebpα does not lead to an immediate decrease in either Hoxa9 or Pu.1 binding, suggesting that C/ebpα may be playing a regulatory as opposed to a scaffolding role at enhancers. Current work focuses on performing ChIP-seq analysis to assess how C/ebpα and Pu.1 affect Hoxa9 and Meis1 binding and epigenetic modifications genome-wide, and in vivo leukemogenesis assays to confirm the requirement of both Pu.1 and C/ebpα in the establishment and maintenance of leukemias with high levels of Hoxa9. Collectively, our findings implicate C/ebpα and Pu.1 as members of a critical transcription factor network required for Hoxa9-mediated transcriptional regulation in leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genome-Wide Mapping of Collier In Vivo Binding Sites Highlights Its Hierarchical Position in Different Transcription Regulatory Networks

PLoS ONE ◽  
2015 ◽  
Vol 10 (7) ◽  
pp. e0133387 ◽  
Author(s):  
Mathilde de Taffin ◽  
Yannick Carrier ◽  
Laurence Dubois ◽  
Laetitia Bataillé ◽  
Anaïs Painset ◽  
...  
Keyword(s):  
Binding Sites ◽  
Regulatory Networks ◽  
In Vivo Binding ◽  
Genome Wide ◽  
Hierarchical Position

scholarly journals A novel k-mer set memory (KSM) motif representation improves regulatory variant prediction

2017 ◽  
Author(s):  
Yuchun Guo ◽  
Kevin Tian ◽  
Haoyang Zeng ◽  
Xiaoyun Guo ◽  
David Kenneth Gifford
Keyword(s):  
Binding Sites ◽  
Regulatory Networks ◽  
Motif Discovery ◽  
De Novo ◽  
Binding Motif ◽  
Large Set ◽  
Public Resource ◽  
Regulatory Variant ◽  
The Impact

ABSTRACTThe representation and discovery of transcription factor (TF) sequence binding specificities is critical for understanding gene regulatory networks and interpreting the impact of disease-associated non-coding genetic variants. We present a novel TF binding motif representation, the K-mer Set Memory (KSM), which consists of a set of aligned k-mers that are over-represented at TF binding sites, and a new method called KMAC for de novo discovery of KSMs. We find that KSMs more accurately predict in vivo binding sites than position weight matrix models (PWMs) and other more complex motif models across a large set of ChIP-seq experiments. KMAC also identifies correct motifs in more experiments than four state-of-the-art motif discovery methods. In addition, KSM derived features outperform both PWM and deep learning model derived sequence features in predicting differential regulatory activities of expression quantitative trait loci (eQTL) alleles. Finally, we have applied KMAC to 1488 ENCODE TF ChIP-seq datasets and created a public resource of KSM and PWM motifs. We expect that the KSM representation and KMAC method will be valuable in characterizing TF binding specificities and in interpreting the effects of non-coding genetic variations.

scholarly journals Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

2020 ◽  
Author(s):  
Ida Höijer ◽  
Josefin Johansson ◽  
Sanna Gudmundsson ◽  
Chen-Shan Chin ◽  
Ignas Bunikis ◽  
...  
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Target Prediction ◽  
Human Cell Line ◽  
Guide Rna ◽  
Target Sites ◽  
Long Read ◽  
Target Binding

AbstractA much-debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. Here we present SMRT-OTS and Nano-OTS, two amplification-free long-read sequencing protocols for detection of gRNA driven digestion of genomic DNA by Cas9. The methods were assessed using the human cell line HEK293, which was first re-sequenced at 18x coverage using highly accurate (HiFi) SMRT reads to get a detailed view of all on- and off-target binding regions. We then applied SMRT-OTS and Nano-OTS to investigate the specificity of three different gRNAs, resulting in a set of 55 high-confidence gRNA binding sites identified by both methods. Twenty-five (45%) of these sites were not reported by off-target prediction software, either because they contained four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. We further discovered that a heterozygous SNP can cause allele-specific gRNA binding. Finally, by performing a de novo genome assembly of the HiFi reads, we were able to re-discover 98.7% of the gRNA binding sites without any prior information about the human reference genome. This suggests that CRISPR-Cas9 off-target sites can be efficiently mapped also in organisms where the genome sequence is unknown. In conclusion, the amplification-free sequencing protocols revealed many gRNA binding sites in vitro that would be difficult to predict based on gRNA sequence alignment to a reference. Nevertheless, it is still unknown whether in vivo off-target editing would occur at these sites.

Sign in / Sign up

Export Citation Format

Share Document