CRISPR Tools for Physiology and Cell State Changes: Potential of Transcriptional Engineering and Epigenome Editing

Given the large amount of genome-wide data that have been collected during the last decades, a good understanding of how and why cells change during development, homeostasis, and disease might be expected. Unfortunately, the opposite is true; triggers that cause cellular state changes remain elusive, and the underlying molecular mechanisms are poorly understood. Although genes with the potential to influence cell states are known, the historic dependency on methods that manipulate gene expression outside the endogenous chromatin context has prevented us from understanding how cells organize, interpret, and protect cellular programs. Fortunately, recent methodological innovations are now providing options to answer these outstanding questions, by allowing to target and manipulate individual genomic and epigenomic loci. In particular, three experimental approaches are now feasible due to DNA targeting tools, namely, activation and/or repression of master transcription factors in their endogenous chromatin context; targeting transcription factors to endogenous, alternative, or inaccessible sites; and finally, functional manipulation of the chromatin context. In this article, we discuss the molecular basis of DNA targeting tools and review the potential of these new technologies before we summarize how these have already been used for the manipulation of cellular states and hypothesize about future applications.

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Genome-wide profiling of an enhancer-associated histone modification reveals the influence of asthma on the epigenome of the airway epithelium

10.1101/282889 ◽

2018 ◽

Cited By ~ 2

Author(s):

Peter McErlean ◽

Audrey Kelly ◽

Jaideep Dhariwal ◽

Max Kirtland ◽

Julie Watson ◽

...

Keyword(s):

Transcription Factors ◽

Histone Modification ◽

Molecular Mechanisms ◽

Airway Disease ◽

Epigenome Editing ◽

Genome Wide ◽

Genes Encoding ◽

Chronic Airway Disease ◽

Dynamic Relationships

AbstractAsthma is a chronic airway disease driven by complex genetic-environmental interactions. The role of epigenetic modifications in bronchial epithelial cells (BECs) in asthma is poorly understood. We undertook genome-wide profiling of the enhancer-associated histone modification H3K27ac in BECs from people with asthma and healthy controls. We identified 49,903 regions exhibiting differential H3K27ac enrichment in asthma, clustered at genes associated with type-2-high asthma (CLCA1) and epithelial processes (EMT). Asthma dramatically influenced the BEC enhancer landscape and we identified asthma-associated Super-Enhancers encompassing genes encoding transcription factors (TP63) and enzymes regulating lipid metabolism (NOX4). We integrated published protein, epigenomic and transcriptomic datasets and identified epithelium-specific transcription factors associated with H3K27ac in asthma (TP73) and dynamic relationships between asthma-associated changes in H3K27ac, DNA methylation, genetic susceptibility and transcriptional profiles. Finally, we used a CRISPR-based approach to recapitulate the H3K27ac-asthma landscape in vitro and provide proof of principal that asthma-associated gene expression (SERPINB2) is driven in part by aberrant histone acetylation, validating the combination of genome-wide and epigenome-editing approaches in deciphering the molecular mechanisms underlying asthma pathogenesis.

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Genetic analysis of amyotrophic lateral sclerosis identifies contributing pathways and cell types

Science Advances ◽

10.1126/sciadv.abd9036 ◽

2021 ◽

Vol 7 (3) ◽

pp. eabd9036

Author(s):

Sara Saez-Atienzar ◽

Sara Bandres-Ciga ◽

Rebekah G. Langston ◽

Jonggeol J. Kim ◽

Shing Wan Choi ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Membrane Trafficking ◽

Molecular Mechanisms ◽

Cell Types ◽

Polygenic Risk Score ◽

Genome Wide ◽

Genome Wide Data ◽

Data Driven Approach ◽

Single Nucleus ◽

Lateral Sclerosis

Despite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely, neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated six differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, MAPKAPK3, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

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Import of Non-Coding RNAs into Human Mitochondria: A Critical Review and Emerging Approaches

Cells ◽

10.3390/cells8030286 ◽

2019 ◽

Vol 8 (3) ◽

pp. 286 ◽

Cited By ~ 15

Author(s):

Damien Jeandard ◽

Anna Smirnova ◽

Ivan Tarassov ◽

Eric Barrey ◽

Alexandre Smirnov ◽

...

Keyword(s):

Molecular Mechanisms ◽

Nuclear Genome ◽

Genetic System ◽

Mitochondrial Import ◽

In Situ Techniques ◽

Genome Wide ◽

Experimental Approaches ◽

Non Coding Rnas ◽

Analyze Data

Mitochondria harbor their own genetic system, yet critically depend on the import of a number of nuclear-encoded macromolecules to ensure their expression. In all eukaryotes, selected non-coding RNAs produced from the nuclear genome are partially redirected into the mitochondria, where they participate in gene expression. Therefore, the mitochondrial RNome represents an intricate mixture of the intrinsic transcriptome and the extrinsic RNA importome. In this review, we summarize and critically analyze data on the nuclear-encoded transcripts detected in human mitochondria and outline the proposed molecular mechanisms of their mitochondrial import. Special attention is given to the various experimental approaches used to study the mitochondrial RNome, including some recently developed genome-wide and in situ techniques.

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Hox transcription factors: an overview of multi-step regulators of gene expression

The International Journal of Developmental Biology ◽

10.1387/ijdb.180294il ◽

2018 ◽

Vol 62 (11-12) ◽

pp. 723-732 ◽

Cited By ~ 4

Author(s):

Julie Carnesecchi ◽

Pedro B. Pinto ◽

Ingrid Lohmann

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Molecular Mechanisms ◽

Target Genes ◽

Cell Fates ◽

Hox Proteins ◽

Regulatory Modules ◽

Genome Wide ◽

Transcription Complexes ◽

Regulatory Functions

Hox transcription factors (TFs) function as key determinants in the specification of cell fates during development. They do so by triggering entire morphogenetic cascades through the activation of specific target genes. In contrast to their fundamental role in development, the molecular mechanisms employed by Hox TFs are still poorly understood. In recent years, a new picture has emerged regarding the function of Hox proteins in gene regulation. Initial studies have primarily focused on understanding how Hox TFs recognize and bind specific enhancers to activate defined Hox targets. However, genome-wide studies on the interactions and dynamics of Hox proteins have revealed a more elaborate function of the Hox factors. It is now known that Hox proteins are involved in several steps of gene expression with potential regulatory functions in the modification of the chromatin landscape and its accessibility, recognition and activation of specific cis-regulatory modules, assembly and activation of promoter transcription complexes and mRNA processing. In the coming years, the characterization of the molecular activity of Hox TFs in these mechanisms will greatly contribute to our general understanding of Hox activity.

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Machine learning approaches to identify sleep genes

10.1101/2021.04.10.439249 ◽

2021 ◽

Author(s):

Yin Yeng Lee ◽

Mehari Endale ◽

Gang Wu ◽

Marc D Ruben ◽

Lauren J Francey ◽

...

Keyword(s):

Machine Learning ◽

Prior Knowledge ◽

Molecular Mechanisms ◽

Genetic Regulation ◽

Learning Approaches ◽

Sleep Regulation ◽

Genome Wide ◽

Genome Wide Data ◽

Key Genes

Genetics impacts sleep, yet, the molecular mechanisms underlying sleep regulation remain elusive. We built machine learning (ML) models to predict genes based on their similarity to known sleep genes. Our predictions fit with prior knowledge of sleep regulation and also identify several key genes/pathways to pursue in follow-up studies. We tested one of our findings, the NF-κB pathway, and showed that its genetic alteration affects sleep duration in mice. Our study highlights the power of ML to integrate prior knowledge and genome-wide data to study genetic regulation of sleep and other complex behaviors.

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Genetic analysis of amyotrophic lateral sclerosis identifies contributing pathways and cell types

10.1101/2020.07.20.211276 ◽

2020 ◽

Author(s):

Sara Saez-Atienzar ◽

Sara Bandres-Ciga ◽

Rebekah G. Langston ◽

Jonggeol J. Kim ◽

Shing Wan Choi ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Membrane Trafficking ◽

Molecular Mechanisms ◽

Cell Types ◽

Polygenic Risk Score ◽

Genome Wide ◽

Genome Wide Data ◽

Data Driven Approach ◽

Single Nucleus ◽

Lateral Sclerosis

ABSTRACTDespite the considerable progress in unraveling the genetic causes of amyotrophic lateral sclerosis (ALS), we do not fully understand the molecular mechanisms underlying the disease. We analyzed genome-wide data involving 78,500 individuals using a polygenic risk score approach to identify the biological pathways and cell types involved in ALS. This data-driven approach identified multiple aspects of the biology underlying the disease that resolved into broader themes, namely neuron projection morphogenesis, membrane trafficking, and signal transduction mediated by ribonucleotides. We also found that genomic risk in ALS maps consistently to GABAergic cortical interneurons and oligodendrocytes, as confirmed in human single-nucleus RNA-seq data. Using two-sample Mendelian randomization, we nominated five differentially expressed genes (ATG16L2, ACSL5, MAP1LC3A, PLXNB2, and SCFD1) within the significant pathways as relevant to ALS. We conclude that the disparate genetic etiologies of this fatal neurological disease converge on a smaller number of final common pathways and cell types.

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Epigenetic control of hematopoiesis: the PU.1 chromatin connection

Biological Chemistry ◽

10.1515/hsz-2014-0195 ◽

2014 ◽

Vol 395 (11) ◽

pp. 1265-1274 ◽

Cited By ~ 27

Author(s):

Boet van Riel ◽

Frank Rosenbauer

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Molecular Mechanisms ◽

Target Genes ◽

Current Knowledge ◽

Dna Methyltransferases ◽

Chromatin Remodelers ◽

Cell Lineages ◽

Protein Partners ◽

Genome Wide

Abstract Purine-rich box1 (PU.1) is a transcription factor that not only has a key role in the development of most hematopoietic cell lineages but also in the suppression of leukemia. To exert these functions, PU.1 can cross-talk with multiple different proteins by forming complexes in order to activate or repress transcription. Among its protein partners are chromatin remodelers, DNA methyltransferases, and a number of other transcription factors with important roles in hematopoiesis. While a great deal of knowledge has been acquired about PU.1 function over the years, it was the development of novel genome-wide technologies, which boosted our understanding of how PU.1 acts on the chromatin to drive its repertoire of target genes. This review summarizes current knowledge and ideas of molecular mechanisms by which PU.1 controls hematopoiesis and suppresses leukemia.

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A single ChIP-seq dataset is sufficient for comprehensive analysis of motifs co-occurrence with MCOT package

Nucleic Acids Research ◽

10.1093/nar/gkz800 ◽

2019 ◽

Vol 47 (21) ◽

pp. e139-e139 ◽

Cited By ~ 6

Author(s):

Victor Levitsky ◽

Elena Zemlyanskaya ◽

Dmitry Oshchepkov ◽

Olga Podkolodnaya ◽

Elena Ignatieva ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Fold Increase ◽

Transcription Factor Binding ◽

Single Chip ◽

Maximum Information ◽

Factor Binding ◽

Protein Protein Interaction ◽

Genome Wide ◽

Genome Wide Data

Abstract Recognition of composite elements consisting of two transcription factor binding sites gets behind the studies of tissue-, stage- and condition-specific transcription. Genome-wide data on transcription factor binding generated with ChIP-seq method facilitate an identification of composite elements, but the existing bioinformatics tools either require ChIP-seq datasets for both partner transcription factors, or omit composite elements with motifs overlapping. Here we present an universal Motifs Co-Occurrence Tool (MCOT) that retrieves maximum information about overrepresented composite elements from a single ChIP-seq dataset. This includes homo- and heterotypic composite elements of four mutual orientations of motifs, separated with a spacer or overlapping, even if recognition of motifs within composite element requires various stringencies. Analysis of 52 ChIP-seq datasets for 18 human transcription factors confirmed that for over 60% of analyzed datasets and transcription factors predicted co-occurrence of motifs implied experimentally proven protein-protein interaction of respecting transcription factors. Analysis of 164 ChIP-seq datasets for 57 mammalian transcription factors showed that abundance of predicted composite elements with an overlap of motifs compared to those with a spacer more than doubled; and they had 1.5-fold increase of asymmetrical pairs of motifs with one more conservative ‘leading’ motif and another one ‘guided’.

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Genome and Epigenome Editing: A Revolution in Science and Medicine

Blood ◽

10.1182/blood.v124.21.sci-10.sci-10 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-10-SCI-10

Author(s):

J. Keith Joung

Keyword(s):

Gene Expression ◽

Board Of Directors ◽

Gene Sequence ◽

New Technologies ◽

Advisory Committees ◽

Epigenome Editing ◽

Genome Wide ◽

Short Palindromic Repeat ◽

Equity Ownership ◽

Or Gene

Abstract Targeted genome and epigenome editing technologies have recently emerged as important tools for biomedical research and as potential reagents for therapies of gene-based diseases. This talk will focus on our recent work on both improving and defining the genome wide specificities of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nucleases, a robust platform for introducing targeted genome sequence alterations. The talk will also briefly describe the creation and validation of new technologies for modifying specific epigenomic marks on histones and DNA that can be used to induce targeted alterations in endogenous human gene expression. Taken together, these methodologies provide transformative tools for understanding human biology and offer promising pathways forward for developing new classes of therapeutics based on targeted alterations of gene sequence and/or gene expression. Disclosures Joung: Editas Medicine: Consultancy, Equity Ownership, Other, Patents & Royalties; Transposagen Biopharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Horizon Discovery: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Genome-wide identification and analysis of the GATA transcription factor gene family in Ustilaginoidea virens

Genome ◽

10.1139/gen-2018-0190 ◽

2019 ◽

Vol 62 (12) ◽

pp. 807-816 ◽

Cited By ~ 1

Author(s):

Mina Yu ◽

Junjie Yu ◽

Huijuan Cao ◽

Mingli Yong ◽

Yongfeng Liu

Keyword(s):

Transcription Factors ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Ustilaginoidea Virens ◽

Genome Wide ◽

Transcriptional Regulatory ◽

Starting Point ◽

Reactive Oxygen Species Stress ◽

False Smut ◽

Transcriptional Regulatory Mechanisms

In filamentous fungi, the conserved transcription factors play important roles in multiple cellular and developmental processes. The GATA proteins, a family of GATA-binding zinc finger transcription factors, play diverse functions in fungi. Ustilaginoidea virens is an economically important pathogen-causing rice false smut worldwide. To gain additional insight into the cellular and molecular mechanisms of this pathogen, in this study, we identified and functionally characterized seven GATA proteins from the U. virens genome (UvGATA). Sequences analysis indicated that these GATA proteins are divided into seven clades. The proteins in each clade contained conserved clade-specific sequences and structures, thus leading to the same motif serving different purposes in various contexts. The expression profiles of UvGATA genes at different infection stages and under H2O2 stress were detected. Results showed that the majority of UvGATA genes performed functions at both processes, thereby confirming the roles of these genes in pathogenicity and reactive oxygen species stress tolerance. This study provided an important starting point to further explore the biological functions of UvGATA genes and increased our understanding of their potential transcriptional regulatory mechanisms in U. virens.

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