Conserved Transcription Factors Control Chromatin Accessibility and Gene Expression to Maintain Cell Fate Stability and Restrict Reprogramming of Differentiated Cells

The comprehensive characterization of mechanisms safeguarding cell fate identity in differentiated cells is crucial for 1) our understanding of how differentiation is maintained in healthy tissues or misregulated in disease states and 2) to improve our ability to use direct reprogramming for regenerative purposes. To uncover novel fate-stabilizing regulators, we employed a genome-wide TF siRNA screen followed by a high-complexity combinatorial evaluation of top performing hits, in a cardiac reprogramming assay in mouse embryonic fibroblasts, and subsequently validated our findings in cardiac, neuronal and iPSCs reprogramming assays in primary human fibroblasts and adult endothelial cells. This approach identified a conserved set of 4 TFs (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming, as demonstrated by up to 6-fold increases in efficiency upon AJSZ knockdown in both lineage- and cell type-independent manners. Mechanistically, ChIP-seq and single-cell ATAC-seq analyses, revealed that AJSZ bind to both open and closed chromatin in a genome-wide and regionalized fashion, thereby limiting reprogramming TFs access to target DNA and ability to remodel the chromatin. In parallel, integration of ChIP-seq and RNA-seq data followed by systematic functional gene testing, identified that AJSZ also promote cell fate stability by proximally downregulating a conserved set of genes involved in the regulation of cell fate specification (MEF2C), proteome remodeling (TPP1, PPIC), ATP homeostasis (EFHD1), and inflammation signaling (IL7R), thereby limiting cells ability to undergo large-scale phenotypic changes. Finally, simultaneous knock-down of AJSZ in combination with cardiac reprogramming TFs overexpression improved heart function by 250% as compared to no treatment and 50% as compared to MGT, 1 month after myocardial infarction. In sum, this study uncovers a novel evolutionarily conserved mechanism mediating cell fate stability in differentiated cells and also identifies AJSZ as promising therapeutic targets for regenerative purposes in adult organs

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STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells

Genome Biology ◽

10.1186/s13059-020-02156-3 ◽

2020 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Tianran Peng ◽

Yanan Zhai ◽

Yaser Atlasi ◽

Menno ter Huurne ◽

Hendrik Marks ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Control Cell ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Mouse Embryonic Stem Cells ◽

Chromatin Signature ◽

Genome Wide ◽

A Genome

Abstract Background Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics. Results Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in “ground-state” (2i+LIF; 2iL) and “metastable” (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency. Conclusions In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.

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Single-cell chromatin and gene-regulatory dynamics of mouse nephron progenitors

10.1101/2021.10.13.464099 ◽

2021 ◽

Author(s):

Sylvia Hilliard ◽

Giovane Tortelote ◽

Hongbing Liu ◽

Chao-Hui Chen ◽

Samir S El-Dahr

Keyword(s):

Transcription Factors ◽

Single Cell ◽

Cell Fate ◽

Cell Types ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Bulk Analysis ◽

Disease States ◽

Differentiated Cells ◽

Nephron Progenitors

Background: Cis-regulatory elements (CREs), such as enhancers and promoters, and their cognate transcription factors play a central role in cell fate specification. Bulk analysis of CREs has provided insights into gene regulation in nephron progenitor cells (NPCs). However, the cellular resolution required to unravel the dynamic changes in regulatory elements associated with cell fate choices remains to be defined. Methods: We integrated single-cell chromatin accessibility (scATAC-seq) and gene expression (scRNA-seq) in embryonic E16.5 (self-renewing) and postnatal P2 (primed) mouse Six2GFP NPCs. This analysis revealed NPC diversity and identified candidate CREs. To validate these findings and gain additional insights into more differentiated cell types, we performed a multiome analysis of E16.5 and P2 kidneys. Results: CRE accessibility recovered the diverse states of NPCs and precursors of differentiated cells. Single-cell types such as podocytes, proximal and distal precursors are marked by differentially accessible CREs. Domains of regulatory chromatin as defined by rich CRE-gene associations identified NPC fate-determining transcription factors (TF). Likewise, key TF expression correlates well with its regulon activity. Young NPCs exhibited enrichment in accessible motifs for bHLH, homeobox, and Forkhead TFs, while older NPCs were enriched in AP-1, HNF1, and HNF4 motif activity. A subset of Forkhead factors exhibiting high chromatin activity in podocyte precursors. Conclusion: Defining the regulatory landscape of nephrogenesis at single-cell resolution informs the basic mechanisms of nephrogenesis and provides a foundation for future studies in disease states characterized by abnormal nephrogenesis.

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A Genome-Wide RNA Interference Screen Identifies a Differential Role of the Mediator CDK8 Module Subunits for GATA/ RUNX-Activated Transcription in Drosophila

Molecular and Cellular Biology ◽

10.1128/mcb.01625-09 ◽

2010 ◽

Vol 30 (11) ◽

pp. 2837-2848 ◽

Cited By ~ 25

Author(s):

Vanessa Gobert ◽

Dani Osman ◽

Stéphanie Bras ◽

Benoit Augé ◽

Muriel Boube ◽

...

Keyword(s):

Cell Differentiation ◽

Rna Interference ◽

Cell Fate ◽

Gata Factor ◽

Hematopoietic System ◽

Crystal Cell ◽

Genome Wide ◽

A Genome

ABSTRACT Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. We used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory “CDK8 module,” composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, our data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, our in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. We thus propose that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, we suggest that the set of conserved factors identified herein may regulate GATA/RUNX activity in mammals.

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Phenotypic Screen and Transcriptomics Approach Complement Each Other in Functional Genomics of Defensive Stink Gland Physiology

10.21203/rs.3.rs-1117784/v1 ◽

2021 ◽

Author(s):

Sabrina Lehmann ◽

Bibi Atika ◽

Daniela Grossmann ◽

Christian Schmitt-Engel ◽

Nadi Strohlein ◽

...

Keyword(s):

Functional Genomics ◽

Large Scale ◽

Reverse Genetics ◽

Expression Profiles ◽

Forward Genetics ◽

Large Set ◽

Knock Down ◽

Genome Wide ◽

Phenotypic Screen ◽

A Genome

Abstract Background Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. Results Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using a reverse genetics strategy based on RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause knock-down gland phenotypes, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. Moreover, of the 29 previously transcriptomics-identified genes causing a gland phenotype, only one gene was recognized by this phenotypic screen despite the fact that 13 of them were covered by the screen. Conclusion Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.

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A New Genome-to-Genome Comparison Approach for Large-Scale Revisiting of Current Microbial Taxonomy

Microorganisms ◽

10.3390/microorganisms7060161 ◽

2019 ◽

Vol 7 (6) ◽

pp. 161 ◽

Cited By ~ 1

Author(s):

Ming-Hsin Tsai ◽

Yen-Yi Liu ◽

Von-Wun Soo ◽

Chih-Chieh Chen

Keyword(s):

Microbial Diversity ◽

Large Scale ◽

Gene Selection ◽

Marker Gene ◽

Genome Comparison ◽

Marker Genes ◽

Species Classification ◽

Genome Wide ◽

A Genome ◽

Comparison Approach

Microbial diversity has always presented taxonomic challenges. With the popularity of next-generation sequencing technology, more unculturable bacteria have been sequenced, facilitating the discovery of additional new species and complicated current microbial classification. The major challenge is to assign appropriate taxonomic names. Hence, assessing the consistency between taxonomy and genomic relatedness is critical. We proposed and applied a genome comparison approach to a large-scale survey to investigate the distribution of genomic differences among microorganisms. The approach applies a genome-wide criterion, homologous coverage ratio (HCR), for describing the homology between species. The survey included 7861 microbial genomes that excluded plasmids, and 1220 pairs of genera exhibited ambiguous classification. In this study, we also compared the performance of HCR and average nucleotide identity (ANI). The results indicated that HCR and ANI analyses yield comparable results, but a few examples suggested that HCR has a superior clustering effect. In addition, we used the Genome Taxonomy Database (GTDB), the gold standard for taxonomy, to validate our analysis. The GTDB offers 120 ubiquitous single-copy proteins as marker genes for species classification. We determined that the analysis of the GTDB still results in classification boundary blur between some genera and that the marker gene-based approach has limitations. Although the choice of marker genes has been quite rigorous, the bias of marker gene selection remains unavoidable. Therefore, methods based on genomic alignment should be considered for use for species classification in order to avoid the bias of marker gene selection. On the basis of our observations of microbial diversity, microbial classification should be re-examined using genome-wide comparisons.

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A Comprehensive Survey on the Terpene Synthase Gene Family Provides New Insight into Its Evolutionary Patterns

Genome Biology and Evolution ◽

10.1093/gbe/evz142 ◽

2019 ◽

Vol 11 (8) ◽

pp. 2078-2098 ◽

Cited By ~ 8

Author(s):

Shu-Ye Jiang ◽

Jingjing Jin ◽

Rajani Sarojam ◽

Srinivasan Ramachandran

Keyword(s):

Gene Family ◽

Large Scale ◽

Family Members ◽

Terpene Synthase ◽

Limited Information ◽

Terpene Synthases ◽

Genome Wide ◽

A Genome ◽

Family Expansion ◽

Insight Into

Abstract Terpenes are organic compounds and play important roles in plant growth and development as well as in mediating interactions of plants with the environment. Terpene synthases (TPSs) are the key enzymes responsible for the biosynthesis of terpenes. Although some species were employed for the genome-wide identification and characterization of the TPS family, limited information is available regarding the evolution, expansion, and retention mechanisms occurring in this gene family. We performed a genome-wide identification of the TPS family members in 50 sequenced genomes. Additionally, we also characterized the TPS family from aromatic spearmint and basil plants using RNA-Seq data. No TPSs were identified in algae genomes but the remaining plant species encoded various numbers of the family members ranging from 2 to 79 full-length TPSs. Some species showed lineage-specific expansion of certain subfamilies, which might have contributed toward species or ecotype divergence or environmental adaptation. A large-scale family expansion was observed mainly in dicot and monocot plants, which was accompanied by frequent domain loss. Both tandem and segmental duplication significantly contributed toward family expansion and expression divergence and played important roles in the survival of these expanded genes. Our data provide new insight into the TPS family expansion and evolution and suggest that TPSs might have originated from isoprenyl diphosphate synthase genes.

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Large-scale GWAS reveals insights into the genetic architecture of same-sex sexual behavior

Science ◽

10.1126/science.aat7693 ◽

2019 ◽

Vol 365 (6456) ◽

pp. eaat7693 ◽

Cited By ~ 53

Author(s):

Andrea Ganna ◽

Karin J. H. Verweij ◽

Michel G. Nivard ◽

Robert Maier ◽

Robbee Wedow ◽

...

Keyword(s):

Sexual Behavior ◽

Genetic Architecture ◽

Large Scale ◽

Genome Wide Association Study ◽

Same Sex ◽

Genome Wide ◽

A Genome ◽

Number Of Sexual Partners ◽

Opposite Sex ◽

Males And Females

Twin and family studies have shown that same-sex sexual behavior is partly genetically influenced, but previous searches for specific genes involved have been underpowered. We performed a genome-wide association study (GWAS) on 477,522 individuals, revealing five loci significantly associated with same-sex sexual behavior. In aggregate, all tested genetic variants accounted for 8 to 25% of variation in same-sex sexual behavior, only partially overlapped between males and females, and do not allow meaningful prediction of an individual’s sexual behavior. Comparing these GWAS results with those for the proportion of same-sex to total number of sexual partners among nonheterosexuals suggests that there is no single continuum from opposite-sex to same-sex sexual behavior. Overall, our findings provide insights into the genetics underlying same-sex sexual behavior and underscore the complexity of sexuality.

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Exploiting regulatory heterogeneity to systematically identify enhancers with high accuracy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808833115 ◽

2018 ◽

Vol 116 (3) ◽

pp. 900-908 ◽

Cited By ~ 4

Author(s):

Hamutal Arbel ◽

Sumanta Basu ◽

William W. Fisher ◽

Ann S. Hammonds ◽

Kenneth H. Wan ◽

...

Keyword(s):

Large Scale ◽

Scale Validation ◽

Expression Patterns ◽

Prediction Method ◽

High Accuracy ◽

Genome Wide ◽

Rank List ◽

A Genome ◽

Improved Accuracy ◽

Genome Wide Scan

Identifying functional enhancer elements in metazoan systems is a major challenge. Large-scale validation of enhancers predicted by ENCODE reveal false-positive rates of at least 70%. We used the pregrastrula-patterning network of Drosophila melanogaster to demonstrate that loss in accuracy in held-out data results from heterogeneity of functional signatures in enhancer elements. We show that at least two classes of enhancers are active during early Drosophila embryogenesis and that by focusing on a single, relatively homogeneous class of elements, greater than 98% prediction accuracy can be achieved in a balanced, completely held-out test set. The class of well-predicted elements is composed predominantly of enhancers driving multistage segmentation patterns, which we designate segmentation driving enhancers (SDE). Prediction is driven by the DNA occupancy of early developmental transcription factors, with almost no additional power derived from histone modifications. We further show that improved accuracy is not a property of a particular prediction method: after conditioning on the SDE set, naïve Bayes and logistic regression perform as well as more sophisticated tools. Applying this method to a genome-wide scan, we predict 1,640 SDEs that cover 1.6% of the genome. An analysis of 32 SDEs using whole-mount embryonic imaging of stably integrated reporter constructs chosen throughout our prediction rank-list showed >90% drove expression patterns. We achieved 86.7% precision on a genome-wide scan, with an estimated recall of at least 98%, indicating high accuracy and completeness in annotating this class of functional elements.

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Chromatin regulators: weaving epigenetic nets

BioMolecular Concepts ◽

10.1515/bmc.2010.023 ◽

2010 ◽

Vol 1 (3-4) ◽

pp. 225-238 ◽

Cited By ~ 1

Author(s):

Inmaculada Hernández-Muñoz

Keyword(s):

Nucleosome Position ◽

Histone Variants ◽

Cellular Memory ◽

Intrinsic Signals ◽

Differentiated Cells ◽

Chromatin Regulators ◽

Genome Wide ◽

Chromatin Regulator ◽

A Genome ◽

Multicellular Organisms

AbstractIn multicellular organisms differentiated cells must maintain their cellular memory, which will be faithfully inherited and maintained by their progeny. In addition, these specialized cells are exposed to specific environmental and cell-intrinsic signals and will have to appropriately respond to them. Some of these stimuli lead to changes in a subset of genes or to a genome-wide reprogramming of the cells that will remain after stimuli removal and, in some instances, will be inherited by the daughter cells. The molecular substrate that integrates cellular memory and plasticity is the chromatin, a complex of DNA and histones unique to eukaryotes. The nucleosome is the fundamental unit of the chromatin and nucleosomal organization defines different chromatin conformations. Chromatin regulators affect chromatin conformation and accessibility by covalently modifying the DNA or the histones, substituting histone variants, remodeling the nucleosome position or modulating chromatin looping and folding. These regulators frequently act in multiprotein complexes and highly specific interplays among chromatin marks and different chromatin regulators allow a remarkable array of possibilities. Therefore, chromatin regulator nets act to propagate the conformation of different chromatin regions through DNA replication and mitosis, and to remodel the chromatin fiber to regulate the accessibility of the DNA to transcription factors and to the transcription and repair machineries. Here, the state-of-the-art of the best-known chromatin regulators is reviewed.

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Genomic variation across a clinical Cryptococcus population linked to disease outcome

10.1101/2021.11.22.469645 ◽

2021 ◽

Author(s):

Poppy Channa Sakti Sephton-Clark ◽

Jennifer Tenor ◽

Dena Toffaletti ◽

Nancy Meyers ◽

Charles Giamberardino ◽

...

Keyword(s):

Patient Outcomes ◽

Large Scale ◽

Genome Wide Association Study ◽

Sugar Transport ◽

Genomic Variation ◽

Clinical Isolates ◽

Genome Wide ◽

A Genome ◽

Gwas Approach

Cryptococcus neoformans is the causative agent of cryptococcosis, a disease with poor patient outcomes, accounting for approximately 180,000 deaths each year. Patient outcomes may be impacted by the underlying genetics of the infecting isolate, however, our current understanding of how genetic diversity contributes to clinical outcomes is limited. Here, we leverage clinical, in vitro growth and genomic data for 284 C. neoformans isolates to identify clinically relevant pathogen variants within a population of clinical isolates from patients with HIV-associated cryptococcosis in Malawi. Through a genome-wide association study (GWAS) approach, we identify variants associated with fungal burden and growth rate. We also find both small and large-scale variation, including aneuploidy, associated with alternate growth phenotypes, which may impact the course of infection. Genes impacted by these variants are involved in transcriptional regulation, signal transduction, glycolysis, sugar transport, and glycosylation. When combined with clinical data, we show that growth within the CNS is reliant upon glycolysis in an animal model, and likely impacts patient mortality, as CNS burden modulates patient outcome. Additionally, we find genes with roles in sugar transport are under selection in the majority of these clinical isolates. Further, we demonstrate that two hypothetical proteins identified by GWAS impact virulence in animal models. Our approach illustrates links between genetic variation and clinically relevant phenotypes, shedding light on survival mechanisms within the CNS and pathways involved in this persistence.

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