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1.
Nucleic Acids Res ; 51(11): 5499-5511, 2023 06 23.
Article in English | MEDLINE | ID: mdl-37013986

ABSTRACT

Classic promoter mutagenesis strategies can be used to study how proximal promoter regions regulate the expression of particular genes of interest. This is a laborious process, in which the smallest sub-region of the promoter still capable of recapitulating expression in an ectopic setting is first identified, followed by targeted mutation of putative transcription factor binding sites. Massively parallel reporter assays such as survey of regulatory elements (SuRE) provide an alternative way to study millions of promoter fragments in parallel. Here we show how a generalized linear model (GLM) can be used to transform genome-scale SuRE data into a high-resolution genomic track that quantifies the contribution of local sequence to promoter activity. This coefficient track helps identify regulatory elements and can be used to predict promoter activity of any sub-region in the genome. It thus allows in silico dissection of any promoter in the human genome to be performed. We developed a web application, available at cissector.nki.nl, that lets researchers easily perform this analysis as a starting point for their research into any promoter of interest.


Subject(s)
Promoter Regions, Genetic , Software , Humans , Binding Sites , Genome, Human/genetics , Protein Binding , Regulatory Sequences, Nucleic Acid
2.
Mol Cell ; 82(13): 2519-2531.e6, 2022 07 07.
Article in English | MEDLINE | ID: mdl-35594855

ABSTRACT

Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterized in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE-promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity to broad promiscuity. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order transcription factors (TF) motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.


Subject(s)
Enhancer Elements, Genetic , Genome , Promoter Regions, Genetic , Transcription Factors , Animals , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , Genome/genetics , Genomics , Mammals/metabolism , Mice , Promoter Regions, Genetic/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
3.
Nat Genet ; 51(7): 1160-1169, 2019 07.
Article in English | MEDLINE | ID: mdl-31253979

ABSTRACT

Most of the millions of SNPs in the human genome are non-coding, and many overlap with putative regulatory elements. Genome-wide association studies (GWAS) have linked many of these SNPs to human traits or to gene expression levels, but rarely with sufficient resolution to identify the causal SNPs. Functional screens based on reporter assays have previously been of insufficient throughput to test the vast space of SNPs for possible effects on regulatory element activity. Here we leveraged the throughput and resolution of the survey of regulatory elements (SuRE) reporter technology to survey the effect of 5.9 million SNPs, including 57% of the known common SNPs, on enhancer and promoter activity. We identified more than 30,000 SNPs that alter the activity of putative regulatory elements, partially in a cell-type-specific manner. Integration of this dataset with GWAS results may help to pinpoint SNPs that underlie human traits.


Subject(s)
Genetic Predisposition to Disease , Genome, Human , Polymorphism, Single Nucleotide , Regulatory Elements, Transcriptional , Transcription Factors/metabolism , Genome-Wide Association Study , Hep G2 Cells , High-Throughput Nucleotide Sequencing , Humans , K562 Cells , Phenotype , Quantitative Trait Loci , Transcription Factors/genetics
4.
Cell ; 177(4): 852-864.e14, 2019 05 02.
Article in English | MEDLINE | ID: mdl-30982597

ABSTRACT

It is largely unclear whether genes that are naturally embedded in lamina-associated domains (LADs) are inactive due to their chromatin environment or whether LADs are merely secondary to the lack of transcription. We show that hundreds of human promoters become active when moved from their native LAD position to a neutral context in the same cells, indicating that LADs form a repressive environment. Another set of promoters inside LADs is able to "escape" repression, although their transcription elongation is attenuated. By inserting reporters into thousands of genomic locations, we demonstrate that escaper promoters are intrinsically less sensitive to LAD repression. This is not simply explained by promoter strength but by the interplay between promoter sequence and local chromatin features that vary strongly across LADs. Enhancers also differ in their sensitivity to LAD chromatin. This work provides a general framework for the systematic understanding of gene regulation by repressive chromatin.


Subject(s)
Gene Expression Regulation/genetics , Nuclear Lamina/genetics , Promoter Regions, Genetic/genetics , Chromatin/genetics , Chromatin/metabolism , Gene Expression/genetics , Genome, Human/genetics , Genomics , Humans , K562 Cells
5.
Mol Cell ; 66(2): 167-168, 2017 Apr 20.
Article in English | MEDLINE | ID: mdl-28431229

ABSTRACT

In this issue of Molecular Cell, Xie et al. (2017) introduce Mosaic-seq, a powerful technology that combines CRISPRi and single-cell RNA-seq. This method enables the high-throughput assessment of contributions of enhancers to gene regulation.


Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation , Promoter Regions, Genetic , RNA/genetics
6.
Genome Res ; 27(7): 1153-1161, 2017 07.
Article in English | MEDLINE | ID: mdl-28420691

ABSTRACT

Housekeeping genes of animal genomes cluster in the same chromosomal regions. It has long been suggested that this organization contributes to their steady expression across all the tissues of the organism. Here, we show that the activity of Drosophila housekeeping gene promoters depends on the expression of their neighbors. By measuring the expression of ∼85,000 reporters integrated in Kc167 cells, we identified the best predictors of expression as chromosomal contacts with the promoters and terminators of active genes. Surprisingly, the chromatin composition at the insertion site and the contacts with enhancers were less informative. These results are substantiated by the existence of genomic "paradoxical" domains, rich in euchromatic features and enhancers, but where the reporters are expressed at low level, concomitant with a deficit of interactions with promoters and terminators. This indicates that the proper function of housekeeping genes relies not on contacts with long distance enhancers but on spatial clustering. Overall, our results suggest that spatial proximity between genes increases their expression and that the linear architecture of the Drosophila genome contributes to this effect.


Subject(s)
Gene Expression Regulation/physiology , Genes, Essential/physiology , Multigene Family/physiology , Animals , Cell Line , Drosophila melanogaster
7.
PLoS One ; 12(2): e0171508, 2017.
Article in English | MEDLINE | ID: mdl-28225770

ABSTRACT

Lineage-selective expression of developmental genes is dependent on the interplay between activating and repressive mechanisms. Gene activation is dependent on cell-specific transcription factors that recognize transcriptional enhancer sequences. Gene repression often depends on the recruitment of Polycomb group (PcG) proteins, although the sequences that underlie the recruitment of PcG proteins, also known as Polycomb response elements (PREs), remain poorly understood in vertebrates. While distal PREs have been identified in mammals, a role for positive-acting enhancers in PcG-mediated repression has not been described. Here we have used a highly efficient procedure based on lentiviral-mediated transgenesis to carry out in vivo fine-mapping of, cis-regulatory sequences that control lineage-specific activation of Neurog3, a master regulator of pancreatic endocrine differentiation. Our findings reveal an enhancer region that is sufficient to drive correct spacio-temporal expression of Neurog3 and demonstrate that this same region serves as a PRE in alternative lineages where Neurog3 is inactive.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Islets of Langerhans/metabolism , Nerve Tissue Proteins/genetics , Polycomb-Group Proteins/genetics , Response Elements/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Islets of Langerhans/cytology , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Polycomb-Group Proteins/metabolism
8.
Nat Biotechnol ; 35(2): 145-153, 2017 02.
Article in English | MEDLINE | ID: mdl-28024146

ABSTRACT

Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of the sequences that could be tested. Here we present 'survey of regulatory elements' (SuRE), a method that assays more than 108 DNA fragments, each 0.2-2 kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library of random genomic fragments upstream of a 20-bp barcode is constructed, and decoded by paired-end sequencing. This library is used to transfect cells, and barcodes in transcribed RNA are quantified by high-throughput sequencing. When applied to the human genome, we achieve 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide in K562 cells. By computational modeling we delineate subregions within promoters that are relevant for their activity. We show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.


Subject(s)
Chromosome Mapping/methods , Genome, Human/genetics , Promoter Regions, Genetic/genetics , Sequence Analysis, DNA/methods , Transcription Initiation, Genetic , Transcriptional Activation/genetics , DNA/genetics , Gene Library , Humans , K562 Cells
9.
Article in English | MEDLINE | ID: mdl-27777628

ABSTRACT

BACKGROUND: Chromatin proteins control gene activity in a concerted manner. We developed a high-throughput assay to study the effects of the local chromatin environment on the regulatory activity of a protein of interest. The assay combines a previously reported multiplexing strategy based on barcoded randomly integrated reporters with Gal4-mediated tethering. We applied the assay to Drosophila heterochromatin protein 1a (HP1a), which is mostly known as a repressive protein but has also been linked to transcriptional activation. RESULTS: Recruitment to over 1000 genomic locations revealed that HP1a is a potent repressor able to silence even highly expressing reporter genes. However, the local chromatin context can modulate HP1a function. In pericentromeric regions, HP1a-induced repression was enhanced by twofold. In regions marked by a H3K36me3-rich chromatin signature, HP1a-dependent silencing was significantly decreased. We found no evidence for an activating function of HP1a in our experimental system. Furthermore, we did not observe stable transmission of repression over mitotic divisions after loss of targeted HP1a. CONCLUSIONS: The multiplexed tethered reporter assay should be applicable to a large number of chromatin proteins and will be a useful tool to dissect combinatorial regulatory interactions in chromatin.


Subject(s)
Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Drosophila Proteins/genetics , Animals , Cell Line , Chromosomal Proteins, Non-Histone/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Silencing , Histones/metabolism , Plasmids/genetics , Plasmids/metabolism , Transcription Elongation, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection
10.
Nucleic Acids Res ; 44(12): 5646-57, 2016 07 08.
Article in English | MEDLINE | ID: mdl-27001518

ABSTRACT

Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages.


Subject(s)
Chromatin/genetics , Chromosome Mapping , DNA Methylation/genetics , Drosophila melanogaster/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/genetics , Animals , Animals, Genetically Modified/genetics , Binding Sites , Cell Lineage/genetics , Gene Expression Regulation , Ligands , Neuroglia/metabolism , Organ Specificity , Protein Binding , Protein Biosynthesis/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/biosynthesis
11.
Trends Cell Biol ; 24(11): 695-702, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25160912

ABSTRACT

Although it was originally believed that enhancers activate only the nearest promoter, recent global analyses enabled by high-throughput technology suggest that the network of enhancer-promoter interactions is far more complex. The mechanisms that determine the specificity of enhancer-promoter interactions are still poorly understood, but they are thought to include biochemical compatibility, constraints imposed by the three-dimensional architecture of chromosomes, insulator elements, and possibly the effects of local chromatin composition. In this review, we assess the current insights into these determinants, and highlight the functional genomic approaches that will lead the way towards better mechanistic understanding.


Subject(s)
Chromatin/genetics , Chromosomes/genetics , Enhancer Elements, Genetic , Insulator Elements , Promoter Regions, Genetic , Animals , Gene Expression Regulation , Humans
12.
Stem Cells Transl Med ; 3(9): 1032-42, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25024431

ABSTRACT

Human embryonic and induced pluripotent stem cells are potential cell sources for regenerative approaches in Parkinson disease. Inductive differentiation protocols can generate midbrain dopamine neurons but result in heterogeneous cell mixtures. Therefore, selection strategies are necessary to obtain uniform dopamine cell populations. Here, we developed a selection approach using lentivirus vectors to express green fluorescent protein under the promoter region of FOXA2, a transcription factor that is expressed in the floor plate domain that gives rise to dopamine neurons during embryogenesis. We first validated the specificity of the vectors in human cell lines against a promoterless construct. We then selected FOXA2-positive neural progenitors from several human pluripotent stem cell lines, which demonstrated a gene expression profile typical for the ventral domain of the midbrain and floor plate, but failed to enrich for dopamine neurons. To investigate whether this was due to the selection approach, we overexpressed FOXA2 in neural progenitors derived from human pluripotent stem cell lines. FOXA2 forced expression resulted in an increased expression of floor plate but not mature neuronal markers. Furthermore, selection of the FOXA2 overexpressing fraction also failed to enrich for dopamine neurons. Collectively, our results suggest that FOXA2 is not sufficient to induce a dopaminergic fate in this system. On the other hand, our study demonstrates that a combined approach of promoter activation and lentivirus vector technology can be used as a versatile tool for the selection of a defined cell population from a variety of human pluripotent stem cell lines.


Subject(s)
Cell Separation/methods , Dopaminergic Neurons/cytology , Hepatocyte Nuclear Factor 3-beta/genetics , Neural Stem Cells/cytology , Pluripotent Stem Cells/cytology , Blotting, Western , Flow Cytometry , Fluorescent Antibody Technique , Genetic Vectors , Green Fluorescent Proteins/genetics , Humans , Lentivirus , Microscopy, Confocal , Promoter Regions, Genetic , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transduction, Genetic
13.
Genes Dev ; 27(1): 52-63, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23271347

ABSTRACT

Polycomb-mediated gene repression is essential for embryonic development, yet its precise role in lineage-specific programming is poorly understood. Here we inactivated Ring1b, encoding a polycomb-repressive complex 1 subunit, in pancreatic multipotent progenitors (Ring1b(progKO)). This caused transcriptional derepression of a subset of direct Ring1b target genes in differentiated pancreatic islet cells. Unexpectedly, Ring1b inactivation in differentiated islet ß cells (Ring1b(ßKO)) did not cause derepression, even after multiple rounds of cell division, suggesting a role for Ring1b in the establishment but not the maintenance of repression. Consistent with this notion, derepression in Ring1b(progKO) islets occurred preferentially in genes that were targeted de novo by Ring1b during pancreas development. The results support a model in which Ring1b bookmarks its target genes during embryonic development, and these genes are maintained in a repressed state through Ring1b-independent mechanisms in terminally differentiated cells. This work provides novel insights into how epigenetic mechanisms contribute to shaping the transcriptional identity of differentiated lineages.


Subject(s)
Gene Expression Regulation, Developmental , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Polycomb Repressive Complex 1/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Differentiation , Cells, Cultured , DNA Methylation , Embryo, Mammalian , Epigenesis, Genetic , Male , Mice , Neurons/metabolism , Polycomb Repressive Complex 1/genetics , Ubiquitin-Protein Ligases/genetics
14.
Cell Metab ; 16(4): 435-48, 2012 Oct 03.
Article in English | MEDLINE | ID: mdl-23040067

ABSTRACT

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and ß cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the ß cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a ß cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to ß cell programming and diabetes pathophysiology.


Subject(s)
Diabetes Mellitus, Type 2/metabolism , Insulin-Secreting Cells/metabolism , RNA, Long Noncoding/metabolism , Animals , Chromatin/chemistry , Chromatin/metabolism , DNA-Binding Proteins , Diabetes Mellitus, Type 2/pathology , Down-Regulation , Gene Expression Profiling , Genetic Loci , Humans , Mice , RNA, Messenger/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism
15.
Dev Cell ; 20(4): 411-2, 2011 Apr 19.
Article in English | MEDLINE | ID: mdl-21497752

ABSTRACT

In this issue of Developmental Cell, Dhawan et al. (2011) show that deletion of the Dnmt1 DNA methyltransferase gene in pancreatic insulin-producing cells makes these cells convert into glucagon-producing cells. This suggests that manipulation of a general epigenetic mechanism may be used to redirect cell fates.

16.
Genome Res ; 20(6): 722-32, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20395405

ABSTRACT

The epigenome changes that underlie cellular differentiation in developing organisms are poorly understood. To gain insights into how pancreatic beta-cells are programmed, we profiled key histone methylations and transcripts in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 differentiated tissues. We report that despite their endodermal origin, beta-cells show a transcriptional and active chromatin signature that is most similar to ectoderm-derived neural tissues. In contrast, the beta-cell signature of trimethylated H3K27, a mark of Polycomb-mediated repression, clusters with pancreatic progenitors, acinar cells and liver, consistent with the epigenetic transmission of this mark from endoderm progenitors to their differentiated cellular progeny. We also identified two H3K27 methylation events that arise in the beta-cell lineage after the pancreatic progenitor stage. One is a wave of cell-selective de novo H3K27 trimethylation in non-CpG island genes. Another is the loss of bivalent and H3K27me3-repressed chromatin in a core program of neural developmental regulators that enables a convergence of the gene activity state of beta-cells with that of neural cells. These findings reveal a dynamic regulation of Polycomb repression programs that shape the identity of differentiated beta-cells.


Subject(s)
Down-Regulation , Gene Expression Regulation, Developmental , Islets of Langerhans/metabolism , Pancreas/embryology , Repressor Proteins/genetics , Animals , Cell Separation , Cells, Cultured , Chromatin Immunoprecipitation , Epigenesis, Genetic , Flow Cytometry , Histones/metabolism , Male , Methylation , Mice , Mice, Inbred C57BL , Pancreas/cytology , Polycomb-Group Proteins
17.
Stem Cells ; 25(2): 411-8, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17038668

ABSTRACT

It is currently not known whether dopamine (DA) neurons derived from human embryonic stem cells (hESCs) can survive in vivo and alleviate symptoms in models of Parkinson disease (PD). Here, we report the use of Noggin (a bone morphogenic protein antagonist) to induce neuroectodermal cell development and increase the yield of DA neurons from hESCs. A combination of stromal-derived inducing activity and Noggin markedly enhanced the generation of neuroepithelial progenitors that could give rise to DA neurons. In addition, Noggin diminished the occurrence of a fibroblast-like Nestin-positive precursor population that differentiated into myocytes. After transplantation of differentiated hESCs to a rodent model of PD, some grafts contained human midbrain-like DA neurons. This protocol demonstrates hESC derivation and survival of human DA neurons appropriate for cell therapy in PD.


Subject(s)
Bone Morphogenetic Proteins/antagonists & inhibitors , Carrier Proteins/pharmacology , Dopamine/metabolism , Embryonic Stem Cells/cytology , Mesencephalon/cytology , Neuroepithelial Cells/cytology , Neurons/cytology , Animals , Biomarkers/metabolism , Cells, Cultured , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/transplantation , Female , Gene Expression Regulation/drug effects , Humans , Mesencephalon/drug effects , Neuroepithelial Cells/drug effects , Neurons/drug effects , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Stem Cell Transplantation
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