scholarly journals Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements

2016 ◽  
Author(s):  
Molly Gasperini ◽  
Gregory M. Findlay ◽  
Aaron McKenna ◽  
Jennifer H. Milbank ◽  
Choli Lee ◽  
...  
Keyword(s):  
Large Scale ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Mendelian Disease ◽  
Regulatory Sequences ◽  
Coding Region ◽  
Endogenous Gene ◽  
Distal Regulatory Elements

AbstractThe extent to which distal non-coding mutations contribute to Mendelian disease remains a major unknown in human genetics. Given that a gene’s in vivo function can be appropriately modeled in vitro, CRISPR/Cas9 genome editing enables the large-scale perturbation of distal non-coding regions to identify functional elements in their native context. However, early attempts at such screens have relied on one individual guide RNA (gRNA) per cell, resulting in sparse mutagenesis with minimal redundancy across regions of interest. To address this, we developed a system that uses pairs of gRNAs to program thousands of kilobase-scale deletions that scan across a targeted region in a tiling fashion (“ScanDel”). As a proof-of-concept, we applied ScanDel to program 4,342 overlapping 1- and 2- kilobase (Kb) deletions that tile a 206 Kb region centered on HPRT1, the gene underlying Lesch-Nyhan syndrome, with median 27-fold redundancy per base. Programmed deletions were functionally assayed by selecting for loss of HPRT1 function with 6-thioguanine. HPRT1 exons served as positive controls, and all were successfully identified as functionally critical by the screen. Remarkably, HPRT1 function appeared robust to deletion of any intergenic or deeply intronic non-coding region across the 206 Kb locus, indicating that proximal regulatory sequences are sufficient for its expression. A sparser mutagenesis screen of the same 206 Kb with individual gRNAs also failed to identify critical distal regulatory elements. Although our screen did find programmed deletions and individual gRNAs with putative functional consequences that targeted exon-proximal non-coding sequences (e.g. the promoter), long-read sequencing revealed that this signal was driven almost entirely by rare, unexpected deletions that extended into exonic sequence. These targeted validation experiments defined a small region surrounding the transcriptional start site as the only non-coding sequence essential to HPRT1 function. Overall, our results suggest that distal regulatory elements are not critical for HPRT1 expression, and underscore the necessity of comprehensive edited-locus genotyping for validating the results of CRISPR screens. The application of ScanDel to additional loci will enable more insight into the extent to which the disruption of distal non-coding elements contributes to Mendelian diseases. In addition, dense, redundant, large-scale deletion scanning with gRNA pairs will facilitate a deeper understanding of endogenous gene regulation in the human genome.

scholarly journals Functional Predictors of Causative Cis-Regulatory Mutations in Mendelian Disease

2020 ◽  
Author(s):  
Hemant Bengani ◽  
Detelina Grozeva ◽  
Lambert Moyon ◽  
Shipra Bhatia ◽  
Susana R Louros ◽  
...  
Keyword(s):  
Target Gene ◽  
Rare Variants ◽  
Regulatory Elements ◽  
Human Populations ◽  
Mendelian Disease ◽  
Loss Of Function ◽  
Coding Region ◽  
Endogenous Gene ◽  
Neurodevelopmental Disease

AbstractUndiagnosed neurodevelopmental disease is significantly associated with rare variants in cis-regulatory elements (CRE) but demonstrating causality is challenging as target gene consequences may differ from a causative variant affecting the coding region. Here, we address this challenge by applying a procedure to discriminate likely diagnostic regulatory variants from those of neutral/low-penetrant effect. We identified six rare CRE variants using targeted and whole genome sequencing in 48 unrelated males with apparent X-linked intellectual disability (XLID) but without detectable coding region variants. These variants segregated appropriately in families and altered conserved bases in predicted CRE targeting known XLID genes. Three were unique and three were rare but too common to be plausibly causative for XLID. We compared the cis-regulatory activity of wild-type and mutant alleles in zebrafish embryos using dual-color fluorescent reporters. Two variants showed striking changes: one plausibly causative (FMR1CRE) and the other likely neutral/low-penetrant (TENM1CRE). These variants were “knocked-in” to mice and both altered embryonic neural expression of their target gene. Only Fmr1CRE mice showed disease-relevant behavioral defects. FMR1CRE is plausibly disease-associated resulting in complex misregulation of Fmr1/FMRP rather than loss-of-function. This is consistent both with absence of Fragile X syndrome in the probands and the observed electrophysiological anomalies in the FMR1CRE mouse brain. Although disruption of in vivo patterns of endogenous gene expression in disease-relevant tissues by CRE variants cannot be used as strong evidence for Mendelian disease association, in conjunction with extreme rarity in human populations and with relevant knock-in mouse phenotypes, such variants can become likely pathogenic.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo

2021 ◽  
Author(s):  
Giuliano Crispatzu ◽  
Rizwan Rehimi ◽  
Tomas Pachano ◽  
Tore Bleckwehl ◽  
Sara de la Cruz Molina ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Regulatory Elements ◽  
Small Subset ◽  
Regulatory Sequences ◽  
Pluripotent Cells ◽  
Developmental Genes ◽  
Topological Features ◽  
Regulatory Properties

AbstractPoised enhancers (PEs) represent a limited and genetically distinct set of distal regulatory elements that control the induction of developmental genes in a hierarchical and non-redundant manner. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally identified and subsequently characterized using embryonic stem cells (ESC) as an in vitro differentiation system, it is currently unknown whether PEs are functionally conserved in vivo. Here, we generate and mine various types of genomic data to show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that, in mouse pluripotent cells, the interactions between PEs and their bivalent target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. proximity to CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation, preferentially within specific vertebrate clades, with only a small subset being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting evolutionary conserved PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential and non-redundant roles during the induction of major developmental genes in vivo.

scholarly journals Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 627-635 ◽  
Author(s):  
D.L. Song ◽  
G. Chalepakis ◽  
P. Gruss ◽  
A.L. Joyner
Keyword(s):  
Dna Binding ◽  
Transgenic Mice ◽  
Binding Sites ◽  
Regulatory Elements ◽  
Developing Brain ◽  
Molecular Evidence ◽  
Regulatory Sequences ◽  
Pax Genes

The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.

Spatial and temporal regulation of a foreign gene by a prestalk-specific promoter in transformed Dictyostelium discoideum

1986 ◽  
Vol 6 (3) ◽  
pp. 811-820
Author(s):  
S Datta ◽  
R H Gomer ◽  
R A Firtel
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Regulatory Elements ◽  
Foreign Gene ◽  
Specific Gene ◽  
Cell Type ◽  
Coding Region ◽  
Endogenous Gene ◽  
Temporal Regulation

We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease. A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene. The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads the way for experiments to identify the cell-type-specific regulatory elements.

scholarly journals Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557 ◽  
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Giuliano Crispatzu ◽  
Rizwan Rehimi ◽  
Tomas Pachano ◽  
Tore Bleckwehl ◽  
Sara Cruz-Molina ◽  
...  
Keyword(s):  
Target Genes ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Pluripotent Cells ◽  
Developmental Genes ◽  
Topological Features ◽  
Regulatory Properties

AbstractPoised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that the interactions between PEs and their target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo.

scholarly journals Spatial and temporal regulation of a foreign gene by a prestalk-specific promoter in transformed Dictyostelium discoideum.

1986 ◽  
Vol 6 (3) ◽  
pp. 811-820 ◽  
Author(s):  
S Datta ◽  
R H Gomer ◽  
R A Firtel
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Regulatory Elements ◽  
Foreign Gene ◽  
Specific Gene ◽  
Cell Type ◽  
Coding Region ◽  
Endogenous Gene ◽  
Temporal Regulation

We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease. A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene. The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads the way for experiments to identify the cell-type-specific regulatory elements.

scholarly journals Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition interface

2020 ◽  
Author(s):  
Rosemary A. Bayne ◽  
Uma Jayachandran ◽  
Aleksandra Kasprowicz ◽  
Stefan Bresson ◽  
David Tollervey ◽  
...  
Keyword(s):  
Stress Responses ◽  
Rna Binding ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Regulatory Sequences ◽  
Rna Recognition ◽  
Rnase Ii ◽  
Sequence Requirements

AbstractThe conserved fungal RNA binding protein Ssd1, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and may act by suppressing translation of associated mRNAs. Previous studies identified motifs that are enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not well understood. Here we present the crystal structure of Ssd1 at 1.9 Å resolution. Active RNase II enzymes have a characteristic, internal RNA binding path, but in Ssd1 this is blocked by remnants of regulatory sequences. Instead, RNA binding activity has likely been relocated to the outer surface of the protein. Using in vivo crosslinking and cDNA analysis (CRAC), we identify Ssd1-RNA binding sites. These are strongly enriched in 5’UTRs of a subset of mRNAs encoding cell wall proteins. Based on these and previous analyses, we identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory elements in the RNase II family.

scholarly journals Mouse GnRH Receptor Gene Expression Is Mediated by the LHX3 Homeodomain Protein

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2180-2185 ◽  
Author(s):  
Shauna M. McGillivray ◽  
Janice S. Bailey ◽  
Rosha Ramezani ◽  
Brian J. Kirkwood ◽  
Pamela L. Mellon
Keyword(s):  
Hormonal Regulation ◽  
Transcriptional Start Site ◽  
Receptor Gene ◽  
Regulatory Elements ◽  
Gnrh Receptor ◽  
Homeodomain Protein ◽  
Pituitary Development ◽  
Basal Expression

Abstract Appropriate expression of GnRH receptor (GnRHR) is necessary for the correct regulation of the gonadotropins, LH and FSH, by GnRH. GnRHR is primarily expressed in the gonadotrope cells of the anterior pituitary, and a number of regulatory elements important for both basal and hormonal regulation of the gene have been identified. Using the gonadotrope-derived cell line, αT3-1, that endogenously expresses GnRHR, we have identified an ATTA element located at −298 relative to the transcriptional start site that is essential for basal expression of the GnRHR gene. LHX3, a member of the LIM homeodomain family, binds the −298 ATTA site in vitro as well as to the endogenous GnRHR promoter in vivo. Additionally, LHX3 specifically activates through this −298 ATTA site in transient transfection assays. LHX3 is essential for pituitary development and has been implicated in the regulation of a number of pituitary specific genes; however, this is the first report identifying its role in the regulation of GnRHR.

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