Developmental effector gene regulation: Multiplexed strategies for functional analysis.
Animals
Chromosomes, Artificial, Bacterial
/ genetics
Gene Expression Regulation, Developmental
/ genetics
Gene Regulatory Networks
/ genetics
Genes, Reporter
/ genetics
Genetic Engineering
/ methods
High-Throughput Nucleotide Sequencing
/ methods
Models, Biological
Sea Urchins
/ embryology
Sequence Analysis, DNA
/ methods
Transcription Factors
/ metabolism
Embryonic gene regulation
Recombineered BACs
Skeletogenic effector genes
Tag vectors
Journal
Developmental biology
ISSN: 1095-564X
Titre abrégé: Dev Biol
Pays: United States
ID NLM: 0372762
Informations de publication
Date de publication:
01 01 2019
01 01 2019
Historique:
received:
22
03
2018
revised:
23
10
2018
accepted:
24
10
2018
pubmed:
6
11
2018
medline:
5
3
2019
entrez:
6
11
2018
Statut:
ppublish
Résumé
The staggering complexity of the genome controls for developmental processes is revealed through massively parallel cis-regulatory analysis using new methods of perturbation and readout. The choice of combinations of these new methods is tailored to the system, question and resources at hand. Our focus is on issues that include the necessity or sufficiency of given cis-regulatory modules, cis-regulatory function in the normal spatial genomic context, and easily accessible high throughput and multiplexed analysis methods. In the sea urchin embryonic model, recombineered BACs offer new opportunities for consecutive modes of cis-regulatory analyses that answer these requirements, as we here demonstrate on a diverse suite of previously unstudied sea urchin effector genes expressed in skeletogenic cells. Positively active cis-regulatory modules were located in single Nanostring experiments per BAC containing the gene of interest, by application of our previously reported "barcode" tag vectors of which> 100 can be analyzed at one time. Computational analysis of DNA sequences that drive expression, based on the known skeletogenic regulatory state, then permitted effective identification of functional target site clusters. Deletion of these sub-regions from the parent BACs revealed module necessity, as simultaneous tests of the same regions in short constructs revealed sufficiency. Predicted functional inputs were then confirmed by site mutations, all generated and tested in multiplex formats. There emerged the simple conclusion that each effector gene utilizes a small subset of inputs from the skeletogenic GRN. These inputs may function to only adjust expression levels or in some cases necessary for expression. Since we know the GRN architecture upstream of the effector genes, we could then conceptually isolate and compare the wiring of the effector gene driver sub-circuits and identify the inputs whose removal abolish expression.
Identifiants
pubmed: 30392838
pii: S0012-1606(18)30208-2
doi: 10.1016/j.ydbio.2018.10.018
pmc: PMC6321769
mid: NIHMS1511624
pii:
doi:
Substances chimiques
Transcription Factors
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
68-79Subventions
Organisme : NICHD NIH HHS
ID : P01 HD037105
Pays : United States
Organisme : NIH HHS
ID : P40 OD010959
Pays : United States
Organisme : NICHD NIH HHS
ID : P41 HD095831
Pays : United States
Organisme : NIH HHS
ID : R24 OD023046
Pays : United States
Organisme : NICHD NIH HHS
ID : P41 HD071837
Pays : United States
Informations de copyright
Copyright © 2018 Elsevier Inc. All rights reserved.
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