Summary Distant-acting tissue-specific enhancers vastly outnumber protein-coding genes in mammalian genomes, but the functional significance of this regulatory complexity remains insufficiently understood1,2. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Surprisingly, none of ten deletions of individual enhancers caused noticeable changes in limb morphology. In contrast, removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain, heart) uncovered more than a thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Taken together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes and provides an effective regulatory buffer preventing deleterious phenotypic consequences upon loss of individual enhancers.
The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP–seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC–seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.
Successful reproduction in mammals requires a competent egg, which is formed during meiosis through two assymetrical cell divisions. Here, we show that a recently identified formin homology (FH) gene, formin-2 (Fmn2), is a maternal-effect gene that is expressed in oocytes and is required for progression through metaphase of meiosis I. Fmn2(-/-) oocytes cannot correctly position the metaphase spindle during meiosis I and form the first polar body. We demonstrate that Fmn2 is required for microtubule-independent chromatin positioning during metaphase I. Fertilization of Fmn2(-/-) oocytes results in polyploid embryo formation, recurrent pregnancy loss and sub-fertility in Fmn2(-/-) females. Injection of Fmn2 mRNA into Fmn2-deficient oocytes rescues the metaphase I block. Given that errors in meiotic maturation result in severe birth defects and are the most common cause of chromosomal aneuploidy and pregnancy loss in humans, studies of Fmn2 may provide a better understanding of infertility and birth defects.
Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to .1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research. C ONVENTIONAL gene targeting technology in mice relies on homologous recombination in embryonic stem (ES) cells to target specific gene sequences, most commonly to disrupt gene function (Doetschman et al. 1987;Kuehn et al. 1987;Thomas and Capecchi 1987). Advantages of gene targeting in ES cells are selective target sequence modification, the ability to insert or delete genetic information, and the stability of the targeted mutations through subsequent generations. There are also potential limitations, including limited rates of germline transmission and strain limitations due to lack of conventional ES cell lines (Ledermann 2000;Mishina and Sakimura 2007). Moving the targeted allele from one strain to another requires 10 generations of backcrosses that take 2-3 years. A minimum of 1 year is necessary for backcrossing if speed congenics is applied (Markel et al. 1997).Zinc-finger nucleases (ZFNs) are fusions of specific DNA-binding zinc finger proteins (ZFPs) and a nuclease domain, such as the DNA cleavage domain of a type II endonuclease, FokI (Kim et al. 1996;Smith et al. 1999;Bibikova et al. 2001). A pair of ZFPs provide target specificity, and their nuclease domains dimerize to cleave the DNA, generating double strand breaks (DSBs) (Mani et al. 2005), which are detrimental to the cell if left unrepaired (Rich et al. 2000). The cell uses two main pathways to repair DSBs: high-fidelity homologous recombination and error-prone nonhomologous end joining (NHEJ) (Lieber 1999;Pardo et al. 2009;Huertas 2010). ZFN-mediated gene disruption results from deletions or insertions frequently introduced by NHEJ. Figure 1 illustrates the cellular events following the injec...
Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.
Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal led to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knock-in mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.
The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tie2 is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2 expressing populations using Tie2-Cre;Rosa26R-EYFP mice. In Tie2-Cre;Rosa26R-EYFP mice, analysis of bone marrow cells showed Cre-mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed sub-classes of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ~84% of each lineage was EYFP+, and nearly all cells that come from Tie2 expressing lineages are CD45+, confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2+ origin. Our findings of high levels of Tie2-Cre recombination in the hematopoietic lineage have implications for the use of the Tie2-Cre mouse as a lineage restricted driver strain.
Enhancers are important noncoding elements, but they have been traditionally hard to characterize experimentally. The development of massively parallel assays allows the characterization of large numbers of enhancers for the first time. Here, we developed a framework using Drosophila STARR-seq to create shape-matching filters based on meta-profiles of epigenetic features. We integrated these features with supervised machine-learning algorithms to predict enhancers. We further demonstrated our model could be transferred to predict enhancers in mammals. We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mouse and transduction-based reporter assays in human cell lines (153 enhancers in total). The results confirmed our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription-factor binding patterns at predicted enhancers versus promoters. We demonstrated that these patterns enable the construction of a secondary model effectively discriminating between enhancers and promoters.
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