Publications by authors named "Len A Pennacchio"

148 Publications

Reactivation of a developmentally silenced embryonic globin gene.

Nat Commun 2021 07 21;12(1):4439. Epub 2021 Jul 21.

MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

The α- and β-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.
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http://dx.doi.org/10.1038/s41467-021-24402-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295333PMC
July 2021

Coding and noncoding variants in EBF3 are involved in HADDS and simplex autism.

Hum Genomics 2021 Jul 13;15(1):44. Epub 2021 Jul 13.

Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.

Background: Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737.

Results: We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10), and combined dataset (p = 1.1 × 10). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs (missense p = 8.12 × 10, loss-of-function p = 2.26 × 10) and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes (p = 7.43 × 10, OR = 1.87) involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity (hypotonia, ataxia, and delayed development syndrome [HADDS]) in comparison to individuals with noncoding DNVs that have autism and hypotonia.

Conclusions: In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs.
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http://dx.doi.org/10.1186/s40246-021-00342-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8278787PMC
July 2021

Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice.

Genome Med 2021 04 26;13(1):69. Epub 2021 Apr 26.

Department of Psychiatry and Behavioral Sciences, University of California, Davis, Davis, CA, USA.

Background: Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the Na1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified.

Methods: To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior.

Results: Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and Na1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits.

Conclusions: This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.
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http://dx.doi.org/10.1186/s13073-021-00884-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080386PMC
April 2021

Ultraconserved enhancer function does not require perfect sequence conservation.

Nat Genet 2021 04 29;53(4):521-528. Epub 2021 Mar 29.

Environmental Genomics & System Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

Ultraconserved enhancer sequences show perfect conservation between human and rodent genomes, suggesting that their functions are highly sensitive to mutation. However, current models of enhancer function do not sufficiently explain this extreme evolutionary constraint. We subjected 23 ultraconserved enhancers to different levels of mutagenesis, collectively introducing 1,547 mutations, and examined their activities in transgenic mouse reporter assays. Overall, we find that the regulatory properties of ultraconserved enhancers are robust to mutation. Upon mutagenesis, nearly all (19/23, 83%) still functioned as enhancers at one developmental stage, as did most of those tested again later in development (5/9, 56%). Replacement of endogenous enhancers with mutated alleles in mice corroborated results of transgenic assays, including the functional resilience of ultraconserved enhancers to mutation. Our findings show that the currently known activities of ultraconserved enhancers do not necessarily require the perfect conservation observed in evolution and suggest that additional regulatory or other functions contribute to their sequence constraint.
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http://dx.doi.org/10.1038/s41588-021-00812-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038972PMC
April 2021

HAND transcription factors cooperatively specify the aorta and pulmonary trunk.

Dev Biol 2021 Aug 20;476:1-10. Epub 2021 Mar 20.

Herman B Wells Center for Pediatric Research Department of Pediatrics, Anatomy, Biochemistry, and Medical and Molecular Genetics, Indiana University School of Medicine, 1044 W. Walnut St., Indianapolis, IN, 46202-5225, USA. Electronic address:

Congenital heart defects (CHDs) affecting the cardiac outflow tract (OFT) constitute a significant cause of morbidity and mortality. The OFT develops from migratory cell populations which include the cardiac neural crest cells (cNCCs) and secondary heart field (SHF) derived myocardium and endocardium. The related transcription factors HAND1 and HAND2 have been implicated in human CHDs involving the OFT. Although Hand1 is expressed within the OFT, Hand1 NCC-specific conditional knockout mice (H1CKOs) are viable. Here we show that these H1CKOs present a low penetrance of OFT phenotypes, whereas SHF-specific Hand1 ablation does not reveal any cardiac phenotypes. Further, HAND1 and HAND2 appear functionally redundant within the cNCCs, as a reduction/ablation of Hand2 on an NCC-specific H1CKO background causes pronounced OFT defects. Double conditional Hand1 and Hand2 NCC knockouts exhibit persistent truncus arteriosus (PTA) with 100% penetrance. NCC lineage-tracing and Sema3c in situ mRNA expression reveal that Sema3c-expressing cells are mis-localized, resulting in a malformed septal bridge within the OFTs of H1CKO;H2CKO embryos. Interestingly, Hand1 and Hand2 also genetically interact within the SHF, as SHF H1CKOs on a heterozygous Hand2 background exhibit Ventricular Septal Defects (VSDs) with incomplete penetrance. Previously, we identified a BMP, HAND2, and GATA-dependent Hand1 OFT enhancer sufficient to drive reporter gene expression within the nascent OFT and aorta. Using these transcription inputs as a probe, we identify a novel Hand2 OFT enhancer, suggesting that a conserved BMP-GATA dependent mechanism transcriptionally regulates both HAND factors. These findings support the hypothesis that HAND factors interpret BMP signaling within the cNCCs to cooperatively coordinate OFT morphogenesis.
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http://dx.doi.org/10.1016/j.ydbio.2021.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8172455PMC
August 2021

ATAC-Seq Reveals an Enhancer That Regulates Sinoatrial Node Development and Function.

Circ Res 2020 12 12;127(12):1502-1518. Epub 2020 Oct 12.

Cardiology Division (G.G., R.M., H.R., C.J., R.S.W., P.K.R.A., A.R., M.B.S., V.V.), University of California, San Francisco.

Rationale: Cardiac pacemaker cells (PCs) in the sinoatrial node (SAN) have a distinct gene expression program that allows them to fire automatically and initiate the heartbeat. Although critical SAN transcription factors, including Isl1 (Islet-1), Tbx3 (T-box transcription factor 3), and Shox2 (short-stature homeobox protein 2), have been identified, the -regulatory architecture that governs PC-specific gene expression is not understood, and discrete enhancers required for gene regulation in the SAN have not been identified.

Objective: To define the epigenetic profile of PCs using comparative ATAC-seq (assay for transposase-accessible chromatin with sequencing) and to identify novel enhancers involved in SAN gene regulation, development, and function.

Methods And Results: We used ATAC-seq on sorted neonatal mouse SAN to compare regions of accessible chromatin in PCs and right atrial cardiomyocytes. PC-enriched assay for transposase-accessible chromatin peaks, representing candidate SAN regulatory elements, were located near established SAN genes and were enriched for distinct sets of TF (transcription factor) binding sites. Among several novel SAN enhancers that were experimentally validated using transgenic mice, we identified a 2.9-kb regulatory element at the locus that was active specifically in the cardiac inflow at embryonic day 8.5 and throughout later SAN development and maturation. Deletion of this enhancer from the genome of mice resulted in SAN hypoplasia and sinus arrhythmias. The mouse SAN enhancer also directed reporter activity to the inflow tract in developing zebrafish hearts, demonstrating deep conservation of its upstream regulatory network. Finally, single nucleotide polymorphisms in the human genome that occur near the region syntenic to the mouse enhancer exhibit significant associations with resting heart rate in human populations.

Conclusions: (1) PCs have distinct regions of accessible chromatin that correlate with their gene expression profile and contain novel SAN enhancers, (2) -regulation of specifically in the SAN depends upon a conserved SAN enhancer that regulates PC development and SAN function, and (3) a corresponding human enhancer may regulate human SAN function.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720845PMC
December 2020

Supervised enhancer prediction with epigenetic pattern recognition and targeted validation.

Nat Methods 2020 08 29;17(8):807-814. Epub 2020 Jul 29.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.

Enhancers are important non-coding elements, but they have traditionally been 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 that 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 mice and transduction-based reporter assays in human cell lines (153 enhancers in total). The results confirmed that 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 that effectively distinguishes enhancers and promoters.
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http://dx.doi.org/10.1038/s41592-020-0907-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073243PMC
August 2020

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

Nature 2020 07 29;583(7818):699-710. Epub 2020 Jul 29.

Department of Biological Science, Florida State University, Tallahassee, FL, USA.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE and Roadmap Epigenomics data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.
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http://dx.doi.org/10.1038/s41586-020-2493-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410828PMC
July 2020

The changing mouse embryo transcriptome at whole tissue and single-cell resolution.

Nature 2020 07 29;583(7818):760-767. Epub 2020 Jul 29.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

During mammalian embryogenesis, differential gene expression gradually builds the identity and complexity of each tissue and organ system. Here we systematically quantified mouse polyA-RNA from day 10.5 of embryonic development to birth, sampling 17 tissues and organs. The resulting developmental transcriptome is globally structured by dynamic cytodifferentiation, body-axis and cell-proliferation gene sets that were further characterized by the transcription factor motif codes of their promoters. We decomposed the tissue-level transcriptome using single-cell RNA-seq (sequencing of RNA reverse transcribed into cDNA) and found that neurogenesis and haematopoiesis dominate at both the gene and cellular levels, jointly accounting for one-third of differential gene expression and more than 40% of identified cell types. By integrating promoter sequence motifs with companion ENCODE epigenomic profiles, we identified a prominent promoter de-repression mechanism in neuronal expression clusters that was attributable to known and novel repressors. Focusing on the developing limb, single-cell RNA data identified 25 candidate cell types that included progenitor and differentiating states with computationally inferred lineage relationships. We extracted cell-type transcription factor networks and complementary sets of candidate enhancer elements by using single-cell RNA-seq to decompose integrative cis-element (IDEAS) models that were derived from whole-tissue epigenome chromatin data. These ENCODE reference data, computed network components and IDEAS chromatin segmentations are companion resources to the matching epigenomic developmental matrix, and are available for researchers to further mine and integrate.
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http://dx.doi.org/10.1038/s41586-020-2536-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410830PMC
July 2020

Spatiotemporal DNA methylome dynamics of the developing mouse fetus.

Nature 2020 07 29;583(7818):752-759. Epub 2020 Jul 29.

Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA.

Cytosine DNA methylation is essential for mammalian development but understanding of its spatiotemporal distribution in the developing embryo remains limited. Here, as part of the mouse Encyclopedia of DNA Elements (ENCODE) project, we profiled 168 methylomes from 12 mouse tissues or organs at 9 developmental stages from embryogenesis to adulthood. We identified 1,808,810 genomic regions that showed variations in CG methylation by comparing the methylomes of different tissues or organs from different developmental stages. These DNA elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. During late stages of fetal development, non-CG methylation accumulated within the bodies of key developmental transcription factor genes, coinciding with their transcriptional repression. Integration of genome-wide DNA methylation, histone modification and chromatin accessibility data enabled us to predict 461,141 putative developmental tissue-specific enhancers, the human orthologues of which were enriched for disease-associated genetic variants. These spatiotemporal epigenome maps provide a resource for studies of gene regulation during tissue or organ progression, and a starting point for investigating regulatory elements that are involved in human developmental disorders.
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http://dx.doi.org/10.1038/s41586-020-2119-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398276PMC
July 2020

An atlas of dynamic chromatin landscapes in mouse fetal development.

Nature 2020 07 29;583(7818):744-751. Epub 2020 Jul 29.

Center for Epigenomics, University of California, San Diego School of Medicine, La Jolla, CA, USA.

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.
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http://dx.doi.org/10.1038/s41586-020-2093-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398618PMC
July 2020

Presynaptic Homeostasis Opposes Disease Progression in Mouse Models of ALS-Like Degeneration: Evidence for Homeostatic Neuroprotection.

Neuron 2020 07 6;107(1):95-111.e6. Epub 2020 May 6.

Department of Biochemistry and Biophysics, Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, CA 94941, USA. Electronic address:

Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.
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http://dx.doi.org/10.1016/j.neuron.2020.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529479PMC
July 2020

Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants.

Cell 2020 03 12;180(6):1262-1271.e15. Epub 2020 Mar 12.

Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute, Berkeley, CA 94720, USA; Comparative Biochemistry Program, University of California, Berkeley, CA 94720, USA. Electronic address:

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 lead 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 knockin 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.
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http://dx.doi.org/10.1016/j.cell.2020.02.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7179509PMC
March 2020

Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons.

Cell Rep 2019 08;28(8):2048-2063.e8

Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA; Development and Stem Cell Biology Program, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.
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http://dx.doi.org/10.1016/j.celrep.2019.07.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750766PMC
August 2019

Stable enhancers are active in development, and fragile enhancers are associated with evolutionary adaptation.

Genome Biol 2019 07 15;20(1):140. Epub 2019 Jul 15.

Computational Biology Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20892, USA.

Background: Despite continual progress in the identification and characterization of trait- and disease-associated variants that disrupt transcription factor (TF)-DNA binding, little is known about the distribution of TF binding deactivating mutations (deMs) in enhancer sequences. Here, we focus on elucidating the mechanism underlying the different densities of deMs in human enhancers.

Results: We identify two classes of enhancers based on the density of nucleotides prone to deMs. Firstly, fragile enhancers with abundant deM nucleotides are associated with the immune system and regular cellular maintenance. Secondly, stable enhancers with only a few deM nucleotides are associated with the development and regulation of TFs and are evolutionarily conserved. These two classes of enhancers feature different regulatory programs: the binding sites of pioneer TFs of FOX family are specifically enriched in stable enhancers, while tissue-specific TFs are enriched in fragile enhancers. Moreover, stable enhancers are more tolerant of deMs due to their dominant employment of homotypic TF binding site (TFBS) clusters, as opposed to the larger-extent usage of heterotypic TFBS clusters in fragile enhancers. Notably, the sequence environment and chromatin context of the cognate motif, other than the motif itself, contribute more to the susceptibility to deMs of TF binding.

Conclusions: This dichotomy of enhancer activity is conserved across different tissues, has a specific footprint in epigenetic profiles, and argues for a bimodal evolution of gene regulatory programs in vertebrates. Specifically encoded stable enhancers are evolutionarily conserved and associated with development, while differently encoded fragile enhancers are associated with the adaptation of species.
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http://dx.doi.org/10.1186/s13059-019-1750-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631995PMC
July 2019

Noncoding deletions reveal a gene that is critical for intestinal function.

Nature 2019 07 19;571(7763):107-111. Epub 2019 Jun 19.

Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.
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http://dx.doi.org/10.1038/s41586-019-1312-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061489PMC
July 2019

TIMELESS mutation alters phase responsiveness and causes advanced sleep phase.

Proc Natl Acad Sci U S A 2019 06 28;116(24):12045-12053. Epub 2019 May 28.

Department of Neurology, University of California, San Francisco, CA 94143;

Many components of the circadian molecular clock are conserved from flies to mammals; however, the role of mammalian Timeless remains ambiguous. Here, we report a mutation in the human (h) gene that causes familial advanced sleep phase (FASP). CRISPR mutant mice exhibit FASP with altered photic entrainment but normal circadian period. We demonstrate that the mutation prevents TIM accumulation in the nucleus and has altered affinity for CRY2, leading to destabilization of PER/CRY complex and a shortened period in nonmature mouse embryonic fibroblasts (MEFs). We conclude that TIM, when excluded from the nucleus, can destabilize the negative regulators of the circadian clock, alter light entrainment, and cause FASP.
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http://dx.doi.org/10.1073/pnas.1819110116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6575169PMC
June 2019

Dynamic BAF chromatin remodeling complex subunit inclusion promotes temporally distinct gene expression programs in cardiogenesis.

Development 2019 07 5;146(19). Epub 2019 Jul 5.

Gladstone Institutes, San Francisco, CA 94158, USA

Chromatin remodeling complexes instruct cellular differentiation and lineage specific transcription. The BRG1/BRM-associated factor (BAF) complexes are important for several aspects of differentiation. We show that the catalytic subunit gene has a specific role in cardiac precursors (CPs) to initiate cardiac gene expression programs and repress non-cardiac expression. Using immunopurification with mass spectrometry, we have determined the dynamic composition of BAF complexes during mammalian cardiac differentiation, identifying several cell-type specific subunits. We focused on the CP- and cardiomyocyte (CM)-enriched subunits BAF60c (SMARCD3) and BAF170 (SMARCC2). and co-regulate gene expression with in CPs, and in CMs their loss results in broadly deregulated cardiac gene expression. BRG1, BAF60c and BAF170 modulate chromatin accessibility, to promote accessibility at activated genes while closing chromatin at repressed genes. BAF60c and BAF170 are required for proper BAF complex composition, and BAF170 loss leads to retention of BRG1 at CP-specific sites. Thus, dynamic interdependent BAF complex subunit assembly modulates chromatin states and thereby participates in directing temporal gene expression programs in cardiogenesis.
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http://dx.doi.org/10.1242/dev.174086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803373PMC
July 2019

Parkinson-Associated SNCA Enhancer Variants Revealed by Open Chromatin in Mouse Dopamine Neurons.

Am J Hum Genet 2018 12 29;103(6):874-892. Epub 2018 Nov 29.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. Electronic address:

The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD), and modulation of risk by common variants in PD has been well established through genome-wide association studies (GWASs). We acquired open chromatin signatures of purified embryonic mouse MB DA neurons because we anticipated that a fraction of PD-associated genetic variation might mediate the variants' effects within this neuronal population. Correlation with >2,300 putative enhancers assayed in mice revealed enrichment for MB cis-regulatory elements (CREs), and these data were reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA, directed reporter expression in catecholaminergic neurons from transgenic mice and zebrafish. Sequencing of this CRE in 986 individuals with PD and 992 controls revealed two common variants associated with elevated PD risk. To assess potential mechanisms of action, we screened >16,000 proteins for DNA binding capacity and identified a subset whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identified a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA might modulate PD risk and highlights the value of cell-context-dependent guided searches for functional non-coding variation.
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http://dx.doi.org/10.1016/j.ajhg.2018.10.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288322PMC
December 2018

Author Correction: Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

Nat Neurosci 2018 Jul;21(7):1015

Ludwig Institute for Cancer Research, La Jolla, CA, USA.

In the version of this article initially published online, the accession code was given as GSE1000333. The correct code is GSE100033. The error has been corrected in the print, HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41593-018-0114-4DOI Listing
July 2018

Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation.

Nat Neurosci 2018 03 12;21(3):432-439. Epub 2018 Feb 12.

Ludwig Institute for Cancer Research, La Jolla, CA, USA.

Analysis of chromatin accessibility can reveal transcriptional regulatory sequences, but heterogeneity of primary tissues poses a significant challenge in mapping the precise chromatin landscape in specific cell types. Here we report single-nucleus ATAC-seq, a combinatorial barcoding-assisted single-cell assay for transposase-accessible chromatin that is optimized for use on flash-frozen primary tissue samples. We apply this technique to the mouse forebrain through eight developmental stages. Through analysis of more than 15,000 nuclei, we identify 20 distinct cell populations corresponding to major neuronal and non-neuronal cell types. We further define cell-type-specific transcriptional regulatory sequences, infer potential master transcriptional regulators and delineate developmental changes in forebrain cellular composition. Our results provide insight into the molecular and cellular dynamics that underlie forebrain development in the mouse and establish technical and analytical frameworks that are broadly applicable to other heterogeneous tissues.
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http://dx.doi.org/10.1038/s41593-018-0079-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862073PMC
March 2018

Enhancer redundancy provides phenotypic robustness in mammalian development.

Nature 2018 02 31;554(7691):239-243. Epub 2018 Jan 31.

Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA.

Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear. 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. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the 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 and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.
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http://dx.doi.org/10.1038/nature25461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808607PMC
February 2018

Ultraconserved Enhancers Are Required for Normal Development.

Cell 2018 01 18;172(3):491-499.e15. Epub 2018 Jan 18.

Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA; U.S. Department of Energy Joint Genome Institute, Walnut Creek, CA 94598, USA; School of Natural Sciences, University of California Merced, Merced, CA 95343, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2017.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786478PMC
January 2018

Relationship between genetic variation at PPP1R3B and levels of liver glycogen and triglyceride.

Hepatology 2018 06 19;67(6):2182-2195. Epub 2018 Apr 19.

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX.

Genetic variation at rs4240624 on chromosome 8 is associated with an attenuated signal on hepatic computerized tomography, which has been attributed to changes in hepatic fat. The closest coding gene to rs4240624, PPP1R3B, encodes a protein that promotes hepatic glycogen synthesis. Here, we performed studies to determine whether the x-ray attenuation associated with rs4240624 is due to differences in hepatic glycogen or hepatic triglyceride content (HTGC). A sequence variant in complete linkage disequilibrium with rs4240624, rs4841132, was genotyped in the Dallas Heart Study (DHS), the Dallas Liver Study, and the Copenhagen Cohort (n = 112,428) of whom 1,539 had nonviral liver disease. The minor A-allele of rs4841132 was associated with increased hepatic x-ray attenuation (n = 1,572; P = 4 × 10 ), but not with HTGC (n = 2,674; P = 0.58). Rs4841132-A was associated with modest, but significant, elevations in serum alanine aminotransferase (ALT) in the Copenhagen Cohort (P = 3 × 10 ) and the DHS (P = 0.004), and with odds ratios for liver disease of 1.13 (95% CI, 0.97-1.31) and 1.23 (1.01-1.51), respectively. Mice lacking protein phosphatase 1 regulatory subunit 3B (PPP1R3B) were deficient in hepatic glycogen, whereas HTGC was unchanged. Hepatic overexpression of PPP1R3B caused accumulation of hepatic glycogen and elevated plasma levels of ALT, but did not change HTGC.

Conclusion: These observations are consistent with the notion that the minor allele of rs4841132 promotes a mild form of hepatic glycogenosis that is associated with hepatic injury. (Hepatology 2018;67:2182-2195).
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http://dx.doi.org/10.1002/hep.29751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991995PMC
June 2018

Genomic Patterns of De Novo Mutation in Simplex Autism.

Cell 2017 Oct 28;171(3):710-722.e12. Epub 2017 Sep 28.

Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA; Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. Electronic address:

To further our understanding of the genetic etiology of autism, we generated and analyzed genome sequence data from 516 idiopathic autism families (2,064 individuals). This resource includes >59 million single-nucleotide variants (SNVs) and 9,212 private copy number variants (CNVs), of which 133,992 and 88 are de novo mutations (DNMs), respectively. We estimate a mutation rate of ∼1.5 × 10 SNVs per site per generation with a significantly higher mutation rate in repetitive DNA. Comparing probands and unaffected siblings, we observe several DNM trends. Probands carry more gene-disruptive CNVs and SNVs, resulting in severe missense mutations and mapping to predicted fetal brain promoters and embryonic stem cell enhancers. These differences become more pronounced for autism genes (p = 1.8 × 10, OR = 2.2). Patients are more likely to carry multiple coding and noncoding DNMs in different genes, which are enriched for expression in striatal neurons (p = 3 × 10), suggesting a path forward for genetically characterizing more complex cases of autism.
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http://dx.doi.org/10.1016/j.cell.2017.08.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679715PMC
October 2017

Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb.

PLoS Comput Biol 2017 Aug 21;13(8):e1005720. Epub 2017 Aug 21.

Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.

Epigenomic mapping of enhancer-associated chromatin modifications facilitates the genome-wide discovery of tissue-specific enhancers in vivo. However, reliance on single chromatin marks leads to high rates of false-positive predictions. More sophisticated, integrative methods have been described, but commonly suffer from limited accessibility to the resulting predictions and reduced biological interpretability. Here we present the Limb-Enhancer Genie (LEG), a collection of highly accurate, genome-wide predictions of enhancers in the developing limb, available through a user-friendly online interface. We predict limb enhancers using a combination of >50 published limb-specific datasets and clusters of evolutionarily conserved transcription factor binding sites, taking advantage of the patterns observed at previously in vivo validated elements. By combining different statistical models, our approach outperforms current state-of-the-art methods and provides interpretable measures of feature importance. Our results indicate that including a previously unappreciated score that quantifies tissue-specific nuclease accessibility significantly improves prediction performance. We demonstrate the utility of our approach through in vivo validation of newly predicted elements. Moreover, we describe general features that can guide the type of datasets to include when predicting tissue-specific enhancers genome-wide, while providing an accessible resource to the general biological community and facilitating the functional interpretation of genetic studies of limb malformations.
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http://dx.doi.org/10.1371/journal.pcbi.1005720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578682PMC
August 2017

Germline Chd8 haploinsufficiency alters brain development in mouse.

Nat Neurosci 2017 Aug 26;20(8):1062-1073. Epub 2017 Jun 26.

Department of Psychiatry and Behavioral Sciences, University of California, Davis, Davis, California, USA.

The chromatin remodeling gene CHD8 represents a central node in neurodevelopmental gene networks implicated in autism. We examined the impact of germline heterozygous frameshift Chd8 mutation on neurodevelopment in mice. Chd8 mice displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased regional brain volume, validating that phenotypes of Chd8 mice overlap pathology reported in humans with CHD8 mutations. We applied network analysis to characterize neurodevelopmental gene expression, revealing widespread transcriptional changes in Chd8 mice across pathways disrupted in neurodevelopmental disorders, including neurogenesis, synaptic processes and neuroimmune signaling. We identified a co-expression module with peak expression in early brain development featuring dysregulation of RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we validated increased neuronal proliferation and developmental splicing perturbation in Chd8 mice. This integrative analysis offers an initial picture of the consequences of Chd8 haploinsufficiency for brain development.
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http://dx.doi.org/10.1038/nn.4592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008102PMC
August 2017

Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites.

Development 2017 04;144(7):1235-1241

Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143-3120, USA

Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here, we identify a bona fide cardiac-specific enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression .
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http://dx.doi.org/10.1242/dev.138487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399617PMC
April 2017
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