Biology subjects

Maurano, M. T.

Publications and source records attributed to Maurano, M. T..

16 recordsLinked to original sources

bioRxiv2026

Genome writing and Targeted Delivery of the NKX6-3/ANK1 gene cluster and its Type 2 Diabetes GWAS Variants to Human iPSCs

Genome-wide association studies (GWAS) identified over 600 loci containing single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes (T2D), most of which reside in non-coding regions. Among the set of T2D SNPs, linking causal genome variants to disease risk experimentally has remained a challenge; however, advances in synthetic mammalian genome writing techniques now enable the delivery of multiple haplotypes to human induced pluripotent stem cells (hiPSCs) to create a series of isogenic cell lines that can be differentiated and phenotyped in vitro. Here, to begin efforts in dissecting a T2D GWAS locus, we engineered an NKX6-3/ANK1 gene cluster knockout hiPSC line and introduced a landing pad facilitating the delivery of synthetic haplotype payloads. We built four haplotypes, including several that are not observed in nature, containing risk SNPs spanning the NKX6-3/ANK1 gene cluster using a method called "variant Switching Auxotrophic markers for Integration" (vSwAP-In), and integrated them precisely into hiPSCs. NKX6-3/ANK1 deletion blocked pancreatic progenitor and skeletal muscle differentiation, suggesting that NKX6-3 and ANK1 are required for early pancreatic and skeletal muscle development, and perhaps related to the existence of two nonoverlapping sets of SNPs in linkage disequilibrium that associate with the expression of the two adjacent genes. When NKX6-3/ANK1 T2D "Risk" haplotypes were reintroduced, skeletal muscle and pancreatic progenitor differentiation capabilities were restored. ANK1 expression was elevated in the ANK1 Risk and All-Risk haplotypes compared to the NKX6-3 Risk and Non-Risk haplotypes, establishing a functional experimental platform to examine risk SNP clusters in their native contexts. Overall, this work establishes a platform for the dissection of GWAS loci using synthetic haplotype genomics in hiPSCs. Significance StatementGenome-wide association studies have been used to identify disease-associated SNPs; however, most SNPs lie in non-coding regions, making functional experimentation difficult to perform. Using vSwAP-In, a yeast-based DNA variant-building method, and mSwAP-In, a mammalian genome engineering approach, we establish a platform for functional GWAS dissection in hiPSCs. This platform allows us to build DNA harboring virtually any combination of disease-risk SNPs, allowing for functional characterization of SNPs without the limitations of linkage disequilibrium. We demonstrate this approach using a Type 2 diabetes GWAS gene cluster, NKX6-3/ANK1.

genetics↗

bioRxiv2025

Genome writing to dissect consequences of SVA retrotransposon disease X-Linked Dystonia Parkinsonism

Human retrotransposon insertions are often associated with diseases. In the case of the neurodegenerative X-Linked Dystonia-Parkinsonism disease, a human-specific SINE-VNTR-Alu subfamily F retrotransposon was inserted in intron 32 of the TAF1 gene. Here, we genomically rewrote a portion of the mouse Taf1 allele with the corresponding 78-kb XDP patient derived TAF1 allele. In mESCs, the presence of the intronic SVAs--rather than the hybrid gene structure--reduces hyTAF1 levels. This leads to transcriptional downregulation of genes with TATA box enriched in their promoters and triggering apoptosis. Chromatin and transcriptome profiling revealed that intronic SVAs are actively transcribed, forming barriers that likely impede transcription elongation. In mice, neuronal lineage TAF1 humanization resulted lethality of male progeny within two months. XDP male mice had severe atrophy centered on the striatum--the same affected brain region in XDP patients. Lastly, CRISPRa-mediated activation of hyTAF1 restored mESC viability, suggesting boosting TAF1 transcription as a therapeutic approach.

genetics↗

bioRxiv2025

Synthetic genomic dissection of enhancer context sensitivity and synergy

Noncoding disease and trait-associated genetic variation is frequently interpreted in the context of genomic regulatory elements such as DNase I hypersensitive sites (DHSs). But while most DHSs lie within a few kilobases of another DHS, regulatory elements are typically analyzed individually without accounting for their neighbors. We characterize multiple heterotypic DHS combinations from different critical mESC regulator loci, all delivered in a constant chromosomal context replacing the Sox2 Locus Control Region (LCR). We employ an optimized high-throughput multiplexed delivery pipeline enabling analysis of 213 distinct payloads in 641 mouse embryonic stem cell (mESC) clones. We identify widespread examples of context-dependent enhancers which have no activity on their own but can more than double the activity of a neighboring DHS. Enhancers exhibit synergy only with certain partners, and deliveries to the Igf2/H19 locus show that synergy is not constrained to a single genomic context. We further show that synergy between neighboring DHSs decays as a characteristic function of distance, with its influence extending up to 4 kilobases. We fine map this context dependency to the contribution of individual transcription factor recognition sequences. Our approach implicates the specific sequence and architectural features underpinning pervasive genomic context effects, and outlines a direction for modeling the functional impact of noncoding regulatory variation on common human traits and diseases.

genomics↗

bioRxiv2025

Iterative improvement of deep learning models using synthetic regulatory genomics

Deep learning models can accurately reconstruct genome-wide epigenetic tracks from the reference genome sequence alone. But it is unclear what predictive power they have on sequence diverging from the reference, such as disease- and trait-associated variants or engineered sequences. Recent work has applied synthetic regulatory genomics to characterized dozens of deletions, inversions, and rearrangements of DNase I hypersensitive sites (DHSs). Here, we use the state-of-the-art model Enformer to predict DNA accessibility and RNA transcription across these engineered sequences when delivered at their endogenous loci. At high level, we observe a good correlation between accessibility predicted by Enformer and experimental data. But model performance was best for sequences that more resembled the reference, such as single deletions or combinations of multiple DHSs. Predictive power was poorer for rearrangements affecting DHS order or orientation. We use these data to fine-tune Enformer, yielding significant reduction in prediction error. We show that this fine-tuning retains strong predictive performance for other tracks. Our results show that current deep learning models perform poorly when presented with novel sequence diverging in certain critical features from their training set. Thus an iterative approach incorporating profiling of synthetic constructs can improve model generalizability and ultimately enable functional classification of regulatory variants identified by population studies.

genomics↗

bioRxiv2023

Genomic context sensitizes regulatory elements to genetic disruption

Enhancer function is frequently investigated piecemeal using truncated reporter assays or single deletion analysis. Thus it remains unclear to what extent enhancer function at native loci relies on surrounding genomic context. Using the Big-IN technology for targeted integration of large DNAs, we analyzed the regulatory architecture of the murine Igf2/H19 locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functional Igf2/H19 locus and engineered mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of the H19 enhancer cluster. Contrasting activity of payloads delivered at the endogenous Igf2/H19 locus or ectopically at Hprt revealed that the Igf2/H19 locus includes additional, previously unknown long-range regulatory elements. Exchanging components of the Igf2/H19 locus with the well-studied Sox2 locus showed that the H19 enhancer cluster functioned poorly out of context, and required its native surroundings to activate Sox2 expression. Conversely, the Sox2 locus control region (LCR) could activate both Igf2 and H19 outside its native context, but its activity was only partially modulated by CTCF occupancy at the ICR. Analysis of regulatory DNA actuation across different cell types revealed that, while the H19 enhancers are tightly coordinated within their native locus, the Sox2 LCR acts more independently. We show that these enhancer clusters typify broader classes of loci genome-wide. Our results show that unexpected dependencies may influence even the most studied functional elements, and our synthetic regulatory genomics approach permits large-scale manipulation of complete loci to investigate the relationship between locus architecture and function. HIGHLIGHTSO_LIComposite enhancer elements are subject to genomic context effects mapped to a specific architecture of their endogenous loci. C_LIO_LIIgf2/H19 expression is affected by long-range regulatory elements beyond the canonically defined locus, and the H19 enhancer cluster in particular relies on the surrounding context at its endogenous locus. C_LIO_LIThe Sox2 LCR functions as an autonomous enhancer without requiring additional surrounding context. C_LIO_LIThe influence of genomic context is buffered at intact loci, but manifests more strongly as key regulatory elements are deleted or repositioned. C_LI

genomics↗

bioRxiv2022

Mouse genomic rewriting and tailoring: synthetic Trp53 and humanized ACE2

Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection.

genomics↗

bioRxiv2022

Genomic analysis of a synthetic reversed sequence reveals default chromatin states in yeast and mammalian cells

Up to 93% of the human genome may show evidence of transcription, yet annotated transcripts account for less than 5%. It is unclear what makes up this major discrepancy, and to what extent the excess transcription has a definable biological function, or is just a pervasive byproduct of non-specific RNA polymerase binding and transcription initiation. Understanding the default state of the genome would be informative in determining whether the observed pervasive activity has a definable function. The genome of any modern organism has undergone billions of years of evolution, making it unclear whether any observed genomic activity, or lack thereof, has been selected for. We sought to address this question by introducing a completely novel 100-kb locus into the genomes of two eukaryotic organisms, S. cerevisiae and M. musculus, and characterizing its genomic activity based on chromatin accessibility and transcription. The locus was designed by reversing (but not complementing) the sequence of the human HPRT1 locus, including [~]30-kb of both upstream and downstream regulatory regions, allowing retention of sequence features like repeat frequency and GC content but ablating coding information and transcription factor binding sites. We also compared this reversed locus with a synthetic version of the normal human HPRT1 locus in both organismal contexts. Despite neither the synthetic HPRT1 locus nor its reverse version coding for any promoters evolved for gene expression in yeast, we observed widespread transcriptional activity of both loci. This activity was observed both when the loci were present as episomes and when chromosomally integrated, although it did not correspond to any of the known HPRT1 functional regulatory elements. In contrast, when integrated in the mouse genome, the synthetic HPRT1 locus showed transcriptional activity corresponding precisely to the HPRT1 coding sequence, while the reverse locus displayed no activity at all. Together, these results show that genomic sequences with no coding information are active in yeast, but relatively inactive in mouse, indicating a potentially major difference in "default genomic states" between these two divergent eukaryotes.

molecular biology↗

bioRxiv2022

Dissection of a complex enhancer cluster at the Sox2 locus

Expression of Sox2 in mouse embryonic stem cells (mESCs) depends on a distal regulatory cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of independence remain a mystery. Here, we comprehensively analyze the regulatory architecture of Sox2 at its endogenous locus using Big-IN to scarlessly integrate DNA payloads ranging up to 143 kb. We analyzed 83 payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed to establish the necessity and sufficiency of genomic features for Sox2 expression. We found that two LCR DHSs comprising a handful of key TF recognition sequences were each sufficient to autonomously sustain significant expression in mESCs. However, three additional LCR DHSs were entirely context-dependent, in that they showed no activity alone but could dramatically augment activity of the core DHSs. Our synthetic regulatory genomics approach demonstrates that composite regulatory elements can be reduced to a tractable set of essential sequence features, and is readily scalable to investigate regulatory architecture at other key loci genome-wide.

genomics↗

bioRxiv2021

The genetic basis of tail-loss evolution in humans and apes

The loss of the tail is one of the main anatomical evolutionary changes to have occurred along the lineage leading to humans and to the "anthropomorphous apes"1,2. This morphological reprogramming in the ancestral hominoids has been long considered to have accommodated a characteristic style of locomotion and contributed to the evolution of bipedalism in humans3-5. Yet, the precise genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Primate genome sequencing projects have made possible the identification of causal links between genotypic and phenotypic changes6-8, and enable the search for hominoid-specific genetic elements controlling tail development9. Here, we present evidence that tail-loss evolution was mediated by the insertion of an individual Alu element into the genome of the hominoid ancestor. We demonstrate that this Alu element - inserted into an intron of the TBXT gene (also called T or Brachyury10-12) - pairs with a neighboring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated a mouse model that mimics the expression of human TBXT products by expressing both full-length and exon-skipped isoforms of the mouse TBXT ortholog. We found that mice with this genotype exhibit the complete absence of a tail or a shortened tail, supporting the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype, albeit with incomplete penetrance. We further noted that mice homozygous for the exon-skipped isoforms exhibited embryonic spinal cord malformations, resembling a neural tube defect condition, which affects [~]1/1000 human neonates13. We propose that selection for the loss of the tail along the hominoid lineage was associated with an adaptive cost of potential neural tube defects and that this ancient evolutionary trade-off may thus continue to affect human health today.

evolutionary biology↗

bioRxiv2021

Transcriptional regulation of HSCs in Aging and MDS reveals DDIT3 as a Potential Driver of Transformation

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) malignancies characterized by ineffective hematopoiesis, with increased incidence in elderly individuals. In this work, we analyzed the transcriptome of human HSCs purified from young and elderly healthy donors, as well as MDS patients, identifying transcriptional alterations following eight different patterns of expression. While aging-associated lesions seemed to predispose HSCs to myeloid transformation, disease-specific alterations may trigger MDS development. Among MDS-specific lesions, we detected the upregulation of the transcription factor DDIT3. Overexpression of DDIT3 in human healthy HSCs induced an MDS-like transcriptional state, and a delay in erythropoiesis. Such effect was associated with downregulation of transcription factors required for normal erythropoiesis, and with a failure in the activation of their transcriptional programs. Moreover, DDIT3 knockdown in CD34+ cells from MDS patients with anemia was able to restore erythropoiesis. These results identify DDIT3 as a driver of dyserythropoiesis, and a potential therapeutic target to restore the inefficient erythropoiesis characterizing MDS patients. STATEMENT OF SIGNIFICANCEThis study defines how human aging and MDS development are characterized by transcriptional alterations in HSCs that follow different patterns, some of which may contribute to myeloid transformation. Among them, we demonstrate how MDS-specific upregulation of DDIT3 in HSCs induces dyserythropoiesis, while its knockdown in HSPCs from MDS patients restores proper erythroid differentiation.

cancer biology↗

bioRxiv2021

Synthetic genomic reconstitution reveals principles of mammalian Hox cluster regulation

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modes controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce synthetic regulatory reconstitution, a novel conceptual framework for studying gene regulation and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130-170 kilobases each) to an ectopic location in the mouse genome. We find that a HoxA cluster lacking distal enhancers recapitulates correct patterns of chromatin remodeling and transcription in response to patterning signals, while distal enhancers are required for full transcriptional output. Synthetic regulatory reconstitution is a generalizable strategy to decipher the regulatory logic of gene expression in complex genomes. One-Sentence SummaryReconstitution of gene regulation using large DNA constructs unravels the regulatory logic of a developmental gene locus.

synthetic biology↗

bioRxiv2021

Genomic context sensitivity of insulator function

Compartmentalization of interactions between genomic regulatory elements and potential target genes is influenced by the binding of insulator proteins such as CTCF, which act as potent enhancer blockers when interposed between an enhancer and a promoter in a reporter assay. But only a minority of CTCF sites genome-wide function as boundary elements, depending on cellular and genomic context. To dissect the influence of genomic context on enhancer blocker activity, we integrated reporter constructs with promoter-only, promoter and enhancer, and enhancer blocker configurations at hundreds of thousands of genomic sites using the Sleeping Beauty transposase. Deconvolution of reporter activity by genomic position revealed strikingly different patterns of reporter function, including a compartment of enhancer blocker reporter integrations with robust expression. The high density of integration sites permits quantitative delineation of characteristic genomic context sensitivity profiles, and their decomposition into sensitivity to both local and distant DNaseI hypersensitive sites. Furthermore, a single-cell expression approach permits direct linkage of reporters integrated into the same clonal lineage with differential endogenous gene expression, revealing that CTCF insulator activity does not completely abrogate reporter effects on endogenous gene expression. Collectively, our results lend new insight to genomic regulatory compartmentalization and its influence on the determinants of promoter-enhancer specificity.

genomics↗

bioRxiv2020

Identification of cellular context sensitive regulatory variation in mouse genomes

Assessment of the functional consequences of disease-associated sequence variation at non-coding regulatory elements is complicated by their high degree of context sensitivity to both the local chromatin and nuclear environments. Allelic profiling of DNA accessibility across individuals has shown that only a select minority of sequence variation affects transcription factor (TF) occupancy, yet the low sequence diversity in human populations means that no experimental assessment is available for the majority of disease-associated variants. Here we describe high-resolution in vivo maps of allelic DNA accessibility in liver, kidney, lung and B cells from 5 increasingly diverged strains of F1 hybrid mice. The high density of heterozygous sites in these hybrids enables precise quantification of the effect size and cell-type specificity of hundreds of thousands of variants throughout the mouse genome. We show that chromatin-altering variants delineate characteristic sensitivity profiles for hundreds of TF motifs. We develop a compendium of TF-specific sensitivity profiles accounting for genomic context effects. Finally, we link these maps of allelic accessibility to allelic transcript levels in the same samples. This work provides a foundation for quantitative prediction of cell-type specific effects of non-coding variation on TF activity, which will dramatically facilitate both fine-mapping and systems-level analyses of common disease-associated variation in human genomes.

genomics↗

bioRxiv2020

An Effector Index to Predict Causal Genes at GWAS Loci

Drug development and biological discovery require effective strategies to map existing genetic associations to causal genes. To approach this problem, we began by identifying a set of positive control genes for 12 common diseases and traits that cause a Mendelian form of the disease or are the target of a medicine used for disease treatment. We then identified a widely-available set of genomic features enriching GWAS-associated single nucleotide variants (SNVs) for these positive control genes. Using these features, we trained and validated the Effector Index (Ei), a causal gene mapping algorithm using the 12 common diseases and traits. The area under Eis receiver operator curve to identify positive control genes was 80% and area under the precision recall curve was 29%. Using an enlarged set of independently curated positive control genes for type 2 diabetes which included genes identified by large-scale exome sequencing, these areas increased to 85% and 61%, respectively. The best predictors were coding or transcript altering SNVs, distance to gene and open chromatin-based metrics. We provide the Ei algorithm for its widespread use and have created a web-portal to facilitate understanding of results. This work outlines a simple, understandable approach to prioritize genes at GWAS loci for functional follow-up and drug development. Author summaryIn order to derive biological insight, or develop drugs based on genome-wide association studies (GWAS) data, causal genes at associated loci need to be identified. GWAS usually identify large genome regions containing many genes, but seldomly identifies specific causal genes. We have developed an algorithm to predict which genes in a region of disease association are likely causal and have named this algorithm the Effector Index. The Effector Index was optimized on diseases that have known causal or drug target genes, and further validated to predict these types of genes in independent datasets. The Effector Index formalizes these predictive features into a tool that can be used by researchers, and results from the traits and diseases studied here are available via the Accelerating Medicine Partnership web-portal at http://hugeamp.org/effectorgenes.html.

bioinformatics↗

bioRxiv2018

Big DNA as a tool to dissect an age-related macular degeneration haplotype

Age-related Macular Degeneration (AMD) is a leading cause of blindness in the developed world, especially in aging populations, and is therefore an important target for new therapeutic development. Recently, there have been several studies demonstrating strong associations between AMD and sites of heritable genetic variation at multiple loci, including a highly significant association at 10q26. The 10q26 risk region contains two genes, HTRA1 and ARMS2, both of which have been separately implicated as causative for the disease, as well as dozens of sites of non-coding variation. To date, no studies have successfully pinpointed which of these variant sites are functional in AMD, nor definitively identified which genes in the region are targets of such regulatory variation. In order to efficiently decipher which sites are functional in AMD phenotypes, we describe a general framework for combinatorial assembly of large synthetic haplotypes along with delivery to relevant disease cell types for downstream functional analysis. We demonstrate the successful and highly efficient assembly of a first-draft 119kb wild-type assemblon covering the HTRA1/ARMS2 risk region. We further propose the parallelized assembly of a library of combinatorial variant synthetic haplotypes covering the region, delivery and analysis of which will identify functional sites and their effects, leading to an improved understanding of AMD development. We anticipate that the methodology proposed here is highly generalizable towards the difficult problem of identifying truly functional variants from those discovered via GWAS or other genetic association studies.

genetics↗

bioRxiv2018

De novo assembly, delivery and expression of a 101 kb human gene in mouse cells

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assembled the 101 kb human HPRT1 gene in yeast, delivered it to mouse embryonic stem cells, and showed expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells. Significance StatementMammalian genomes consist of a tiny proportion of relatively well-characterized coding regions and vast swaths of poorly characterized "dark matter" containing critical but much less well-defined regulatory sequences. Given the dominant role of noncoding DNA in common human diseases and traits, the interconnectivity of regulatory elements, and the importance of genomic context, de novo design, assembly, and delivery can enable large-scale manipulation of these elements on a locus scale. Here we outline a pipeline for de novo assembly, delivery and expression of mammalian genes replete with native regulatory sequences. We expect this pipeline will be useful for dissecting the function of non-coding sequence variation in mammalian genomes.

genetics↗