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Genomes contain both a genetic code specifying amino acids and a regulatory code specifying transcription factor (TF) recognition sequences. We used genomic deoxyribonuclease I footprinting to map nucleotide resolution TF occupancy across the human exorne in 81 diverse cell types. We found that -15% of human codons are dual-use codons (\"duons\") that simultaneously specify both amino acids and TF recognition sites. Duons are highly conserved and have shaped protein evolution, and TF-imposed constraint appears to be a major driver of codon usage bias. Conversely, the regulatory code has been selectively depleted of TFs that recognize stop codons. More than 17% of single-nucleotide variants within duons directly alter TF binding. Pervasive dual encoding of amino acid and regulatory information appears to be a fundamental feature of genome evolution.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis –regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein–DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency. DNase I footprinting in 41 cell and tissue types reveals millions of short sequence elements encoding an expansive repertoire of conserved recognition sequences for DNA-binding proteins. ENCODE: transcription-factor footprints DNaseI footprinting detects DNA sequences that are protected from cleavage by DNaseI because they are bound by regulatory factors. Studying these footprints in 41 diverse cell and tissue types, the authors describe millions of short sequence elements that are conserved recognition sequences for DNA-binding proteins. The effort nearly doubles the size of the human cis -regulatory lexicon and provides insight into chromatin states and levels of evolutionary conservation. A large collection of novel regulatory-factor recognition motifs that closely parallel major regulators of development, differentiation and pluripotency is also described.

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity. Mouse genomic footprinting reveals conservation of transcription factor (TF) recognition repertoires and trans-regulatory circuitry despite massive turnover of DNA elements that contact TFs in vivo. Trans-acting networks in the mouse epigenome Having generated genomic DNase I footprinting data of the mouse genome across 25 cell and tissue types, these authors use these data to quantify cis-versus-trans regulatory contributions to mammalian regulatory evolution. They describe more than 600 motifs that collectively are over 95% similar to that recognized in vivo by human transcription factors (TFs). Despite substantial turnover of the cis-regulatory landscape around each TF gene, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Conservation between mouse and human TF regulatory networks is particularly similar at the highest organization level. The work was performed as part of the mouse ENCODE project.

An empirical approach for identifying optimal proteotypic peptides and fragmentation patterns from in vitro –synthesized proteins, for targeted proteomics applications, is described. We report a method for high-throughput, cost-efficient empirical discovery of optimal proteotypic peptides and fragment ions for targeted proteomics applications using in vitro –synthesized proteins. We demonstrate the approach using human transcription factors, which are typically difficult, low-abundance targets and empirically derived proteotypic peptides for 98% of the target proteins. We show that targeted proteomic assays developed using our approach facilitate robust in vivo quantification of human transcription factors.

Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells. Isaac et al. present mtFiber-seq, a method that measures individual mitochondrial genome packaging at nucleotide resolution. They show that most nucleoids are in an inaccessible state, modulated by the abundance of the DNA-binding protein TFAM.

Much of our current understanding of rare human diseases is driven by coding genetic variants. However, non-coding genetic variants play a pivotal role in numerous rare human diseases, resulting in diverse functional impacts ranging from altered gene regulation, splicing, and/or transcript stability. With the increasing use of genome sequencing in clinical practice, it is paramount to have a clear framework for understanding how non-coding genetic variants cause disease. To this end, we have synthesized the literature on hundreds of non-coding genetic variants that cause rare Mendelian conditions via the disruption of gene regulatory patterns and propose a functional classification system. Specifically, we have adapted the functional classification framework used for coding variants (i.e., loss-of-function, gain-of-function, and dominant-negative) to account for features unique to non-coding gene regulatory variants. We identify that non-coding gene regulatory variants can be split into three distinct categories by functional impact: (1) non-modular loss-of-expression (LOE) variants; (2) modular loss-of-expression (mLOE) variants; and (3) gain-of-ectopic-expression (GOE) variants. Whereas LOE variants have a direct corollary with coding loss-of-function variants, mLOE and GOE variants represent disease mechanisms that are largely unique to non-coding variants. These functional classifications aim to provide a unified terminology for categorizing the functional impact of non-coding variants that disrupt gene regulatory patterns in Mendelian conditions.

Specific peptide ligand recognition by modular interaction domains is essential for the fidelity of information flow through the signal transduction networks that control cell behavior in response to extrinsic and intrinsic stimuli. Src homology 2 (SH2) domains recognize distinct phosphotyrosine peptide motifs, but the specific sites that are phosphorylated and the complement of available SH2 domains varies considerably in individual cell types. Such differences are the basis for a wide range of available protein interaction microstates from which signaling can evolve in highly divergent ways. This underlying complexity suggests the need to broadly map the signaling potential of systems as a prerequisite for understanding signaling in specific cell types as well as various pathologies that involve signal transduction such as cancer, developmental defects and metabolic disorders. This report describes interactions between SH2 domains and potential binding partners that comprise initial signaling downstream of activated fibroblast growth factor (FGF), insulin (Ins), and insulin-like growth factor-1 (IGF-1) receptors. A panel of 50 SH2 domains screened against a set of 192 phosphotyrosine peptides defines an extensive potential interactome while demonstrating the selectivity of individual SH2 domains. The interactions described confirm virtually all previously reported associations while describing a large set of potential novel interactions that imply additional complexity in the signaling networks initiated from activated receptors. This study of pTyr ligand binding by SH2 domains provides valuable insight into the selectivity that underpins complex signaling networks that are assembled using modular protein interaction domains.

Thyrotropin (TSH) is the master regulator of thyroid gland growth and function. Resistance to TSH (RTSH) describes conditions with reduced sensitivity to TSH. Dominantly inherited RTSH has been linked to a locus on chromosome 15q, but its genetic basis has remained elusive. Here we show that non-coding mutations in a (TTTG) 4 short tandem repeat (STR) underlie dominantly inherited RTSH in all 82 affected participants from 12 unrelated families. The STR is contained in a primate-specific Alu retrotransposon with thyroid-specific cis -regulatory chromatin features. Fiber-seq and RNA-seq studies revealed that the mutant STR activates a thyroid-specific enhancer cluster, leading to haplotype-specific upregulation of the bicistronic MIR7-2 / MIR1179 locus 35 kb downstream and overexpression of its microRNA products in the participants’ thyrocytes. An imbalance in signaling pathways targeted by these micro-RNAs provides a working model for this cause of RTSH. This finding broadens our current knowledge of genetic defects altering pituitary–thyroid feedback regulation. Short tandem repeat mutations in a primate Alu element on chromosome 15q cause activation of a thyroid-specific enhancer, upregulating MIR7-2 / MIR1179 . This results in defective thyroid proliferation and thyrotropin resistance.

Resolving the molecular basis of a Mendelian condition remains challenging owing to the diverse mechanisms by which genetic variants cause disease. To address this, we developed a synchronized long-read genome, methylome, epigenome and transcriptome sequencing approach, which enables accurate single-nucleotide, insertion–deletion and structural variant calling and diploid de novo genome assembly. This permits the simultaneous elucidation of haplotype-resolved CpG methylation, chromatin accessibility and full-length transcript information in a single long-read sequencing run. Application of this approach to an Undiagnosed Diseases Network participant with a chromosome X;13-balanced translocation of uncertain significance revealed that this translocation disrupted the functioning of four separate genes ( NBEA , PDK3 , MAB21L1 and RB1 ) previously associated with single-gene Mendelian conditions. Notably, the function of each gene was disrupted via a distinct mechanism that required integration of the four ‘omes’ to resolve. These included fusion transcript formation, enhancer adoption, transcriptional readthrough silencing and inappropriate X-chromosome inactivation of autosomal genes. Overall, this highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes. Simultaneous profiling of the genome, methylome, epigenome and transcriptome using single-molecule chromatin fiber sequencing and multiplexed arrays isoform sequencing identifies the genetic and molecular basis of an undiagnosed Mendelian disease case with an X;13-balanced translocation.