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‘Pioneer’ transcription factors are required for stem-cell pluripotency, cell differentiation and cell reprogramming 1 , 2 . Pioneer factors can bind nucleosomal DNA to enable gene expression from regions of the genome with closed chromatin. SOX2 is a prominent pioneer factor that is essential for pluripotency and self-renewal of embryonic stem cells 3 . Here we report cryo-electron microscopy structures of the DNA-binding domains of SOX2 and its close homologue SOX11 bound to nucleosomes. The structures show that SOX factors can bind and locally distort DNA at superhelical location 2. The factors also facilitate detachment of terminal nucleosomal DNA from the histone octamer, which increases DNA accessibility. SOX-factor binding to the nucleosome can also lead to a repositioning of the N-terminal tail of histone H4 that includes residue lysine 16. We speculate that this repositioning is incompatible with higher-order nucleosome stacking, which involves contacts of the H4 tail with a neighbouring nucleosome. Our results indicate that pioneer transcription factors can use binding energy to initiate chromatin opening, and thereby facilitate nucleosome remodelling and subsequent transcription. Cryo-electron microscopy structures of the DNA-binding domains of the pioneer transcription factor SOX2 and its close homologue SOX11 elucidate the role of these factors in initiating chromatin opening and nucleosome remodelling.

Nucleosomes cover most of the genome and are thought to be displaced by transcription factors in regions that direct gene expression. However, the modes of interaction between transcription factors and nucleosomal DNA remain largely unknown. Here we systematically explore interactions between the nucleosome and 220 transcription factors representing diverse structural families. Consistent with earlier observations, we find that the majority of the studied transcription factors have less access to nucleosomal DNA than to free DNA. The motifs recovered from transcription factors bound to nucleosomal and free DNA are generally similar. However, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many transcription factors preferentially bind close to the end of nucleosomal DNA, or to periodic positions on the solvent-exposed side of the DNA. In addition, several transcription factors usually bind to nucleosomal DNA in a particular orientation. Some transcription factors specifically interact with DNA located at the dyad position at which only one DNA gyre is wound, whereas other transcription factors prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals notable differences in the binding of transcription factors to free and nucleosomal DNA, and uncovers a diverse interaction landscape between transcription factors and the nucleosome. A method for systematically exploring interactions between the nucleosome and transcription factors identifies five major interaction patterns.

We have developed CRISPR-assisted RNA–protein interaction detection method (CARPID), which leverages CRISPR–CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific long non-coding RNAs (lncRNAs) in the native cellular context. We applied CARPID to the nuclear lncRNA XIST, and it captured a list of known interacting proteins and multiple previously uncharacterized binding proteins. We generalized CARPID to explore binders of the lncRNAs DANCR and MALAT1, revealing the method’s wide applicability in identifying RNA-binding proteins. CARPID uses CRISPR technology to navigate biotin ligase to specific lncRNAs, which allows proximal labeling and thus the querying of RNA–protein interactions in living cells.

Understanding tumorigenesis and inherited risk for cancer requires a multiomic approach Developments in modern genomics tools have led to rapid progress in our understanding of the genetic basis of cancer. Recent large-scale efforts have primarily focused on two types of analysis: mapping acquired somatic mutations by whole-exome and whole-genome sequencing ( 1 , 2 ), and identification of common inherited variants that increase cancer risk using genome-wide association studies (GWAS) ( 3 ). Despite the power of these technologies, we are still far from understanding how the variants and mutations found in individual tumors precisely drive the oncogenic process. A large number of genetic variants increase risk for cancer, but most explain only a very small fraction of the risk. Furthermore, although acquired somatic mutations are found in almost all tumors, most do not carry complete sets of mutations that, according to our present mechanistic understanding, would be sufficient to cause cancer. On page 420 of this issue, Corces et al. ( 4 ) show how a third type of genomics approach—functional genomic analyses of primary human tumors—can begin to bridge this gap in our mechanistic understanding of the tumorigenic process.

When the DNA bases cytosine and guanine are next to each other, a methyl group is generally added to the pyrimidine, generating a mCpG dinucleotide. This modification alters DNA structure but can also affect function by inhibiting transcription factor (TF) binding. Yin et al. systematically analyzed the effect of CpG methylation on the binding of 542 human TFs (see the Perspective by Hughes and Lambert). In addition to inhibiting binding of some TFs, they found that mCpGs can promote binding of others, particularly TFs involved in development, such as homeodomain proteins. Science , this issue p. eaaj2239 ; see also p. 489 Genome-scale analysis reveals positive and negative binding of transcription factors to methylated CpG dinucleotides. The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF–TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression. Analysis of massively parallel reporter assays measuring the transcriptional activity of DNA sequences indicates that most transcription factor (TF) activity is additive and does not rely on specific TF–TF interactions. Individual TFs can have different gene regulatory activities.

Clonal hematopoiesis driven by somatic heterozygous TET2 loss is linked to malignant degeneration via consequent aberrant DNA methylation, and possibly to cardiovascular disease via increased cytokine and chemokine expression as reported in mice. Here, we discover a germline TET2 mutation in a lymphoma family. We observe neither unusual predisposition to atherosclerosis nor abnormal pro-inflammatory cytokine or chemokine expression. The latter finding is confirmed in cells from three additional unrelated TET2 germline mutation carriers. The TET2 defect elevates blood DNA methylation levels, especially at active enhancers and cell-type specific regulatory regions with binding sequences of master transcription factors involved in hematopoiesis. The regions display reduced methylation relative to all open chromatin regions in four DNMT3A germline mutation carriers, potentially due to TET2-mediated oxidation. Our findings provide insight into the interplay between epigenetic modulators and transcription factor activity in hematological neoplasia, but do not confirm the putative role of TET2 in atherosclerosis. Somatic heterozygous TET2 loss drives clonal hematopoiesis, which is linked to malignant cell degeneration and potentially cardiovascular disease. Here, the authors investigate the molecular impact of a germline TET2 mutation in a lymphoma family, finding elevated blood DNA methylation levels and no predisposition to atherosclerosis

Unique molecular identifiers (UMIs) associate distinct sequences with every DNA or RNA molecule and can be counted after amplification to quantify molecules in the original sample. Using UMIs, the authors obtain a digital karyotype of an individual with Down's syndrome and quantify mRNA in Drosophila melanogaster cells. Counting individual RNA or DNA molecules is difficult because they are hard to copy quantitatively for detection. To overcome this limitation, we applied unique molecular identifiers (UMIs), which make each molecule in a population distinct, to genome-scale human karyotyping and mRNA sequencing in Drosophila melanogaster . Use of this method can improve accuracy of almost any next-generation sequencing method, including chromatin immunoprecipitation–sequencing, genome assembly, diagnostics and manufacturing-process control and monitoring.

The Zoonomia Project is investigating the genomics of shared and specialized traits in eutherian mammals. Here we provide genome assemblies for 131 species, of which all but 9 are previously uncharacterized, and describe a whole-genome alignment of 240 species of considerable phylogenetic diversity, comprising representatives from more than 80% of mammalian families. We find that regions of reduced genetic diversity are more abundant in species at a high risk of extinction, discern signals of evolutionary selection at high resolution and provide insights from individual reference genomes. By prioritizing phylogenetic diversity and making data available quickly and without restriction, the Zoonomia Project aims to support biological discovery, medical research and the conservation of biodiversity. A whole-genome alignment of 240 phylogenetically diverse species of eutherian mammal—including 131 previously uncharacterized species—from the Zoonomia Project provides data that support biological discovery, medical research and conservation.

Background DNA methylation is an important epigenetic modification which has numerous roles in modulating genome function. Its levels are spatially correlated across the genome, typically high in repressed regions but low in transcription factor (TF) binding sites and active regulatory regions. However, the mechanisms establishing genome-wide and TF binding site methylation patterns are still unclear. Results Here we use a comparative approach to investigate the association of DNA methylation to TF binding evolution in mammals. Specifically, we experimentally profile DNA methylation and combine this with published occupancy profiles of five distinct TFs (CTCF, CEBPA, HNF4A, ONECUT1, FOXA1) in the liver of five mammalian species (human, macaque, mouse, rat, dog). TF binding sites are lowly methylated, but they often also have intermediate methylation levels. Furthermore, biding sites are influenced by the methylation status of CpGs in their wider binding regions even when CpGs are absent from the core binding motif. Employing a classification and clustering approach, we extract distinct and species-conserved patterns of DNA methylation levels at TF binding regions. CEBPA, HNF4A, ONECUT1, and FOXA1 share the same methylation patterns, while CTCF's differ. These patterns characterize alternative functions and chromatin landscapes of TF-bound regions. Leveraging our phylogenetic framework, we find DNA methylation gain upon evolutionary loss of TF occupancy, indicating coordinated evolution. Furthermore, each methylation pattern has its own evolutionary trajectory reflecting its genomic contexts. Conclusions Our epigenomic analyses indicate a role for DNA methylation in TF binding changes across species including that specific DNA methylation profiles characterize TF binding and are associated with their regulatory activity, chromatin contexts, and evolutionary trajectories.