Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.

Summary Transcription factors related to the insect sex determination gene Doublesex (DMRT proteins) control sex determination and/or sexual differentiation in diverse metazoans, and are implicated in transitions between sex-determining mechanisms during vertebrate evolution [1]. In mice Dmrt1 is required for male gonadal differentiation in somatic cells and germ cells [2-4]. DMRT1 also maintains male gonadal sex: its loss, even in adults, can trigger sexual fate reprogramming in which male Sertoli cells transdifferentiate into their female equivalents – granulosa cells – and testicular tissue reorganizes to a more ovarian morphology [5]. Here we use a conditional Dmrt1 transgene to show that Dmrt1 is not only necessary but also sufficient to specify male cell identity in the mouse gonad. DMRT1 expression in the ovary silenced the female sex-maintenance gene Foxl2 and reprogrammed juvenile and adult granulosa cells into Sertoli-like cells, triggering formation of structures resembling male seminiferous tubules. DMRT1 can silence Foxl2 even in the absence of the testis-determining genes Sox8 and Sox9. mRNA profiling found that DMRT1 activates many testicular genes and downregulates ovarian genes and single cell RNA-seq in transdifferentiating cells identified dynamically expressed candidate mediators of this process. Strongly upregulated genes were highly enriched on chromosome X, consistent with sexually antagonistic functions. This study provides an in vivo example of single gene reprogramming of cell sexual identity. Our findings suggest a reconsideration of mechanisms involved in human disorders of sexual development (DSD) and empirically support evolutionary models where loss or gain of Dmrt1 function promotes establishment of new vertebrate sex determination systems.

Summary Mammalian sex determination initiates in the fetal gonad with specification of bipotential precursor cells into male Sertoli cells or female granulosa cells. This choice was long presumed to be irreversible, but genetic analysis in the mouse recently revealed that sexual fates must be maintained throughout life. Somatic cells in the testis or ovary, even in adults, can be induced to transdifferentiate to their opposite-sex equivalents by loss of a single transcription factor, DMRT1 in the testis or FOXL2 in the ovary. Here we ask what mechanism DMRT1 prevents from triggering transdifferentiation. We find that DMRT1 blocks testicular retinoic acid (RA) signaling from activating genes normally involved in female sex determination and ovarian development and show that inappropriate activation of these genes can drive sexual transdifferentiation. By preventing activation of potential feminizing genes, DMRT1 allows Sertoli cells to participate in RA signaling, which is essential for reproduction, without being sexually reprogrammed.

DNA is naturally well suited to serve as a digital medium for in vivo molecular recording. However, contemporary DNA-based memory devices are constrained in terms of the number of distinct ‘symbols’ that can be concurrently recorded and/or by a failure to capture the order in which events occur1. Here we describe DNA Typewriter, a general system for in vivo molecular recording that overcomes these and other limitations. For DNA Typewriter, the blank recording medium (‘DNA Tape’) consists of a tandem array of partial CRISPR–Cas9 target sites, with all but the first site truncated at their 5′ ends and therefore inactive. Short insertional edits serve as symbols that record the identity of the prime editing guide RNA2 mediating the edit while also shifting the position of the ‘type guide’ by one unit along the DNA Tape, that is, sequential genome editing. In this proof of concept of DNA Typewriter, we demonstrate recording and decoding of thousands of symbols, complex event histories and short text messages; evaluate the performance of dozens of orthogonal tapes; and construct ‘long tape’ potentially capable of recording as many as 20 serial events. Finally, we leverage DNA Typewriter in conjunction with single-cell RNA-seq to reconstruct a monophyletic lineage of 3,257 cells and find that the Poisson-like accumulation of sequential edits to multicopy DNA tape can be maintained across at least 20 generations and 25 days of in vitro clonal expansion.