Chromatin architecture has been implicated in cell-type-specific gene regulatory programs; yet, how chromatin remodels during development remains to be fully elucidated. Here, by interrogating chromatin reorganization during human pluripotent stem cell (PSC) differentiation, we discover a role for the primate-specific endogenous retrotransposon HERV-H in creating topologically associating domains (TAD) in human PSCs. Deleting these HERV-H elements eliminates their corresponding TAD boundaries and reduces transcription of upstream genes, while de novo insertion of HERV-Hs can introduce new TAD boundaries. HERV-H’s ability to create these TAD boundaries depends on high transcription, as transcriptional repression of HERV-H elements prevents formation of these boundaries. This ability is not limited to human PSCs, as these actively transcribed HERV-Hs and their corresponding TAD boundaries also appear in PSCs from other hominids but not in more distantly related species lacking HERV-Hs. Overall, our results provide direct evidence for retrotransposons in actively shaping cell-type- and species-specific chromatin architecture.

To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.

While enhancers for embryonic stem cell (ESC)-expressed genes and lineage-determining factors are characterized by conventional marks of enhancer activation in ESCs1,2,3, it remains unclear whether enhancers destined to regulate cell-type-restricted transcription units might also have some currently overlooked, distinct signatures in ESCs. Here, we report that cell-type-restricted enhancers, are unexpectedly premarked and activated as transcription units by the binding of a single, or two, ESC transcription factors, although not exhibiting traditional enhancer epigenetic marks in ESCs, thus uncovering the initial temporal origins of cell-type-restricted enhancers. This premarking is required for future cell-type-restricted enhancer activity in the differentiated cells, with the strength of the ESCs signature being functionally important for subsequent robustness of cell-type-restricted enhancer activation. This model has been experimentally validated in macrophage-restricted enhancers and neural precursor cells (NPCs)-restricted enhancers using ESCs-derived macrophages or NPCs, edited to contain specific ESC transcription factor motif deletions. The ESC transcription factor-determined DNA hydroxyl-methylation of the enhancers in ESCs may serve as a potential molecular memory for subsequent enhancer activation in the mature macrophage. These findings suggest that the massive repertoire of cell-type-restricted enhancers are essentially hierarchically and obligatorily “premarked” by binding of a defining ESC transcription factor in ESCs, dictating robustness of enhancer activation in mature cells.

29Restructuring of chromatin architecture is an essential process for establishing cell type-30 specific gene regulatory programs in eukaryotic cells including cardiomyocytes 1-3 . 31 Supporting its importance, recent studies have reported that a substantial number of 32 mutations discovered in congenital heart disease (CHD) patients reside in genes 33 encoding chromatin remodeling factors 4-6 ; yet, how chromatin structure reorganizes to 34 assemble gene regulatory networks crucial for controlling human cardiomyocyte 35 development remains to be elucidated. Here, through comprehensively analyzing high- 36 resolution genomic maps that detail the dynamic changes of chromatin architecture, 37 chromatin accessibility and modifications, and gene expression during human 38 pluripotent stem cell (PSC) cardiomyocyte differentiation, we reveal novel molecular 39 insights into how human PSC chromatin architecture is iteratively remodeled to build 40 gene regulatory networks directing cardiac lineage specification. Specifically, we 41 uncover a new class of human PSC-specific topologically associating domain (TAD) 42 that is created by the active transcription of the primate-specific endogenous 43 retrotransposon HERV-H. Silencing of specific HERV-Hs during the initial stages of 44 human PSC differentiation or by genome-editing results in the elimination of 45 corresponding TAD boundaries and reduced transcription of genes upstream of HERV-46Hs. Supporting their role in maintaining pluripotency, we discovered that deletion of 47 specific HERV-Hs leads to more efficient human PSC cardiomyocyte differentiation. 48Using chromatin interaction maps from these analyses, we also assigned potential 49 target genes to distal regulatory elements involved in cardiac differentiation. Genome-50 editing of enhancers harboring cardiac-disease risk loci associated with congenital and 51 adult heart diseases further confirmed that these loci regulate predicted target genes. 52 Our results highlight a novel role for HERV-Hs in establishing human-specific PSC 53 chromatin architecture, delineate the dynamic gene regulatory networks during 54 cardiomyocyte development and inform how non-coding genetic variants contribute to 55 congenital and adult heart diseases.56 3 MAIN TEXT 57 The three-dimensional organization of chromosomes enables long-range 58 communications between enhancers and promoters that are critical for building complex 59 gene regulatory networks in multicellular species 1,3 . In somatic cells, interphase 60 chromosomes occupy separate nuclear spaces known as chromosome territories 7 . 61 Each chromosome is folded into a dynamic but non-random hierarchical structure 62 characterized by stretches of transcriptionally active, megabase-long compartments that 63 are interspersed with stretches of transcriptionally inactive compartments 8 . These 64 compartments can be further partitioned into topologically associating domains (TADs), 65 which exhibit high levels of intra-domain interactions and relatively low levels of inter...