SUMMARYDefects in patterning during human embryonic development frequently result in craniofacial abnormalities. The gene regulatory programs that build the craniofacial complex are likely controlled by information located between genes and within intronic sequences. However, systematic identification of regulatory sequences important for forming the human face has not been performed. Here, we describe comprehensive epigenomic annotations from human embryonic craniofacial tissues and systematic comparisons with multiple tissues and cell types. We identified thousands of tissue-specific craniofacial regulatory sequences and likely causal regions for rare craniofacial abnormalities. We demonstrate significant enrichment of common variants associated with orofacial clefting in enhancers active early in embryonic development, while those associated with normal facial variation are enriched near the end of the embryonic period. These data are provided in easily accessible formats for both craniofacial researchers and clinicians to aid future experimental design and interpretation of noncoding variation in those affected by craniofacial abnormalities.

Rationale: There is growing evidence that common variants and rare sequence alterations in regulatory sequences can result in birth defects or predisposition to disease. Congenital heart defects (CHDs) are the most common birth defect and have a clear genetic component, yet only a third of cases can be attributed to structural variation in the genome or a mutation in a gene. The remaining unknown cases could be caused by alterations in regulatory sequences. Objective: Identify regulatory sequences and gene expression networks that are active during organogenesis of the human heart. Determine if these sites and networks are enriched for disease relevant genes and associated genetic variation. Methods and Results: We characterized chromatin state and gene expression dynamics during human heart organogenesis. We profiled seven histone modifications in embryonic hearts from each of nine distinct Carnegie stages (CS13-14, CS16-21, and CS23), annotated chromatin states, and compared these maps to over 100 human tissues and cell types. We also generated RNA-seq data, performed differential expression, and constructed weighted gene co-expression networks. We identified 177,412 heart enhancers, 12,395 had not been previously annotated as strong enhancers. We identified 92% of all functionally validated heart positive enhancers (n=281, 7.5x enrichment, p<2.2x10 -16 ). Integration of these data demonstrated novel heart enhancers are enriched near genes expressed more strongly in cardiac tissue and are enriched for variants associated with electrocardiogram measures and atrial fibrillation. Our gene expression network analysis identified gene modules strongly enriched for heart related functions, regulatory control by heart specific enhancers, and putative disease genes. Conclusions: Well-connected "hub" genes with heart-specific expression targeted by embryonic heart-specific enhancers are likely disease candidates. Our functional annotations will allow for better interpretation of whole genome sequencing data in the large number of patients affected by CHDs.

SummaryPKR (protein kinase R) is a key component of the interferon antiviral defense pathway. Upon binding dsRNA, PKR undergoes autophosphorylation reactions that activate the kinase. PKR contains an Nterminal double-stranded RNA binding domain (dsRBD), which consists of two tandem dsRNA binding motifs, and a C-terminal kinase domain. We have used small angle X-ray and neutron scattering to define the conformation of latent PKR in solution. Guinier analysis indicates a radius of gyration of about 35 Å. The p(r) distance distribution function exhibits a peak near 30 Å with a broad shoulder extending to longer distances. Good fits to the scattering data require models that incorporate multiple compact and extended conformations of the two interdomain linker regions. Thus, PKR belongs to the growing family of proteins that contain intrinsically unstructured regions. We propose that the flexible linkers may allow PKR to productively dimerize upon interaction with RNA activators that have diverse structures.

dStem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G 2 , mitotic, and G 1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G 2 -M-G 1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. Human embryonic stem cells (hESCs) are an increasingly powerful tool for regenerative medicine. They recapitulate, in vitro, the molecular phenomena that take place during the first stages of embryonic development. Like their in vivo counterparts, ESCs proliferate rapidly and are able to form the three embryonic germ layers (1). This highly self-renewing and pluripotent state is sustained by a unique epigenetic landscape, consisting of transcription factors, chromatin remodeling complexes, and histone modifications that provide the transcriptional plasticity required for rapid response to differentiation cues (2).Histone H3 lysine 4 and 27 trimethylations (H3K4me3 and H3K27me3, respectively) are key histone modifications that are involved in transcriptional regulation (3, 4). H3K4me3 near transcriptional start sites (TSSs) marks regions of active transcription or transcriptional readiness (5). H3K27me3 modification, in contrast, is a well-established negative regulator of gene expression that repels transcriptional activators and attracts chromatin repressors that promote chromatin compaction (6). Genomic regions that host both histone marks, so-called bivalent domains, were first observed in ESCs, primarily near promoters of genes with developmental functions (7-9). Significant effort has gone into understanding the biological role of bivalency; the consensus is that, in ESCs, it represses transcription but poises genes for rapid expression during lineage commitment (10). Although this proposition is not yet supported with direct evidence, it has become clear that bivalent domains are essential for maintaining ESC pluripotency and self-renewing capacity (10). Despite the extensive...

The Runx1 transcription factor, known for its essential role normal hematopoiesis, was reported in limited studies to be mutated or associated with human breast tumor tissues. Runx 1 increases concomitant with disease progression in the MMTV-PyMT transgenic mouse model of breast cancer. Compelling questions relate to mechanisms that regulate Runx1 expression in breast cancer. Here, we tested the hypothesis that dysregulation of Runx1-targeting microRNAs (miRNAs) allows for pathologic increase of Runx1 during breast cancer progression. Microarray profiling of the MMTV-PyMT model revealed significant down-regulation of numerous miRNAs predicted to target Runx1. One of these, miR-378, was inversely correlated with Runx1 expression during breast cancer progression in mouse, and in human breast cancer cell lines MCF7 and triple negative MDA-MB-231 that represent early and late stage disease, respectively. MiR-378 is nearly absent in MDA-MB-231 cells. Luciferase reporter assays revealed that miR-378 binds the Runx1 3′UTR and inhibits Runx1 expression. Functionally, we demonstrated that ectopic expression of miR-378 in MDA-MB-231 cells inhibited Runx1 and suppressed migration and invasion; while inhibition of miR-378 in MCF7 cells increased Runx1 levels and cell migration. Depletion of Runx1 in late stage breast cancer cells resulted in increased expression of both the miR-378 host gene PPARGC1B and pre-miR-378, suggesting a feedback loop. Taken together, our study identifies a novel and clinically relevant mechanism for regulation of Runx1 in breast cancer that is mediated by a PPARGC1B-miR-378-Runx1 regulatory pathway. Our results highlight the translational potential of miRNA replacement therapy for inhibiting Runx1 in breast cancer.