Embryonic stem (ES) cells are regulated by a network of transcription factors that maintain the pluripotent state. Differentiation relies on down-regulation of pluripotency transcription factors disrupting this network. While investigating transcriptional regulation of the pluripotency transcription factor Kruppel-like factor 4 (Klf4), we observed that homozygous deletion of distal enhancers caused a 17-fold decrease in Klf4 transcript but surprisingly decreased protein levels by less than twofold, indicating that posttranscriptional control of KLF4 protein overrides transcriptional control. The lack of sensitivity of KLF4 to transcription is due to high protein stability (half-life >24 h). This stability is context-dependent and is disrupted during differentiation, as evidenced by a shift to a half-life of <2 h. KLF4 protein stability is maintained through interaction with other pluripotency transcription factors (NANOG, SOX2, and STAT3) that together facilitate association of KLF4 with RNA polymerase II. In addition, the KLF4 DNA-binding and transactivation domains are required for optimal KLF4 protein stability. Posttranslational modification of KLF4 destabilizes the protein as cells exit the pluripotent state, and mutations that prevent this destabilization also prevent differentiation. These data indicate that the core pluripotency transcription factors are integrated by posttranslational mechanisms to maintain the pluripotent state and identify mutations that increase KLF4 protein stability while maintaining transcription factor function.

During gestation, uterine smooth muscle cells transition from a state of quiescence to one of contractility, but the molecular mechanisms underlying this transition at a genomic level are not well-known. To better understand these events, we evaluated the epigenetic landscape of the mouse myometrium during the pregnant, laboring, and postpartum stages. We generated gestational time point-specific enrichment profiles for histone H3 acetylation on lysine residue 27 (H3K27ac), histone H3 trimethylation of lysine residue 4 (H3K4me3), and RNA polymerase II (RNAPII) occupancy by chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq), as well as gene expression profiles by total RNA-sequencing (RNA-seq). Our findings reveal that 533 genes, including known contractility-driving genes (Gap junction alpha 1 [Gja1], FBJ osteosarcoma oncogene [Fos], Fos-like antigen 2 [Fosl2], Oxytocin receptor [Oxtr], and Prostaglandin G/H synthase 2 (Ptgs2), for example), are upregulated at day 19 during active labor because of an increase in transcription at gene bodies. Labor-associated promoters and putative intergenic enhancers, however, are epigenetically activated as early as day 15, by which point the majority of genome-wide H3K27ac or H3K4me3 peaks present in term laboring tissue is already established. Despite this early exhibited histone signature, increased noncoding enhancer RNA (eRNA) production at putative intergenic enhancers and recruitment of RNAPII to the gene bodies of labor-associated loci were detected only during labor. Our findings indicate that epigenetic activation of the myometrial genome precedes active labor by at least 4 days in the mouse model, suggesting that the myometrium is poised for rapid activation of contraction-associated genes in order to exit the state of quiescence.

25During gestation, uterine smooth muscle cells transition from a state of quiescence to one of 26 contractility, but the molecular mechanisms underlying this transition at a genomic level are not well-27 known. To better understand these events, we evaluated the epigenetic landscape of the mouse 28 myometrium during pregnancy, labor and post-partum. We established gestational timepoint-specific 29 enrichment profiles involving histone H3K27 acetylation (H3K27ac), H3K4 tri-methylation (H3K4me3), 30 and RNA polymerase II (RNAPII) occupancy by chromatin immunoprecipitation sequencing (ChIP-seq), as 31 well as gene expression profiles by total RNA-sequencing (RNA-seq). Our findings reveal that 533 genes, 32 including known contractility-driving genes (Gja1, Fos, Oxtr, Ptgs2), are upregulated during active labor 33 due to an increase in transcription at gene bodies. Their promoters and putative intergenic enhancers, 34 however, are epigenetically activated by H3K27ac as early as day 15, four days prior to the onset of 35 active labor on day 19. In fact, we find that the majority of genome-wide H3K27ac or H3K4me3 peaks 36 identified during active labor are present in the myometrium on day 15. Despite the early presence of 37 H3K27ac at labor-associated genes, both an increase in non-coding enhancer RNA (eRNA) production, 38 and in recruitment of RNAPII to corresponding genes occur during active labor, at labor upregulated 39 gene loci. Our findings indicate that epigenetic activation of the myometrial genome precedes active 40 labor by at least four days in the mouse model, suggesting the myometrium is poised for rapid activation 41 of contraction-associated genes in order to exit the state of quiescence. 42 43 65 [16]. Selective reduction of GJA1 production in the uterine smooth muscle cells of two different mouse 66 models has been shown to significantly prolong the quiescent state during pregnancy and thereby delay 67 the onset of labor [17,18]. Reporter expression downstream of a synthetic Gja1 promoter is increased 68 by co-expression of constructs encoding members of the activator protein 1 (AP-1) transcription factor 69 FOS and JUN sub-families [19-21]. Furthermore, increased levels of FOS and FOSL2 in particular within 70 the nuclei of myometrial cells during labor raises the possibility that the FOS:JUN family acts to

Enhancers are cis-regulatory sequences located distally to target genes. These sequences consolidate developmental and environmental cues to coordinate gene expression in a tissue-specific manner. Enhancer function and tissue specificity depend on the expressed set of transcription factors, which recognize binding sites and recruit cofactors that regulate local chromatin organization and gene transcription. Unlike other genomic elements, enhancers are challenging to identify because they function independently of orientation, are often distant from their promoters, have poorly defined boundaries and display no reading frame. In addition, there are no defined genetic or epigenetic features that are unambiguously associated with enhancer activity. Over recent years there have been developments in both empirical assays and computational methods for enhancer prediction. We review genome-wide tools, CRISPR advancements and high-throughput screening approaches that have improved our ability to both observe and manipulate enhancers in vitro at the level of primary genetic sequences, chromatin states and spatial interactions. We also highlight contemporary animal models and their importance to enhancer validation. Together, these experimental systems and techniques complement one another and broaden our understanding of enhancer function in development, evolution, and disease.

Quebec Platelet Disorder (QPD) is an autosomal dominant bleeding disorder with a unique, platelet-dependent gain-of-function defect in fibrinolysis, without systemic fibrinolysis. The hallmark feature of QPD is a &gt;100-fold overexpression of PLAU specifically in megakaryocytes. This overexpression leads to &gt;100-fold increased platelet stores of urokinase plasminogen activator (PLAU/uPA), subsequent plasmin-mediated degradation of diverse a-granule proteins, and platelet-dependent, accelerated fibrinolysis. The causative mutation is a 78kb tandem duplication of PLAU. How this duplication causes megakaryocyte-specific PLAU overexpression is unknown. To investigate the mechanism that causes QPD, we used epigenomic profiling, comparative genomics, and chromatin conformation capture approaches to study PLAU regulation in cultured megakaryocytes from QPD participants and unaffected controls. We show that the QPD duplication leads to ectopic interactions between PLAU and a conserved megakaryocyte enhancer found within the same topologically associating domain (TAD). Our results support a unique disease mechanism whereby the reorganization of subTAD genome architecture results in a dramatic, cell-type specific blood disorder phenotype.