SOX4 exerts contrasting regulatory effects on labor-associated gene promoters in myometrial cells.

The uterine muscular layer, or myometrium, undergoes profound changes in global gene expression during its progression from a quiescent state during pregnancy to a contractile state at the onset of labor. In this study, we investigate the role of SOX family transcription factors in myometrial cells and provide evidence for the role of SOX4 in regulating labor-associated genes. We show that Sox4 has elevated expression in the murine myometrium during a term laboring process and in two mouse models of preterm labor. Additionally, SOX4 differentially affects labor-associated gene promoter activity in cooperation with activator protein 1 (AP-1) dimers. SOX4 exerted no effect on the Gja1 promoter; a JUND-specific activation effect at the Fos promoter; a positive activation effect on the Mmp11 promoter with the AP-1 dimers; and surprisingly, we noted that the reporter expression of the Ptgs2 promoter in the presence of JUND and FOSL2 was repressed by the addition of SOX4. Our data indicate SOX4 may play a diverse role in regulating gene expression in the laboring myometrium in cooperation with AP-1 factors. This study enhances our current understanding of the regulatory network that governs the transcriptional changes associated with the onset of labor and highlights a new molecular player that may contribute to the labor transcriptional program.

Identification of CD38, CD97, and CD278 on the HIV surface using a novel flow virometry screening assay.

While numerous cellular proteins in the HIV envelope are known to alter virus infection, methodology to rapidly phenotype the virion surface in a high throughput, single virion manner is lacking. Thus, many human proteins may exist on the virion surface that remain undescribed. Herein, we developed a novel flow virometry screening assay to discover new proteins on the surface of HIV particles. By screening a CD4+ T cell line and its progeny virions, along with four HIV isolates produced in primary cells, we discovered 59 new candidate proteins in the HIV envelope that were consistently detected across diverse HIV isolates. Among these discoveries, CD38, CD97, and CD278 were consistently present at high levels on virions when using orthogonal techniques to corroborate flow virometry results. This study yields new discoveries about virus biology and demonstrates the utility and feasibility of a novel flow virometry assay to phenotype individual virions.

MYB and ELF3 differentially modulate labor-inducing gene expression in myometrial cells.

Spontaneous uterine contractions are initiated when smooth muscle cells (SMCs) within the uterine muscle, or myometrium, transition from a functionally dormant to an actively contractile phenotype at the end of the pregnancy period. We know that this process is accompanied by gestational time point-specific differences in the SMC transcriptome, which can be modulated by the activator protein 1 (AP-1), nuclear factor kappa beta (NF-κß), estrogen receptor (ER), and progesterone receptor (PR) transcription factors. Less is known, however, about the additional proteins that might assist these factors in conferring the transcriptional changes observed at labor onset. Here, we present functional evidence for the roles of two proteins previously understudied in the SMC context-MYB and ELF3-which can contribute to the regulation of labor-driving gene transcription. We show that the MYB and ELF3 genes exhibit elevated transcript expression levels in mouse and human myometrial tissues during spontaneous term labor. The expression of both genes was also significantly increased in mouse myometrium during preterm labor induced by the progesterone antagonist mifepristone (RU486), but not during infection-simulating preterm labor induced by intrauterine infusion of lipopolysaccharide (LPS). Furthermore, both MYB and ELF3 proteins affect labor-driving gene promoter activity, although in surprisingly opposing ways: Gja1 and Fos promoter activation increases in the presence of MYB and decreases in the presence of ELF3. Collectively, our study adds to the current understanding of the transcription factor network that defines the transcriptomes of SMCs during late gestation and implicates two new players in the control of labor timing.

Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

Transcriptional control of parturition: insights from gene regulation studies in the myometrium.

The onset of labour is a culmination of a series of highly coordinated and preparatory physiological events that take place throughout the gestational period. In order to produce the associated contractions needed for foetal delivery, smooth muscle cells in the muscular layer of the uterus (i.e. myometrium) undergo a transition from quiescent to contractile phenotypes. Here, we present the current understanding of the roles transcription factors play in critical labour-associated gene expression changes as part of the molecular mechanistic basis for this transition. Consideration is given to both transcription factors that have been well-studied in a myometrial context, i.e. activator protein 1, progesterone receptors, oestrogen receptors, and nuclear factor kappa B, as well as additional transcription factors whose gestational event-driving contributions have been demonstrated more recently. These transcription factors may form pregnancy- and labour-associated transcriptional regulatory networks in the myometrium to modulate the timing of labour onset. A more thorough understanding of the transcription factor-mediated, labour-promoting regulatory pathways holds promise for the development of new therapeutic treatments that can be used for the prevention of preterm labour in at-risk women.

Transcriptional enhancers: from prediction to functional assessment on a genome-wide scale.

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.

The pregnant myometrium is epigenetically activated at contractility-driving gene loci prior to the onset of labor in mice.

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 up-regulated 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.