Negative elongation factor is essential for endometrial function

Abstract

Pausing of RNA polymerase II (Pol II) during early transcription, mediated by the negative elongation factor (NELF) complex, allows cells to coordinate and appropriately respond to signals by modulating the rate of transcriptional pause release. Promoter proximal enrichment of Pol II occurs at uterine genes relevant to reproductive biology; thus, we hypothesized that pausing might impact endometrial response by coordinating hormonal signals involved in establishing and maintaining pregnancy. We deleted the NELF‐B subunit in the mouse uterus using PgrCre (NELF‐B UtcKO). Resulting females were infertile. Uterine response to the initial decidual stimulus of NELF‐B UtcKO was similar to that of control mice; however, subsequent full decidual response was not observed. Cultured NELF‐B UtcKO stromal cells exhibited perturbances in extracellular matrix components and also expressed elevated levels of the decidual prolactin Prl8a2, as well as altered levels of transcripts encoding enzymes involved in prostaglandin synthesis and metabolism. Because endometrial stromal cell decidualization is also critical to human reproductive health and fertility, we used small interfering to suppress NELF‐B or NELF‐E subunits in cultured human endometrial stromal cells, which inhibited decidualization, as reflected by the impaired induction of decidual markers PRL and IGFBP1. Overall, our study indicates NELF‐mediated pausing is essential to coordinate endometrial responses and that disruption impairs uterine decidual development during pregnancy.—Hewitt, S. C., Li, R., Adams, N., Winuthayanon, W., Hamilton, K. J., Donoghue, L. J., Lierz, S. L., Garcia, M., Lydon, J. P., DeMayo, F. J., Adelman, K., Korach, K. S. Negative elongation factor is essential for endometrial function. FASEB J. 33, 3010–3023 (2019). http://www.fasebj.org

RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription

“…Surprisingly, degrading NELF transiently in the context of pluripotency transitions from naïve to the formative state caused a hyper-induction of genes associated with the formative state accompanied by hyper-silencing of downregulated genes. This is in agreement with recent studies suggesting that absence of NELF perturbs fate transition events more so than steady-state cellular function in a variety of contexts (Yu et al 2020;Robinson et al 2021;Hewitt et al 2019). Accordingly, we observed increased recruitment of NELF to chromatin during pluripotency state transitions.…”

“…We recently showed that NELF is required at this specific stage prior to ZGA to regulate the major ZGA wave in mouse embryos (Abuhashem and Hadjantonakis 2021). At the cellular level, several studies investigating tissue-specific Nelfb knockouts have revealed that functional defects are observed when Nelfb -/- tissues are challenged by an external stimulus, such as an injury or an infection or the need to regenerate in the context of muscle stem cells, the uterine and intestinal walls, and in macrophages (Hewitt et al 2019; Ou et al 2021; Robinson et al 2021; Yu et al 2020). Our model, suggesting that Pol II pausing acts to fine-tune transcription during state transitions, explains the defects observed in both the present and previous studies.…”

Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

“…Among these, two proteins and their corresponding phosphosites, signal transducer and activator of transcription 1 (STAT1) S727 and tumor protein p53 binding protein 1 (TP53BP1) S265, were previously identified as Mediator kinase targets in other cellular contexts (39,41), substantiating our phosphoproteomics approach in MM SP cells and also providing positive controls for site-specific Mediator kinase activity in our validation assays. An additional three proteins carrying four phosphosites (CUX1, S1237; FOXK1, S416 and S420 [mouse equivalents S402 and S406]; and negative elongation factor complex member B [NELFB], S557), none of which have previously been linked with Mediator kinase, were chosen for further analysis based on their established associations with UFs (CUX1 [25,46]), muscle stem cell biology (FOXK1 [29,32]), or RNA Pol II transcriptional elongation (NELFB [47,48]) with which Mediator kinase itself has been implicated (16,19). For each of the five candidate substrates tested, purified recombinant WT or phosphomutant (Ser to Ala) derivatives were assessed for their abilities to serve as direct Mediator kinase substrates in vitro.…”

Estrogen receptor α (ERα)-binding super-enhancers drive key mediators that control uterine estrogen responses in mice

“…gous (ER␣ ϩ/Ϫ) males and females) treated for 2 h with V or E2. RNA was assessed by microarray as described previously (66) and analyzed in Partek using analysis of variance to find DEG and then filtering for signal intensity Ͼ100 in at least one sample and then filtering for super-enhancer-associated genes. Data from ovariectomized adults (68) (GSE100131) and data from 21-day-old mice (GSE148006) have been deposited in GEO.…”