Despite much research, our understanding of the architecture and
-regulatory elements of human promoters is still lacking. Here, we devised a high-throughput assay to quantify the activity of ...approximately 15,000 fully designed sequences that we integrated and expressed from a fixed location within the human genome. We used this method to investigate thousands of native promoters and preinitiation complex (PIC) binding regions followed by in-depth characterization of the sequence motifs underlying promoter activity, including core promoter elements and TF binding sites. We find that core promoters drive transcription mostly unidirectionally and that sequences originating from promoters exhibit stronger activity than those originating from enhancers. By testing multiple synthetic configurations of core promoter elements, we dissect the motifs that positively and negatively regulate transcription as well as the effect of their combinations and distances, including a 10-bp periodicity in the optimal distance between the TATA and the initiator. By comprehensively screening 133 TF binding sites, we find that in contrast to core promoters, TF binding sites maintain similar activity levels in both orientations, supporting a model by which divergent transcription is driven by two distinct unidirectional core promoters sharing bidirectional TF binding sites. Finally, we find a striking agreement between the effect of binding site multiplicity of individual TFs in our assay and their tendency to appear in homotypic clusters throughout the genome. Overall, our study systematically assays the elements that drive expression in core and proximal promoter regions and sheds light on organization principles of regulatory regions in the human genome.
Despite extensive research, our understanding of the rules according to which cis-regulatory sequences are converted into gene expression is limited. We devised a method for obtaining parallel, ...highly accurate gene expression measurements from thousands of designed promoters and applied it to measure the effect of systematic changes in the location, number, orientation, affinity and organization of transcription-factor binding sites and nucleosome-disfavoring sequences. Our analyses reveal a clear relationship between expression and binding-site multiplicity, as well as dependencies of expression on the distance between transcription-factor binding sites and gene starts which are transcription-factor specific, including a striking ∼10-bp periodic relationship between gene expression and binding-site location. We show how this approach can measure transcription-factor sequence specificities and the sensitivity of transcription-factor sites to the surrounding sequence context, and compare the activity of 75 yeast transcription factors. Our method can be used to study both cis and trans effects of genotype on transcriptional, post-transcriptional and translational control.
Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, ...our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs, we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences serving as promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences and allows the characterization of TF binding determinants within and outside of core binding sites.
Purpose
To assess the effects of letrozole or tamoxifen coadministration on fertility preservation treatment outcomes.
Methods
Retrospective cohort study of 118 breast cancer patients undergoing ...fertility preservation treatment between 2008 and 2018. Patients who received letrozole (
n
= 36) or tamoxifen (
n
= 30) were compared to controls (
n
= 52) who underwent standard ovarian stimulation protocols. The primary outcome measures included the number of retrieved oocytes, mature oocytes (MII), fertilization, and top-quality embryo rates. The secondary outcome measures included duration of stimulation, gonadotropin dose and peak estradiol level.
Results
The number of oocytes retrieved, MII oocytes, fertilization rate, duration of stimulation, or gonadotropin dose were similar in the letrozole and tamoxifen groups, compared to controls. Top-quality embryo rate was lower in the tamoxifen group compared to controls (25% vs 39.4%, respectively,
P
= 0.034). The abnormal fertilization rate was higher in the letrozole group compared to controls (7.8% vs 3.60%, respectively,
P
= 0.015). A stepwise logistic regression analysis revealed that letrozole and peak estradiol were significantly associated with abnormal fertilization (OR 11.94; 95% CI 2.35–60.4,
P
= 0.003 for letrozole and OR 1.075; 95% CI 1.024–1.12,
P
= 0.004 per 100 unit change in estradiol).
Conclusions
There may be a negative effect of letrozole or tamoxifen on fertilization and embryo quality, in fertility preservation cycles. Further studies are needed to confirm these findings.
Purpose:
To compare the morphokinetic parameters of pre-implantation development between embryos of women of advanced maternal age (AMA) and young women.
Methods:
Time-lapse microscopy was used to ...compare morphokinetic variables between 495 embryos of AMA women ≥ age 42 years and 653 embryos of young patients (<age 38 years) who underwent IVF in our unit. Developmental events annotated and analyzed include observed cell divisions in correlation to the timing of fertilization, synchrony of the second (s2) and third cell cycles (s3) and the duration to the second (cc2) and third cleavages (cc3).
Results:
No significant differences were observed in cleavage times between the embryos of AMA and the control embryos. Interestingly, the older embryos appear to be more prone to developmental arrest (a higher percentage of embryos of older women arrested at 4–7 cells resulting in less embryos reaching the 8-cell stage (66% vs. 72%, respectively), though this difference did not reach a significance at least during the first 3 days of development (
p
> 0.05).
Conclusions:
While early morphokinetic parameters do not reflect dynamics unique to embryos of older women, a tendency toward developmental arrest was observed, which would likely be even more pronounced at later stages of development.
Abstract
Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to ...capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.
To explore the possibility that endometrial injury modulates the expression of specific genes that may increase uterine receptivity.
Controlled clinical study.
Clinical IVF unit and academic research ...center.
IVF patients with 28- to 30-day menstrual cycles.
Endometrial biopsies from two groups of patients were collected on days 20–21 of their spontaneous menstrual cycle. The experimental, but not the control, group underwent biopsies on days 11–13 and 21–24 of their preceding cycle.
Global endometrial gene expression and specific analysis of uroplakin Ib (UPIb) mRNA level throughout the menstrual cycle.
Local injury modulated the expression of a wide variety of genes. One of the prominently up-regulated genes was the bladder transmembranal protein, UPIb, whose expression by the endometrium is shown here for the first time. Endometrial UPIb mRNA increases after biopsy in the same cycle wct 2with an additional elevation in the following cycle. Immunohistochemical analysis localized the UPIb protein to the glandular-epithelial cells. Genes encoding other membrane proteins such as adipose differentiation-related protein and mucin 1, transmembrane, were also up-regulated.
The biopsy-induced increase in the expression of UPIb and other genes encoding membrane proteins supports the possible importance of the membrane structure and stability during implantation. The specific role of UPIb in uterine receptivity should be elucidated.
Quantitative and qualitative spermatogenic impairments are major causes of men’s infertility. Although in vitro fertilization (IVF) is effective, some couples persistently fail to conceive. To ...identify causal variants in patients with severe male infertility factor and repeated IVF failures, we sequenced the exome of two consanguineous family members who underwent several failed IVF cycles and were diagnosed with low sperm count and motility. We identified a rare homozygous nonsense mutation in a previously uncharacterized gene, RNF212B, as the causative variant. Recurrence was identified in another unrelated, infertile patient who also faced repeated failed IVF treatments. scRNA-seq demonstrated meiosis-specific expression of RNF212B. Sequence analysis located a protein domain known to be associated with aneuploidy, which can explain multiple IVF failures. Accordingly, FISH analysis revealed a high aneuploidy rate in the patients' sperm cells and their IVF embryos. Finally, inactivation of the Drosophila orthologs significantly reduced male fertility. Given that members of the evolutionary conserved RNF212 gene family are involved in meiotic recombination and crossover maturation, our findings indicate a critical role of RNF212B in meiosis, genome stability, and in human fertility. Since recombination is completely absent in Drosophila males, our findings may indicate an additional unrelated role for the RNF212-like paralogs in spermatogenesis.
We report the discovery of a pathogenic variant in the uncharacterized human gene RNF212B, which belongs to an evolutionary conserved gene family involved in meiotic recombination. RNF212B loss of function is associated with sperm abnormalities and broad aneuploidy in patient-derived sperm cells and embryos, resulting in multiple repeated IVF failures.
The coordinated function of the different compartments of the follicle, the oocyte and the somatic cumulus/granulosa cells, is enabled by the presence of a network of cell-to-cell communication ...generated by gap junctions. Connexin 43 (Cx43) is the most abundant gap junction protein expressed by the ovarian follicle. The expression of Cx43 is subjected to the control of gonadotropins as follows: FSH up-regulates, whereas LH down-regulates its levels. The aim of this study was to explore the mechanism by which LH reduces the levels of Cx43 and to identify the signal transduction pathway involved in this process. The effect of LH was studied in vitro using isolated intact ovarian follicles. The possible mediators of LH-induced Cx43 down-regulation were examined by incubating the follicles with LH in the presence or absence of inhibitors of protein kinase A (PKA) and of MAPK signaling pathways. Our experiments revealed a 3-h half-life of Cx43 in both control and LH-treated follicles, suggesting that LH did not affect the rate of Cx43 degradation. We further demonstrated that the level of Cx43 mRNA was not significantly influenced by this gonadotropin. However, upon LH administration, 35Smethionine incorporation into Cx43 protein was remarkably reduced. The LH-induced arrest of Cx43 synthesis was counteracted by inhibitors of both the PKA and the MAPK cascades. We show herein that LH inhibits Cx43 expression by reducing its rate of translation and that this effect is mediated by both PKA and MAPK.
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