Systematic Investigation of Transcription Factor Activity in the Context of Chromatin Using Massively Parallel Binding and Expression Assays.

Precise gene expression patterns are established by transcription factor (TFs) binding to regulatory sequences. While these events occur in the context of chromatin, our understanding of how TF-nucleosome interplay affects gene expression is highly limited. Here, we present an assay for high-resolution measurements of both DNA occupancy and gene expression on large-scale libraries of systematically designed regulatory sequences. Our assay reveals occupancy patterns at the single-cell level. It provides an accurate quantification of the fraction of the population bound by a nucleosome and captures distinct, even adjacent, TF binding events. By applying this assay to over 1,500 promoter variants in yeast, we reveal pronounced differences in the dependency of TF activity on chromatin and classify TFs by their differential capacity to alter chromatin and promote expression. We further demonstrate how different regulatory sequences give rise to nucleosome-mediated TF collaborations that quantitatively account for the resulting expression.

BlastAssist: a deep learning pipeline to measure interpretable features of human embryos.

STUDY QUESTION: Can the BlastAssist deep learning pipeline perform comparably to or outperform human experts and embryologists at measuring interpretable, clinically relevant features of human embryos in IVF? SUMMARY ANSWER: The BlastAssist pipeline can measure a comprehensive set of interpretable features of human embryos and either outperform or perform comparably to embryologists and human experts in measuring these features. WHAT IS KNOWN ALREADY: Some studies have applied deep learning and developed 'black-box' algorithms to predict embryo viability directly from microscope images and videos but these lack interpretability and generalizability. Other studies have developed deep learning networks to measure individual features of embryos but fail to conduct careful comparisons to embryologists' performance, which are fundamental to demonstrate the network's effectiveness. STUDY DESIGN, SIZE, DURATION: We applied the BlastAssist pipeline to 67 043 973 images (32 939 embryos) recorded in the IVF lab from 2012 to 2017 in Tel Aviv Sourasky Medical Center. We first compared the pipeline measurements of individual images/embryos to manual measurements by human experts for sets of features, including: (i) fertilization status (n = 207 embryos), (ii) cell symmetry (n = 109 embryos), (iii) degree of fragmentation (n = 6664 images), and (iv) developmental timing (n = 21 036 images). We then conducted detailed comparisons between pipeline outputs and annotations made by embryologists during routine treatments for features, including: (i) fertilization status (n = 18 922 embryos), (ii) pronuclei (PN) fade time (n = 13 781 embryos), (iii) degree of fragmentation on Day 2 (n = 11 582 embryos), and (iv) time of blastulation (n = 3266 embryos). In addition, we compared the pipeline outputs to the implantation results of 723 single embryo transfer (SET) cycles, and to the live birth results of 3421 embryos transferred in 1801 cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: In addition to EmbryoScope™ image data, manual embryo grading and annotations, and electronic health record (EHR) data on treatment outcomes were also included. We integrated the deep learning networks we developed for individual features to construct the BlastAssist pipeline. Pearson's χ2 test was used to evaluate the statistical independence of individual features and implantation success. Bayesian statistics was used to evaluate the association of the probability of an embryo resulting in live birth to BlastAssist inputs. MAIN RESULTS AND THE ROLE OF CHANCE: The BlastAssist pipeline integrates five deep learning networks and measures comprehensive, interpretable, and quantitative features in clinical IVF. The pipeline performs similarly or better than manual measurements. For fertilization status, the network performs with very good parameters of specificity and sensitivity (area under the receiver operating characteristics (AUROC) 0.84-0.94). For symmetry score, the pipeline performs comparably to the human expert at both 2-cell (r = 0.71 ± 0.06) and 4-cell stages (r = 0.77 ± 0.07). For degree of fragmentation, the pipeline (acc = 69.4%) slightly under-performs compared to human experts (acc = 73.8%). For developmental timing, the pipeline (acc = 90.0%) performs similarly to human experts (acc = 91.4%). There is also strong agreement between pipeline outputs and annotations made by embryologists during routine treatments. For fertilization status, the pipeline and embryologists strongly agree (acc = 79.6%), and there is strong correlation between the two measurements (r = 0.683). For degree of fragmentation, the pipeline and embryologists mostly agree (acc = 55.4%), and there is also strong correlation between the two measurements (r = 0.648). For both PN fade time (r = 0.787) and time of blastulation (r = 0.887), there's strong correlation between the pipeline and embryologists. For SET cycles, 2-cell time (P < 0.01) and 2-cell symmetry (P < 0.03) are significantly correlated with implantation success rate, while other features showed correlations with implantation success without statistical significance. In addition, 2-cell time (P < 5 × 10-11), PN fade time (P < 5 × 10-10), degree of fragmentation on Day 3 (P < 5 × 10-4), and 2-cell symmetry (P < 5 × 10-3) showed statistically significant correlation with the probability of the transferred embryo resulting in live birth. LIMITATIONS, REASONS FOR CAUTION: We have not tested the BlastAssist pipeline on data from other clinics or other time-lapse microscopy (TLM) systems. The association study we conducted with live birth results do not take into account confounding variables, which will be necessary to construct an embryo selection algorithm. Randomized controlled trials (RCT) will be necessary to determine whether the pipeline can improve success rates in clinical IVF. WIDER IMPLICATIONS OF THE FINDINGS: BlastAssist provides a comprehensive and holistic means of evaluating human embryos. Instead of using a black-box algorithm, BlastAssist outputs meaningful measurements of embryos that can be interpreted and corroborated by embryologists, which is crucial in clinical decision making. Furthermore, the unprecedentedly large dataset generated by BlastAssist measurements can be used as a powerful resource for further research in human embryology and IVF. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Harvard Quantitative Biology Initiative, the NSF-Simons Center for Mathematical and Statistical Analysis of Biology at Harvard (award number 1764269), the National Institute of Heath (award number R01HD104969), the Perelson Fund, and the Sagol fund for embryos and stem cells as part of the Sagol Network. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.

A clinical predictive model for live birth in women of advanced age undergoing PGT cycles.

PURPOSE: The trend of delaying childbirth has resulted in a growing number of advanced-aged women who are opting for preimplantation genetic testing (PGT) to screen for monogenic diseases or structural chromosomal rearrangements (PGT-M and PGT-SR). This increase in demand necessitates the development of a clinical predictive model for live birth outcomes in these women. Therefore, the objective of this study is to construct a comprehensive predictive model that assesses the likelihood of achieving a successful live birth in advanced-aged women undergoing PGT-M and PGT-SR treatments. METHODS: A retrospective cohort study of 37-45-year-old women undergoing preimplantation genetic testing for monogenic disease or structural chromosomal rearrangement cycles from 2010 to 2021 was conducted at a university hospital reproductive centre. The purpose was to develop a clinical predictive model for live birth in these women. The main outcome studied was the cumulative live birth rate in the first or subsequent cycles. Developing a decision tree enabled a comprehensive study of clinical parameters and expected outcomes. RESULTS: The analysis included 158 women undergoing 753 preimplantation genetic testing cycles. The cumulative live birth rate was 37.342% (59/158). Decision tree analysis revealed that women aged ≤ 40.1 or women > 40.1 with one or more top-quality transferable embryos in their first cycle had the best chance for a live baby (56% and 41%, respectively). Those older than 40.1 without top-quality embryos and seven or fewer dominant follicles had no live births. A Kaplan-Meier curve showed that for autosomal dominant diseases, there was a negligible increase in live birth rate after three cycles, compared to six cycles in autosomal recessive inheritance. CONCLUSION: In older women, the chance of delivering after repeated cycles is higher in those with at least one top-quality unaffected embryo in their first preimplantation genetic testing cycle. Additional preimplantation genetic testing cycles after three in carriers of an autosomal dominant disorder and six in those with an autosomal recessive disorder should be considered prudently.

The association between a carrier state of FMR1 premutation and numeric sex chromosome variations.

PURPOSE: Women carriers of FMR1 premutation are at increased risk of early ovarian dysfunction and even premature ovarian insufficiency. The aim of this study was to examine a possible association between FMR1 permutation and numeric sex chromosome variations. METHODS: A retrospective case-control study conducted in the reproductive center of a university-affiliated medical center. The primary outcome measure was the rate of sex chromosomal numerical aberrations, as demonstrated by haplotype analyses, in FMR1 premutation carriers compared to X-linked preimplantation genetic testing for monogenic/single gene defect (PGT-M) cycles for other indications that do not affect the ovarian follicles and oocytes. RESULTS: A total of 2790 embryos with a final genetic analysis from 577 IVF PGT-M cycles were included in the final analysis. Mean age was similar between the groups, however, FMR1 carriers required more gonadotropins, and more women were poor responders with three or less oocytes collected. The ratio of embryos carrying a numeric sex chromosome variation was similar: 8.3% (138/1668) of embryos in the FMR1 group compared to 7.1% (80/1122) in the controls. A subgroup analysis based on age and response to stimulation has not demonstrated a significant difference either. CONCLUSIONS: Although carriers of FMR1 premutation exhibit signs of reduced ovarian response, it does not seem to affect the rate of numeric sex chromosomal variation compared to women undergoing PGT-M for other indications. This suggests that the mechanism for chromosomal number aberrations in women at advanced maternal age are different to those FMR1 premutation carriers with poor ovarian reserve.

Systematic interrogation of human promoters.

Despite much research, our understanding of the architecture and cis-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.

Similar fertilization rates and preimplantation embryo development among testosterone-treated transgender men and cisgender women.

RESEARCH QUESTION: What are the effects of testosterone treatment on oocyte fertilization and preimplantation embryo development among transgender men who have undergone fertility preservation? DESIGN: A retrospective study was undertaken in a university-affiliated tertiary hospital between April 2016 and November 2021. Embryos were divided into three groups by source: 210 embryos from 7 testosterone-exposed transgender men, 135 from 10 cisgender women who cryopreserved embryos, and 276 from 24 cisgender women who underwent fertility treatment. Statistical analyses compared assisted reproductive technology outcomes between the group of transgender men and both groups of cisgender women. Morphokinetic and morphological parameters were compared between the embryos derived from these three groups. RESULTS: The transgender men (30.2 ± 3.5 years of age) were significantly younger than the cisgender women who cryopreserved embryos (35.1 ± 1.8 years; P = 0.005) and the cisgender women who underwent fertility treatment (33.8 ± 3.2 years; P = 0.017). After adjusting for participant age, the fertilization rate was comparable between the transgender men and both groups of cisgender women (P = 0.391 and 0.659). There were no significant differences between the transgender men and the cisgender women who preserved fertility in terms of number of cryopreserved embryos (7.2 ± 5.1 and 3.5 ± 2.6; P = 0.473) or the distribution of embryo age at cryopreservation (P = 0.576). All morphokinetic parameters evaluated by time-lapse imaging, as well as the morphological characteristics, were comparable for the embryos in all three groups. CONCLUSIONS: Testosterone exposure among transgender men has no adverse impact upon fertilization rates or preimplantation embryo development and quality.

A decision tree analysis applied to women aged 43-45: who should be referred for ovum donation?

RESEARCH QUESTION: In women at the advanced age of 43-45 years undergoing repeated IVF cycles with autologous oocytes, who has the highest chance for birth and who should be referred early to receive donor oocytes? DESIGN: A retrospective cohort study was conducted at a university hospital reproductive centre. The computerized database of 394 women aged 43-45 years undergoing 1528 non-donor IVF or intracytoplasmic sperm injection cycles between 2010 and 2019 was analysed. A decision tree was developed, enabling a comprehensive study of a set of clinical parameters and the expected outcomes. RESULTS: The cumulative clinical pregnancy rate was 15.0% (59/394) and the cumulative live birth rate was 8.4% (33/394). The decision tree developed to predict women who should be offered egg donation included age, poor ovarian response to stimulation, the number of top-quality embryos, dominant follicles, previous pregnancy or live birth, fertilized oocytes and body mass index. The model showed that a good ovarian response in the first cycle was the best predictor for live birth (13.3% gave birth). However, among women with poor responses, 7.1% of those who were younger than 43.5 years gave birth, and none of the women who were older than 43.5 years did. CONCLUSIONS: Women over 43.5 years old with fewer than four oocytes collected in their first IVF cycle should be offered ovum donation, since their live birth rate in subsequent cycles is negligible.

The impact of fragile X premutation carrier status on embryo morphokinetic development.

RESEARCH QUESTION: Does inheritance of the fragile X mental retardation 1 (FMR1) premutation allele affect embryo morphokinetic development? DESIGN: A retrospective cohort analysis of 529 embryos from 126 IVF cycles of 39 FMR1 premutation female carriers undergoing preimplantation genetic testing for monogenic/single gene defects (PGT-M). Morphological and morphokinetic parameters obtained using a time-lapse monitoring system were compared between embryos that inherited the FMR1 premutation allele (FMR1 group, n = 271) and those who received the normal allele (normal group, n = 258). The following embryo outcome measures were compared: morphokinetic parameters up to day 3, start of blastulation time (tSB) for day 5 embryos and the rate of top-quality embryos on days 3 and 5. RESULTS: No differences were found in morphokinetic parameters between the groups from the time of intracytoplasmic sperm injection (ICSI) until a biopsy on day 3. The blastulation rate in the two groups was comparable. However, the start of blastulation was delayed in FMR1 embryos compared to that in the genetically normal embryos (median tSB: 104.2 h [99.3-110.3] versus 101.6 h [94.5-106.7], P = 0.01). In addition, the rate of top-quality FMR1 embryos was lower than that of genetically normal embryos (25.6% versus 38.8%, P = 0.04). CONCLUSION: Embryos that inherit the FMR1 premutation allele are of lower quality at the blastocyst stage compared with those that do not inherit the mutated allele.

Maternal underweight does not adversely affect the outcomes of IVF/ICSI and frozen embryo transfer cycles or early embryo development.

OBJECTIVE: To compare assisted reproductive technology (ART) outcomes and preimplantation embryo development between underweight and normal-weight women. METHODS: This retrospective cohort study included 26 underweight women (body mass index [BMI] < 18.50 kg/m2) and 104 normal-weight women (BMI >20 and <24.9 kg/m2) who underwent a total of 204 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and 358 fresh/frozen embryo transfers (ET) in our institution between January 2016 and December 2018. Statistical analyses compared selected ART outcomes (ovarian stimulation, fertilization, and pregnancy) between both weight groups. Morphokinetic and morphological parameters were also compared between 346 and 1467 embryos of underweight and normal-weight women, respectively. RESULTS: The mean ± standard deviation age of the underweight and normal-weight women was similar (31.6 ± 4.17 vs 32.4 ± 3.59 years; p = .323). There were no differences in the peak estradiol levels, the number of retrieved oocytes, the number of metaphase II oocytes, and the oocyte maturity rates between the two groups. The IVF/ICSI fertilization rates and the number of embryos suitable for transfer or cryopreservation were similar for both groups. All morphokinetic parameters that were evaluated by means of time-lapse imaging as well as the morphological characteristics were comparable between low and normal BMI categories. There were no significant differences in pregnancy achievement, clinical pregnancy, live births, and miscarriage rates between the suboptimal and optimal weight women. CONCLUSION: Underweight status has no adverse impacts on the outcomes of IVF/ICSI with either fresh or frozen ET or on preimplantation embryo development and quality.

A decision tree model for predicting live birth in FMR1 premutation carriers undergoing preimplantation genetic testing for monogenic/single gene defects.

RESEARCH QUESTION: Can patient selection for successful preimplantation genetic testing for women who are fragile X (FMR1) premutation carriers be optimized using a decision tree analysis? This decision support tool enables a comprehensive study of a set of clinical parameters and the expected outcomes. DESIGN: A retrospective case-control study analysing the results of 264 fresh and 21 frozen preimplantation genetic testing for monogenic disorders/single gene defects (PGT-M) cycles in 64 FMR1 premutation carriers. Primary outcome was live birth per cycle start. Live birth rate was calculated for the start of the ovarian stimulation cycle. Fresh and frozen embryo transfers from the same cycle were included. RESULTS: The decision tree model showed that the number of cytosine guanine (CGG) repeats was only a moderate predictor for live birth, whereas an age younger than 36 years was the best predictor for live birth, followed by a collection of 14 or more oocytes. These findings were supported by the results of the logistic regression, which found that only age and oocyte number were significantly associated with live birth (P = 0.005 and 0.017, respectively). CONCLUSIONS: The number of CGG repeats is a relatively poor predictor for live birth in PGT-M cycles. FMR1 premutation carriers are no different from non-carriers. Age is the best identifier of live birth, followed by the number of retrieved oocytes.

Effects of letrozole or tamoxifen coadministered with a standard stimulation protocol on fertility preservation among breast cancer patients.

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.

In-vitro maturation of oocytes recovered during cryopreservation of pre-pubertal girls undergoing fertility preservation.

RESEARCH QUESTION: In-vitro maturation (IVM) of oocytes recovered during ovarian tissue cryopreservation (OTC) is often practised, although it is still considered experimental. To date, only a few studies have examined the success of this maturation process in pre-menarche girls. The aim of this study was to examine the outcomes of IVM of oocytes recovered during OTC in pre-menarche patients scheduled for onco-therapy. DESIGN: A retrospective cohort study in a tertiary university-affiliated hospital. A total of 93 patients aged 0-25 years who underwent OTC as part of onco-fertility preservation between 2007 and 2019 were included in the study. Oocytes were recovered from the medium after OTC and matured over 48 h. Oocyte development and maturation rate were recorded and compared between different age groups. RESULTS: Patient's age was not correlated linearly with the total number of mature oocytes R = 0.2. The absolute maturation rate in post-menarche and pre-menarche patients differed significantly (38.0% versus 25.3%, respectively; P > 0.001), whereas the degeneration rate of the oocytes did not (39.8% versus 33.5%; P = 0.167). The pre-menarche group had significantly lower mean number of metaphase II oocytes compared with the post-menarche group (2.8 [±2.3] versus 5.6 [±4.6]; P = 0.01; 95% CI -4.62 to -0.46). Oocytes recovered from patients aged 1-5 years demonstrated low maturation rate. CONCLUSIONS: Oocytes recovered from pre-menarche girls, and especially those younger than the age of 5 years who undergo fertility preservation, have a lower chance of reaching maturity in IVM compared with older women. This may indicate a need for alternative methods for preserving fertility in these young patients.

Derivation of novel human ground state naive pluripotent stem cells.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3ß signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Time-lapse imaging reveals delayed development of embryos carrying unbalanced chromosomal translocations.

PURPOSE: The purpose of the study was to compare the morphokinetic parameters of embryos carrying balanced chromosomal translocations with those carrying unbalanced chromosomal translocations using time-lapse microscopy. METHODS: The study group included 270 embryos that underwent biopsies on day 3 for preimplantation genetic diagnosis (PGD) for chromosomal translocations in our unit between 2013 and 2015. All embryos were incubated under time-lapse microscopy and evaluated for timing of developmental events up to day 5. The timing of these events was compared between balanced and unbalanced embryos, potentially viable and nonviable variants, and maternal versus paternal inheritance of the translocation. RESULTS: The PGD analysis found that 209 (77%) of the 270 biopsied embryos carried an unbalanced translocation. Embryos carrying unbalanced translocations, which are expected to lead to implantation failure or miscarriage, cleaved less synchronously and were delayed in time of cleavage to the 4-cell stage (t4) and in time of start of blastulation (tSB) compared with balanced embryos (P < 0.05). Furthermore, embryos carrying nonviable translocations demonstrated a significant delay at the time of pronuclei fading (tPNf) compared with those carrying potentially viable translocations (P < 0.05). Embryos whose unbalanced translocations were of maternal origin were significantly delayed in most of the morphokinetic parameters (including tPNf, t2, t3, t4, t6, t7, t8, cc2, s2, and tSB) compared with embryos carrying balanced translocations (P < 0.05). CONCLUSIONS: Embryos carrying unbalanced chromosomal translocations mainly of maternal origin undergo delayed development and asynchronous cleavage that may lead to implantation failure or miscarriage.

Unraveling determinants of transcription factor binding outside the core binding site.

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.

Probing the effect of promoters on noise in gene expression using thousands of designed sequences.

Genetically identical cells exhibit large variability (noise) in gene expression, with important consequences for cellular function. Although the amount of noise decreases with and is thus partly determined by the mean expression level, the extent to which different promoter sequences can deviate away from this trend is not fully known. Here, we present a high-throughput method for measuring promoter-driven noise for thousands of designed synthetic promoters in parallel. We use it to investigate how promoters encode different noise levels and find that the noise levels of promoters with similar mean expression levels can vary more than one order of magnitude, with nucleosome-disfavoring sequences resulting in lower noise and more transcription factor binding sites resulting in higher noise. We propose a kinetic model of gene expression that takes into account the nonspecific DNA binding and one-dimensional sliding along the DNA, which occurs when transcription factors search for their target sites. We show that this assumption can improve the prediction of the mean-independent component of expression noise for our designed promoter sequences, suggesting that a transcription factor target search may affect gene expression noise. Consistent with our findings in designed promoters, we find that binding-site multiplicity in native promoters is associated with higher expression noise. Overall, our results demonstrate that small changes in promoter DNA sequence can tune noise levels in a manner that is predictable and partly decoupled from effects on the mean expression levels. These insights may assist in designing promoters with desired noise levels.

The effect of a germline mutation in the APC gene on ß-catenin in human embryonic stem cells.

BACKGROUND: Most cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-ß-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs. METHODS: Two FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) ß-catenin expression was analyzed by Western blot analysis; 2) Wnt-ß-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of ß-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed. RESULTS: We have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced ß-catenin/TCF-mediated activity. Additionally, ß-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in ß-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population. CONCLUSIONS: Our results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur during early stages of malignant transformation.

Impaired function of trophoblast cells derived from translocated hESCs may explain pregnancy loss in women with balanced translocation (11;22).

PURPOSE: The aim of the study was to study whether the trophoblasts carrying unbalanced translocation 11,22 [t(11;12)] display abnormal expression of trophoblastic genes and impaired functional properties that may explain implantation failure. METHODS: t(11;22) hESCs and control hESCs were differentiated in vitro into trophoblast cells in the presence of BMP4, and trophoblast vesicles (TBVs) were created in suspension. The expression pattern of extravillous trophoblast (EVT) genes was compared between translocated and control TBVs. The functional properties of the TBVs were evaluated by their attachment to endometrium cells (ECC1) and invasion through trans-well inserts. RESULTS: TBVs derived from control hESCs expressed EVT genes from functioning trophoblast cells. In contrast, TBVs differentiated from the translocated hESC line displayed impaired expression of EVT genes. Moreover, the number of TBVs that were attached to endometrium cells was significantly lower compared to the controls. Correspondingly, invasiveness of trophoblast-differentiated translocated cells was also significantly lower than that of the control cells. CONCLUSIONS: These results may explain the reason for implantation failure in couple carriers of t(11;22). They also demonstrate that translocated hESCs comprise a valuable in vitro human model for studying the mechanisms underlying implantation failure.

Blastomere biopsy for PGD delays embryo compaction and blastulation: a time-lapse microscopic analysis.

PURPOSE: The purpose of the study was to explore the effect of blastomere biopsy for preimplantation genetic diagnosis (PGD) on the embryos' dynamics, further cleavage, development, and implantation. METHODS: The study group included 366 embryos from all PGD treatments (September 2012 to June 2014) cultured in the EmbryoScope™ time-lapse monitoring system. The control group included all intracytoplasmic sperm injection (ICSI) embryos cultured in EmbryoScope™ until day 5 during the same time period (385 embryos). Time points of key embryonic events were analyzed with an EmbryoViewer™. RESULTS: Most (88 %) of the embryos were biopsied at ≥8 cells. These results summarize the further dynamic development of the largest cohort of biopsied embryos and demonstrate that blastomere biopsy of cleavage-stage embryos significantly delayed compaction and blastulation compared to the control non-biopsied embryos. This delay in preimplanation developmental events also affected postimplantation development as observed when the dynamics of non-implanted embryos (known implantation data (KID) negative) were compared to those of implanted embryos (KID positive). CONCLUSION: Analysis of morphokinetic parameters enabled us to explore how blastomere biopsy interferes with the dynamic sequence of developmental events. Our results show that biopsy delays the compaction and the blastulation of the embryos, leading to a decrease in implantation.