The intrinsically disordered CARDs-Helicase linker in RIG-I is a molecular gate for RNA proofreading.

The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics.

The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination.

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.

Genome-wide analysis of histone modifications can contribute to the identification of candidate cis-regulatory regions in the threespine stickleback fish.

BACKGROUND: Cis-regulatory mutations often underlie phenotypic evolution. However, because identifying the locations of promoters and enhancers in non-coding regions is challenging, we have fewer examples of identified causative cis-regulatory mutations that underlie naturally occurring phenotypic variations than of causative amino acid-altering mutations. Because cis-regulatory elements have epigenetic marks of specific histone modifications, we can detect cis-regulatory elements by mapping and analyzing them. Here, we investigated histone modifications and chromatin accessibility with cleavage under targets and tagmentation (CUT&Tag) and assay for transposase-accessible chromatin-sequencing (ATAC-seq). RESULTS: Using the threespine stickleback (Gasterosteus aculeatus) as a model, we confirmed that the genes for which nearby regions showed active marks, such as H3K4me1, H3K4me3, and high chromatin accessibility, were highly expressed. In contrast, the expression levels of genes for which nearby regions showed repressive marks, such as H3K27me3, were reduced, suggesting that our chromatin analysis protocols overall worked well. Genomic regions with peaks of histone modifications showed higher nucleotide diversity within and between populations. By comparing gene expression in the gills of the marine and stream ecotypes, we identified several insertions and deletions (indels) with transposable element fragments in the candidate cis-regulatory regions. CONCLUSIONS: Thus, mapping and analyzing histone modifications can help identify cis-regulatory elements and accelerate the identification of causative mutations in the non-coding regions underlying naturally occurring phenotypic variations.

eSCAN: scan regulatory regions for aggregate association testing using whole-genome sequencing data.

Multiple statistical methods for aggregate association testing have been developed for whole-genome sequencing (WGS) data. Many aggregate variants in a given genomic window and ignore existing knowledge to define test regions, resulting in many identified regions not clearly linked to genes, and thus, limiting biological understanding. Functional information from new technologies (such as Hi-C and its derivatives), which can help link enhancers to their effector genes, can be leveraged to predefine variant sets for aggregate testing in WGS data. Here, we propose the eSCAN (scan the enhancers) method for genome-wide assessment of enhancer regions in sequencing studies, combining the advantages of dynamic window selection in SCANG (SCAN the Genome), a previously developed method, with the advantages of incorporating putative regulatory regions from annotation. eSCAN, by searching in putative enhancers, increases statistical power and aids mechanistic interpretation, as demonstrated by extensive simulation studies. We also apply eSCAN for blood cell traits using NHLBI Trans-Omics for Precision Medicine WGS data. Results from real data analysis show that eSCAN is able to capture more significant signals, and these signals are of shorter length (indicating higher resolution fine-mapping capability) and drive association of larger regions detected by other methods.

A genome-wide association study reveals candidate genes and regulatory regions associated with birth weight in pigs.

Piglet birth weight is associated with preweaning survival, and its related traits have been included in the breeding program. Thus, understanding its genetic basis is essential. This study identified four birth weight-associated genomic regions on chromosomes 2, 4, 5, and 7 through genome-wide association study analysis in 7286 pigs from three different pure breeds using the FarmCPU model. The genetic and phenotypic variance explained by the four candidate regions is 8.42% and 1.85%, respectively. Twenty-eight candidate genes were detected, of which APPL2, TGFBI, MACROH2A1, and SEC22B have been reported to affect body growth or development. In addition, 21 H3K4me3-enriched peaks overlapped with the birth weight-associated genomic regions were identified by integrating the genome-wide association study results with our previous ChIP-seq and RNA-seq data generated in the pig placenta, a fetal organ relevant to birth weight, and three of the regulatory regions influence TGFBI, MACROH2A1, and SEC22B expression. This study provides new insights into understanding the mechanisms for birth weight. Further investigating the variants in the regulatory regions would help identify the functional variants for birth weight in pigs.

Establishment of chromatin accessibility by the conserved transcription factor Grainy head is developmentally regulated.

The dramatic changes in gene expression required for development necessitate the establishment of cis-regulatory modules defined by regions of accessible chromatin. Pioneer transcription factors have the unique property of binding closed chromatin and facilitating the establishment of these accessible regions. Nonetheless, much of how pioneer transcription factors coordinate changes in chromatin accessibility during development remains unknown. To determine whether pioneer-factor function is intrinsic to the protein or whether pioneering activity is developmentally modulated, we studied the highly conserved, essential transcription factor Grainy head (Grh). Prior work established that Grh is expressed throughout Drosophila development and is a pioneer factor in the larva. We demonstrated that Grh remains bound to mitotic chromosomes, a property shared with other pioneer factors. By assaying chromatin accessibility in embryos lacking maternal and/or zygotic Grh at three stages of development, we discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. Our data reveal that the pioneering activity of Grh is temporally regulated and likely influenced by additional factors expressed at a given developmental stage.

Transgenic HPV11-E2 protein modulates URR activity in vivo.

In vitro experiments have shown that the E2 protein of human papillomaviruses (HPV) binds to the upstream regulatory region (URR) of the viral genome and modulates transcription. Additionally, it seems to be a necessary component for viral DNA replication together with E1. We have developed a transgenic mouse model containing the URR region of the low-risk virus HPV11 that regulates the expression of the lacZ reporter gene. Most interestingly, in these mice, the transgene was exclusively expressed in the bulge region of the hair follicle but not in any other tissues. Further experimental data indicate that in double transgenic mice that also express the HPV11-E2 protein under the control of the Ubiquitin C-promoter, the transcription of the reporter gene is modulated. When E2 is present, the expression of the reporter gene also occurs exclusively in the bulge region of the hair follicles as it does in the single transgenic mice, but the expression of the lacZ driven by the URR is increased and the statistical spread is greater. Even if the expression of the reporter gene occurs in the hair follicles of the dorsal skin of an animal uniform, E2 obviously has the capacity for both to induce and to repress the URR activity in vivo.

Boosting tissue-specific prediction of active cis-regulatory regions through deep learning and Bayesian optimization techniques.

BACKGROUND: Cis-regulatory regions (CRRs) are non-coding regions of the DNA that fine control the spatio-temporal pattern of transcription; they are involved in a wide range of pivotal processes such as the development of specific cell-lines/tissues and the dynamic cell response to physiological stimuli. Recent studies showed that genetic variants occurring in CRRs are strongly correlated with pathogenicity or deleteriousness. Considering the central role of CRRs in the regulation of physiological and pathological conditions, the correct identification of CRRs and of their tissue-specific activity status through Machine Learning methods plays a major role in dissecting the impact of genetic variants on human diseases. Unfortunately, the problem is still open, though some promising results have been already reported by (deep) machine-learning based methods that predict active promoters and enhancers in specific tissues or cell lines by encoding epigenetic or spectral features directly extracted from DNA sequences. RESULTS: We present the experiments we performed to compare two Deep Neural Networks, a Feed-Forward Neural Network model working on epigenomic features, and a Convolutional Neural Network model working only on genomic sequence, targeted to the identification of enhancer- and promoter-activity in specific cell lines. While performing experiments to understand how the experimental setup influences the prediction performance of the methods, we particularly focused on (1) automatic model selection performed by Bayesian optimization and (2) exploring different data rebalancing setups for reducing negative unbalancing effects. CONCLUSIONS: Results show that (1) automatic model selection by Bayesian optimization improves the quality of the learner; (2) data rebalancing considerably impacts the prediction performance of the models; test set rebalancing may provide over-optimistic results, and should therefore be cautiously applied; (3) despite working on sequence data, convolutional models obtain performance close to those of feed forward models working on epigenomic information, which suggests that also sequence data carries informative content for CRR-activity prediction. We therefore suggest combining both models/data types in future works.

Fine-tuning grain amylose contents by genome editing of Waxy cis-regulatory region in rice.

Rice grain amylose contents (ACs) is a key quantitative trait influencing eating and cooking quality. Regulating the expression level of Waxy, a key gene controlling ACs, and in turn fine-tuning the grain ACs, is an ideal approach to improve grain quality of rice varieties. Based on CRISPR/Cas9 genome editing technology, we designed eight targets in the cis-regulatory region of Wxa background, screened phenotypic changes of the transgenic lines and generated eight novel Waxy alleles with altered grain ACs. Among the eight alleles, we found that a 407-bp non-homologous substitution (NHS) in the 5'UTR-intron caused by genome editing regulated Waxy expression and decreased grain ACs by 2.9%. Moreover, embedding the 407-bp NHS into the cis-regulatory region of Wxb allele can also affect gene activity. Our work suggested the effect of 5'UTR-intron on Waxy gene expression regulation, and provided a potentially useful allele in breeding that can finely adjust rice grain ACs.

Analysis of genotype-phenotype correlations in PAX6-associated aniridia.

BackgroundAniridia is a severe autosomal dominant panocular disorder associated with pathogenic sequence variants of the PAX6 gene or 11p13 chromosomal aberrations encompassing the coding and/or regulatory regions of the PAX6 gene in a heterozygous state. Patients with aniridia display several ocular anomalies including foveal hypoplasia, cataract, keratopathy, and glaucoma, which can vary in severity and combination.MethodsA cohort of 155 patients from 125 unrelated families with identified point PAX6 pathogenic variants (118 patients) or large chromosomal 11p13 deletions (37 patients) was analyzed. Genetic causes were divided into 6 types. The occurrence of 6 aniridic eye anomalies was analyzed. Fisher's exact test was applied for 2×2 contingency tables assigning numbers of patients with/without each sign and each type of the PAX6 variants or 11p13 deletions with Benjamini-Hochberg correction. The age of patients with different types of mutation did not differ.ResultsPatients with 3'-cis-regulatory region deletions had a milder aniridia phenotype without keratopathy, nystagmus, or foveal hypoplasia. The phenotypes of the patients with other rearrangements involving 11p13 do not significantly differ from those associated with point pathogenic variants in the PAX6 gene. Missense mutations and genetic variants disrupting splicing are associated with a severe aniridia phenotype and resemble loss-of-function mutations. It is particularly important that in all examined patients, PAX6 mutations were found to be associated with multiple eye malformations. The age of patients with keratopathy, cataract, and glaucoma was significantly higher than the age of patients without these signs.ConclusionWe got clear statistically significant genotype-phenotype correlations in congenital aniridia and evident that aniridia severity indeed had worsened with age.

Ectopic GRHL2 Expression Due to Non-coding Mutations Promotes Cell State Transition and Causes Posterior Polymorphous Corneal Dystrophy 4.

In a large family of Czech origin, we mapped a locus for an autosomal-dominant corneal endothelial dystrophy, posterior polymorphous corneal dystrophy 4 (PPCD4), to 8q22.3-q24.12. Whole-genome sequencing identified a unique variant (c.20+544G>T) in this locus, within an intronic regulatory region of GRHL2. Targeted sequencing identified the same variant in three additional previously unsolved PPCD-affected families, including a de novo occurrence that suggests this is a recurrent mutation. Two further unique variants were identified in intron 1 of GRHL2 (c.20+257delT and c.20+133delA) in unrelated PPCD-affected families. GRHL2 is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT) and is a direct transcriptional repressor of ZEB1. ZEB1 mutations leading to haploinsufficiency cause PPCD3. We previously identified promoter mutations in OVOL2, a gene not normally expressed in the corneal endothelium, as the cause of PPCD1. OVOL2 drives mesenchymal-to-epithelial transition (MET) by directly inhibiting EMT-inducing transcription factors, such as ZEB1. Here, we demonstrate that the GRHL2 regulatory variants identified in PPCD4-affected individuals induce increased transcriptional activity in vitro. Furthermore, although GRHL2 is not expressed in corneal endothelial cells in control tissue, we detected GRHL2 in the corneal "endothelium" in PPCD4 tissue. These cells were also positive for epithelial markers E-Cadherin and Cytokeratin 7, indicating they have transitioned to an epithelial-like cell type. We suggest that mutations inducing MET within the corneal endothelium are a convergent pathogenic mechanism leading to dysfunction of the endothelial barrier and disease.

Two promoter regions confer heat-induced activation of SlDREBA4 in Solanum lycopersicum.

Dehydration-responsive element binding (DREB) transcription factors activate the expression of downstream functional genes in combination with a dehydration-responsive element (DRE), and thereby improve the resistance of plants to abiotic stresses such as heat. However, the upstream regulatory mechanism of DREB genes under heat is unclear. A DREBA4 subfamily transcription factor (SlDREBA4), which is heat-responsive and improves heat resistance, was isolated from Solanum lycopersicum 'Microtom'. In this study, promoter truncation experiments were performed to verify changes in ß-glucuronidase (GUS) enzyme activity and GUS gene expression levels in transgenic plants with different lengths of promoter fragments under heat and to identify specific regions in the promoter that respond to heat. Our results showed that the GUS reporter gene was constitutively expressed in tissues of the full-length promoter transgenic 'Microtom' plants, with higher expression in conducting tissues of root, stem, and leaf, as well as sepals of flowers and fruits. Under heat treatment, GUS enzyme activity and GUS gene expression levels in tissues of the full-length promoter transgenic plants increased. Promoter deletion analysis identified two positive regulatory regions (-1095 to -730 bp and -162 to -38 bp) responsible for the promoter's response to heat. These results indicated that the heat shock element (HSE) and MYC recognition sequences may cooperate in heat-induced activation of SlDREBA4 promoter.

Nucleosome Positioning around Transcription Start Site Correlates with Gene Expression Only for Active Chromatin State in Drosophila Interphase Chromosomes.

We analyzed the whole-genome experimental maps of nucleosomes in Drosophila melanogaster and classified genes by the expression level in S2 cells (RPKM value, reads per kilobase million) as well as the number of tissues in which a gene was expressed (breadth of expression, BoE). Chromatin in 5'-regions of genes we classified on four states according to the hidden Markov model (4HMM). Only the Aquamarine chromatin state we considered as Active, while the rest three states we defined as Non-Active. Surprisingly, about 20/40% of genes with 5'-regions mapped to Active/Non-Active chromatin possessed the minimal/at least modest RPKM and BoE. We found that regardless of RPKM/BoE the genes of Active chromatin possessed the regular nucleosome arrangement in 5'-regions, while genes of Non-Active chromatin did not show respective specificity. Only for genes of Active chromatin the RPKM/BoE positively correlates with the number of nucleosome sites upstream/around TSS and negatively with that downstream TSS. We propose that for genes of Active chromatin, regardless of RPKM value and BoE the nucleosome arrangement in 5'-regions potentiates transcription, while for genes of Non-Active chromatin, the transcription machinery does not require the substantial support from nucleosome arrangement to influence gene expression.

Progressive multifocal leukoencephalopathy in Zambia is caused by JC virus with prototype regulatory region.

There are only few documented cases of progressive multifocal leukoencephalopathy (PML) in Africa. Whether this is caused by a lack of JC virus (JCV) spread or alteration in the JCV genome is unknown. We characterized the clinical presentation, laboratory findings, and JCV regulatory region (RR) pattern of the first documented PML cases in Zambia as well as JCV seroprevalence among HIV+ and HIV- Zambians. We identified PML patients with positive JCV DNA PCR in their cerebrospinal fluid (CSF) among subjects enrolled in an ongoing tuberculous meningitis study from 2014 to 2016 in Lusaka. JCV regulatory region was further characterized by duplex PCR in patients' urine and CSF. Of 440 HIV+ patients, 14 (3%) had detectable JCV DNA in their CSF (age 18-50; CD4+ T cells counts 15-155 × 106/µl) vs 0/60 HIV- patients. The main clinical manifestations included altered mental status and impaired consciousness consistent with advanced PML. While prototype JCV was identified by duplex PCR assay in the CSF samples of all 14 PML patients, only archetype JCV was detected in their urine. All PML Zambian patients tested were seropositive for JCV compared to 46% in a control group of HIV+ and HIV- Zambian patients without PML. PML occurs among HIV-infected individuals in Zambia and is caused by CNS infection with prototype JCV, while archetype JCV strains are present in their urine. JCV seroprevalence is comparable in Zambia and the USA, and PML should be included in the differential diagnosis of immunosuppressed individuals presenting with neurological dysfunction in Zambia.

Genome-wide prediction of cis-regulatory regions using supervised deep learning methods.

BACKGROUND: In the human genome, 98% of DNA sequences are non-protein-coding regions that were previously disregarded as junk DNA. In fact, non-coding regions host a variety of cis-regulatory regions which precisely control the expression of genes. Thus, Identifying active cis-regulatory regions in the human genome is critical for understanding gene regulation and assessing the impact of genetic variation on phenotype. The developments of high-throughput sequencing and machine learning technologies make it possible to predict cis-regulatory regions genome wide. RESULTS: Based on rich data resources such as the Encyclopedia of DNA Elements (ENCODE) and the Functional Annotation of the Mammalian Genome (FANTOM) projects, we introduce DECRES based on supervised deep learning approaches for the identification of enhancer and promoter regions in the human genome. Due to their ability to discover patterns in large and complex data, the introduction of deep learning methods enables a significant advance in our knowledge of the genomic locations of cis-regulatory regions. Using models for well-characterized cell lines, we identify key experimental features that contribute to the predictive performance. Applying DECRES, we delineate locations of 300,000 candidate enhancers genome wide (6.8% of the genome, of which 40,000 are supported by bidirectional transcription data), and 26,000 candidate promoters (0.6% of the genome). CONCLUSION: The predicted annotations of cis-regulatory regions will provide broad utility for genome interpretation from functional genomics to clinical applications. The DECRES model demonstrates potentials of deep learning technologies when combined with high-throughput sequencing data, and inspires the development of other advanced neural network models for further improvement of genome annotations.

Implication of non-coding PAX6 mutations in aniridia.

There is an increasing implication of non-coding regions in pathological processes of genetic origin. This is partly due to the emergence of sophisticated techniques that have transformed research into gene expression by allowing a more global understanding of the genome, both at the genomic, epigenomic and chromatin levels. Here, we implemented the analysis of PAX6, whose coding loss-of-function variants are mainly implied in aniridia, by studying its non-coding regions (untranslated regions, introns and cis-regulatory sequences). In particular, we have taken advantage of the development of high-throughput approaches to screen the upstream and downstream regulatory regions of PAX6 in 47 aniridia patients without identified mutation in the coding sequence. This was made possible through the use of custom targeted resequencing and/or CGH array to analyze the entire PAX6 locus on 11p13. We found candidate variants in 30 of the 47 patients. 9/30 correspond to the well-known described 3' deletions encompassing SIMO and other enhancer elements. In addition, we identified numerous different variants in various non-coding regions, in particular untranslated regions. Among these latter, most of them demonstrated an in vitro functional effect using a minigene strategy, and 12/21 are thus considered as causative mutations or very likely to explain the phenotypes. This new analysis strategy brings molecular diagnosis to more than 90% of our aniridia patients. This study revealed an outstanding mutation pattern in non-coding PAX6 regions confirming that PAX6 remains the major gene for aniridia.

Analysis of variability of urinary excreted JC virus strains in patients infected with HIV and healthy donors.

In immunocompromised individuals, especially in patients with T cell immunodeficiency, reactivation of JCPyV can cause serious life-threatening diseases. Nowadays, HIV infection is one of the most important factor for reactivation of JCPyV and the development of of the progressive multifocal leukoencephalopathy (PML). Mutations in the outer loops of the VP1 region can lead to the selection of the viral variants with changed tropism and increased pathological potential. The aims of this study were to determine sequence variation and amino acid changes within VP1 loops and the structure of non-coding control region (NCCR) of urinary excreted JCPyV isolates among HIV-infected patients and healthy donors. Single urine samples from 114 HIV-infected patients and 120 healthy donors were collected. PCR was performed for amplification of VP1 and NCCR. Amplified fragments were directly sequenced and analyzed by using bioinformatics tools. Nucleotide substitutions were detected within DE and EF loops and in the ß-sheets of both studied groups. In HIV-infected patients group, 70% of mutations were detected within receptor domains. Among healthy donors, one mutation was identified within ß-sheets while the remaining were located within receptor domains. The most prevalent mutation was L157V in both groups. Analysis of NCCR revealed that all isolates had archetype structure with some minor changes. Since single point mutations at specific place within outer loop of VP1 region can cause formation of variants with changed receptor specificity, identification of these mutations in HIV-infected patients can help to single out those with higher risk for development of polyomavirus-associated diseases.

Central Precocious Puberty Caused by a Heterozygous Deletion in the MKRN3 Promoter Region.

CONTEXT: Loss-of-function mutations in the coding region of MKRN3, a maternally imprinted gene at chromosome 15q11.2, are a common cause of familial central precocious puberty (CPP). Whether MKRN3 alterations in regulatory regions can cause CPP has not been explored to date. We aimed to investigate potential pathogenic variants in the promoter region of MKRN3 in patients with idiopathic CPP. PATIENTS/METHODS: A cohort of 110 patients with idiopathic CPP was studied. Family history of precocious sexual development was present in 25%. Mutations in the coding region of MKRN3 were excluded in all patients. Genomic DNA was extracted from peripheral blood leukocytes, and 1,100 nucleotides (nt) of the 5'-regulatory region of MKRN3 were amplified and sequenced. Luciferase assays were performed in GT1-7 cells transiently transfected with plasmids containing mutated and wild-type MKRN3 promoter. RESULTS: We identified a rare heterozygous 4-nt deletion (c.-150_-147delTCAG; -38 to -41 nt upstream to the transcription start site) in the proximal promoter region of MKRN3 in a girl with CPP. In silico analysis predicted that this deletion would lead to the loss of a binding site for a downstream res-ponsive element antagonist modulator (DREAM), a potential transcription factor for MKRN3 and GNRH1 expression. Luciferase assays demonstrated a significant reduction of MKRN3 promoter activity in transfected cells with a c.-150_- 147delTCAG construct plasmid in both homozygous and heterozygous states when compared with cells transfected with the corresponding wild-type MKRN3 promoter region. CONCLUSION: A rare genetic alteration in the regulatory region of MKRN3 causes CPP.

Secretory expression of negative regulatory region of human Notch1 in Escherichia coli and preparation of a functional polyclonal antibody.

Notch signaling is a highly conserved pathway existed in multicellular organisms. It plays roles in normal human body development, human cancer initiation, progression and metastasis. The Notch negative regulatory region (NRR) is critical for Notch signaling, and cleavage at the S2 site in the NRR ultimately leads to the activation of Notch signaling. To study the function of human NRR1, we expressed the recombinant human NRR1 (rhNRR1) domain in Escherichia coli. After purification, rhNRR1 was obtained with approximately 94% purity according to SDS-PAGE analysis. Furthermore, the polyclonal anti-rhNRR1 serum raised by immunizing mouse with the purified rhNRR1 was able to reduce the generation of active form of Notch1 intracellular domain in HeLa cells, which implied the raised antibody could recognize and bind the natural conformation of Notch1 NRR. Preparation of rhNRR1 by this way is convenient, time-consuming, and could be used to the preparation of anti-NRR1 therapeutic antibody.

Genetic screening of regulatory regions of pituitary transcription factors in patients with idiopathic pituitary hormone deficiencies.

PURPOSE: Mutation frequencies of PROP1, POU1F1 and HESX1 in patients with combined pituitary hormone deficiencies (CPHD) vary substantially between populations. They are low in sporadic CPHD patients in Western Europe. However, most clinicians still routinely send DNA of their CPHD patients for genetic screening of these pituitary transcription factors. Before we can recommend against screening of PROP1, POU1F1 and HESX1 as part of routine work-up for Western-European sporadic CPHD patients, it is crucial to rule out possible defects in regulatory regions of these genes, which could also disturb the complex process of pituitary organogenesis. METHODS: The regulatory regions of PROP1, POU1F1 and HESX1 are not covered by Whole Exome Sequencing as they are largely located outside the coding regions. Therefore, we manually sequenced the regulatory regions, previously defined in the literature, of PROP1, POU1F1 and HESX1 among 88 Dutch patients with CPHD. We studied promoter SNPs in relation to phenotypic data. RESULTS: We found six known SNPs in the PROP1 promoter. In the POU1F1 promoter, we found one new variant and two known SNPs. We did not find any variant in the HESX1 promoter. CONCLUSION: Although the new POU1F1 variant might explain the phenotype of one patient, the general conclusion of this study is that variants in regulatory regions of PROP1, POU1F1 and HESX1 are rare in patients with sporadic CPHD in the Netherlands. We recommend that genetic screening of these pituitary transcription factors should no longer be part of routine work-up for Western-European, and especially Dutch, sporadic CPHD patients.