Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages.

Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.

Comprehensive multiparameter genetic analysis improves circulating tumor DNA detection in head and neck cancer patients.

INTRODUCTION: Tumor-specific genetic aberrations in cell-free DNA (cfDNA) from plasma are promising biomarkers for diagnosis of recurrent head and neck squamous cell carcinoma (HNSCC). However, the sensitivity when using somatic mutations only in cfDNA is suboptimal. Here, we combined detection of copy number aberrations (CNAs), human papillomavirus (HPV) DNA and somatic mutations in a single sequencing workflow. METHODS: Pretreatment plasmas of 40 patients and 20 non-cancer controls were used for analysis. Plasma DNA underwent low-coverage whole genome sequencing (lcWGS) to detect both CNAs and HPV-DNA, and deep sequencing to detect mutations in 12 frequently altered cancer driver genes in HNSCC using the same sequencing library. A specific analysis pipeline line was developed for data mining. The corresponding tumors were analyzed using slightly adapted protocols. RESULTS: Using the developed method, somatic mutations and CNAs were detected in plasma DNA of HNSCC patients in 67% and 52%, respectively. HPV-DNA in plasma was detected in 100% of patients with HPV-positive tumors, and not in plasma of patients with HPV-negative tumors or non-cancer controls. Combined analysis increased the detection rate of tumor DNA in plasma to 78%. The detection rate was significantly associated with the stage of disease of the tumor. Neither HPV status nor location of the primary tumor influenced detection of CNAs or somatic mutations in plasma. CONCLUSIONS: This study demonstrates that the combined analysis of CNAs, HPV and somatic mutations in plasma of HNSCC patients is feasible and contributes to a higher sensitivity of the assay compared to single modality analyses.

The effect of maternal NODAL on STOX1 expression in extravillous trophoblasts is mediated by IGF1.

The number of molecules identified to be involved in communication between placenta and decidua is fast expanding. Previously, we showed that NODAL expressed in maternal endometrial stromal cells is able to affect NODAL and STOX1 expression in placental extravillous trophoblasts. The effect of maternal NODAL on placental NODAL expression is achieved via Activin A, while preliminary data suggests that maternal NODAL affects STOX1 expression in trophoblasts potentially via IGF1. In the current study, T-HESC endometrial stromal cells were treated with siRNAs against NODAL after which IGF1 mRNA expression was determined by quantitative RT-PCR, while IGF1 secretion was measured by ELISA. Recombinant IGF1 and inhibitors of the MAPK and PI3K/AKT pathways were added to SGHPL-5 extravillous trophoblasts after which the effects on STOX1 mRNA and STOX1 protein expression were determined. The effect of IGF1 and the MAPK and PI3K/AKT inhibitors on the invasive capacity of SGHPL-5 cells was investigated by performing invasion assays. We found that T-HESC cells treated with NODAL siRNAs showed significant upregulation of IGF1 mRNA expression and IGF1 protein secretion. Addition of IGF1 to SGHPL-5 cell media significantly upregulated STOX1 mRNA and protein expression. Using inhibitors of the PI3K/AKT and MAPK pathway showed that the effect of IGF1 on STOX1 expression is accomplished via MAPK signaling. Secondly, PI3K inhibition independently leads to reduced STOX1 expression which can be rescued by adding IGF1. IGF1 was unable to influence the invasive capacity of SGHPL-5 cells, while inhibiting the PI3K/AKT pathway did reduce the invasion of these cells. To conclude, here we show that downregulated NODAL expression in endometrial stromal cells, previously associated with pre-eclampsia like symptoms in mice, increases IGF1 secretion. Increased levels of IGF1 lead to increased expression levels of STOX1 in extravillous trophoblasts via the MAPK pathway, hereby identifying a novel signaling cascade involved in maternal-fetal communication.

Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing.

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

Cardiac expression of deiodinase type 3 (Dio3) following myocardial infarction is associated with the induction of a pluripotency microRNA signature from the Dlk1-Dio3 genomic region.

The adult heart has almost completely lost the proliferative potential of the fetal heart. Instead, loss of cardiomyocytes due to myocardial infarction (MI) leads to a limited, and often insufficient, hypertrophic response of cardiomyocytes in the spared myocardium. This response is still characterized by a partial reexpression of the fetal gene program. Because of the suggested involvement of microRNAs (miRNAs) in cardiac remodeling, we examined the miRNA expression profile of the spared left ventricular myocardium using a MI mouse model. C57Bl/6J mice of either sex were randomly assigned to the sham-operated group or MI group. MI was induced by ligation of the left coronary artery. One week after surgery RNA was isolated from the left ventricle. MiRNA analysis was performed using the Taqman Megaplex rodent array. Unexpectedly, we found a set of 29 up-regulated miRNAs originating from the Dlk1-Dio3 genomic imprinted region, which has been identified as a hallmark of pluripotency and proliferation. This miRNA signature was associated with a 6-fold increase in expression of the deiodinase type 3 gene (Dio3) located in this region. Dio3 is a fetally expressed thyroid hormone-inactivating enzyme associated with cell proliferation, which was shown to be up-regulated in cardiomyocytes creating a local hypothyroid condition in the spared myocardium in this model. These data suggest that a regenerative process is initiated, but not completed, in adult cardiomyocytes after MI. The identified miRNA signature could provide new ways to manipulate the in vivo response of adult cardiomyocytes to stress and to increase the regenerative capacity of the injured myocardium.

STOX1A induces phosphorylation of tau proteins at epitopes hyperphosphorylated in Alzheimer's disease.

Intraneuronal fibrillary tangles are a major hallmark of several neurodegenerative diseases including Alzheimer's disease. The major constituents of these hallmarks are hyper-phosphorylated tau. In this study we used a neuronal cellular model which over-expresses transcription factor STOX1A in combination with the longest human tau isoform to test the effect of STOX1A on tau phosphorylation. Our results show that STOX1A induces phosphorylation of the longest human tau isoform at phospho-epitopes typically found in neurofibrillary tangles in Alzheimer's disease. In conclusion, our results show a STOX1A-dependent effect on tau phosphorylation found in neurodegenerative diseases such as Alzheimer's disease.

Direct downregulation of CNTNAP2 by STOX1A is associated with Alzheimer's disease.

STOX1A is a transcription factor which is functionally and structurally similar to the forkhead box protein family. STOX1A has been shown to be associated with pre-eclampsia, a pregnancy associated disease, and to have potential implications in late onset Alzheimer's disease. However, the exact function of STOX1A and its target genes are still largely unknown. Therefore, in this study we performed chromatin immunoprecipitation coupled to shotgun cloning to discover novel STOX1A target genes. Our results show that CNTNAP2, a member of the neurexin family, is directly downregulated by STOX1A. Additionally, we show that CNTNAP2 expression is downregulated in the hippocampus of Alzheimer's disease patients where STOX1A expression has been shown to be upregulated. In conclusion, these results further indicate the potential involvement of STOX1A and its target genes in the etiology of Alzheimer's disease.

Naturally occurring variation in trophoblast invasion as a source of novel (epigenetic) biomarkers.

During the first trimester of pregnancy fetal trophoblasts invade the maternal decidua, thereby remodeling the maternal spiral arteries. This process of trophoblast invasion is very similar to cancer cell invasion, with multiple signaling pathways shared between the two. Pregnancy-related diseases, e.g., pre-eclampsia, and cancer metastasis start with a decrease or increase in cellular invasion, respectively. Here, we investigate if first trimester placental explants can be used to identify epigenetic factors associated with changes in cellular invasion and their potential use as biomarkers. We show that the outgrowth potential of first trimester explants significantly correlates with promoter methylation of PRKCDBP and MMP2, two genes known to be differentially methylated in both placenta and cancer. The increase in methylation percentage of placental cells coincides with an increase in invasion potential. Subsequently, as a non-invasive marker must be detectable in blood, plasma samples of pregnant and non-pregnant women were analyzed. The MMP2 promoter showed high methylation levels in non-pregnant plasma samples, which decreased in pregnant plasma samples which also contain placental DNA. The decrease in methylated plasma DNA during pregnancy is most likely due to the fractional increase in unmethylated placental DNA. This suggests that the level of unmethylated DNA has the potential to be used as an invasion marker, where higher levels of unmethylated DNA indicate a lower invasion potential of trophoblasts. These proof of principle data provide evidence that human first trimester placental explants are an excellent ex vivo model system to identify (epigenetic) factors and thus potential biomarkers associated with changes in cellular invasion, e.g., to detect pregnancy-related diseases or cancer metastasis. To identify novel biomarkers the next step is to correlate naturally occurring variation in invasion potential to changes in (epigenetic) factors by genome-wide approaches such as massively parallel sequencing.

Transcription factor STOX1A promotes mitotic entry by binding to the CCNB1 promotor.

BACKGROUND: In this study we investigated the involvement of the transcription factor STOX1A in the regulation of the cell cycle. METHODOLOGY/PRINCIPAL FINDINGS: We found that several major cell cycle regulatory genes were differentially expressed upon STOX1A stimulation and knockdown in the neuroblastoma cell line SH-SY5Y. This includes STOX1A dependent differential regulation of cyclin B1 expression, a cyclin which is known to regulate mitotic entry during the cell cycle. The differential regulation of cyclin B1 expression by STOX1A is direct as shown with chromatin immunoprecipitation. Results furthermore suggest that mitotic entry is enhanced through the direct upregulation of cyclin B1 expression effectuated by STOX1A. CONCLUSIONS: In conclusion we hereby show that STOX1A is directly involved in the regulation of the cell cycle.

SFRS7-mediated splicing of tau exon 10 is directly regulated by STOX1A in glial cells.

BACKGROUND: In this study, we performed a genome-wide search for effector genes bound by STOX1A, a winged helix transcription factor recently demonstrated to be involved in late onset Alzheimer's disease and affecting the amyloid processing pathway. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that out of 218 genes bound by STOX1A as identified by chromatin-immunoprecipitation followed by sequencing (ChIP-Seq), the serine/arginine-rich splicing factor 7 (SFRS7) was found to be induced, both at the mRNA and protein levels, by STOX1A after stable transfection in glial cells. The increase in SFRS7 was followed by an increase in the 4R/3R ratios of the microtubule-associated protein tau (MAPT) by differential exon 10 splicing. Secondly, STOX1A also induced expression of total tau both at the mRNA and protein levels. Upregulation of total tau expression (SFRS7-independent) and tau exon 10 splicing (SFRS7-dependent), as shown in this study to be both affected by STOX1A, is known to have implications in neurodegeneration. CONCLUSIONS: Our data further supports the functional importance and central role of STOX1A in neurodegeneration.

The STOX1 genotype associated with pre-eclampsia leads to a reduction of trophoblast invasion by alpha-T-catenin upregulation.

By using complementary in vitro and ex vivo approaches, we show that the risk allele (Y153H) of the pre-eclampsia susceptibility gene STOX1 negatively regulates trophoblast invasion by upregulation of the cell-cell adhesion protein alpha-T-catenin (CTNNA3). This is effectuated at the crucial epithelial-mesenchymal transition of proliferative into invasive extravillous trophoblast. This STOX1-CTNNA3 interaction is direct and includes Akt-mediated phosphorylated control of nucleo-cytoplasmic shuttling and ubiquitin-mediated degradation as shared with the FOX multigene family. This, to our knowledge, is the first time a genotype associated with pre-eclampsia has been shown to directly limit first trimester extravillous trophoblast invasion, the earliest hallmark of pre-eclampsia.

Decreased plasma levels of metastin in early pregnancy are associated with small for gestational age neonates.

OBJECTIVE: To investigate whether pregnancies with small for gestational age (SGA) neonates, defined as customized birth weight below the 10th centile, are associated with altered levels of metastin in maternal plasma in the first trimester. STUDY DESIGN: Maternal blood was obtained between 8 and 14 weeks of pregnancy. Levels of metastin were measured in pregnancies with (n = 31) or without SGA-neonates (n = 31), matched for gestational age at venipuncture. Measurement of beta-hCG was included to study the influence of gestational age and placental volume on plasma levels of the measured markers. RESULTS: Metastin was significantly lower in SGA-pregnancies compared to an equal number of matched uneventful pregnancies (metastin: 1376 +/- 1317 pmol/L vs 2035 +/- 1260 pmol/L, p = 0.035; mean +/- standard deviation). beta-hCG levels were not different. CONCLUSION: Metastin is significantly lower in maternal plasma in the first trimester, in pregnancies with SGA-neonates. It might therefore be used in combination with other markers for risk estimation of growth impairment in the first trimester.

Measurement of allelic-expression ratios in trisomy 21 placentas by quencher extension of heterozygous samples identified by partially denaturing HPLC.

BACKGROUND: Measuring the allelic ratios of placental transcripts in maternal plasma permits noninvasive prenatal detection of chromosomal aneuploidy. Current methods, however, require highly specialized equipment (MALDI-TOF), limiting the widespread implementation of this powerful RNA single-nucleotide polymorphism (SNP) strategy in routine diagnostic settings. We adapted and applied the Transgenomic WAVE System and quencher extension (QEXT) for this purpose. METHODS: The expressed SNP (rs2187247) in exon 2 of the placentally expressed C21orf105 gene (chromosome 21 open reading frame 105) on chromosome 21 was tested in a trisomy 21 model system in which we obtained RNA selectively released from syncytiotrophoblasts of normal and trisomy 21 placentas during first trimester. RESULTS: In identifying heterozygous samples, we observed an exact correspondence between sequencing results and results obtained with the WAVE System. With respect to the analysis time required, the WAVE System was superior. In addition, the real-time QEXT assay (as optimized and validated with calibration standards consisting of 262-bp C21orf105 cDNA amplicons) accurately measured allele ratios after we optimized fragment purification, concentrations of input DNA and quencher label, and calculations of reporter signals. Finally, the optimized and validated QEXT assay correctly distinguished normal placentas from trisomy 21 placentas in tests of the following clinically relevant combinations: diploid homozygous (CC), diploid heterozygous (AC), triploid homozygous (AAA), and triploid heterozygous (AAC or ACC). CONCLUSION: The QEXT method, which is directly adaptable to current real-time PCR equipment, along with rapid identification of informative samples with the WAVE System, may facilitate routine implementation of the RNA-SNP assay for noninvasive aneuploidy diagnostics.

Genetics of preeclampsia: paradigm shifts.

Segregation of preeclampsia into early-onset, placental and late-onset, maternal subtypes along with the acknowledgement of the contribution of epigenetics in placentally expressed genes proved to be a key first step in the identification of essential gene variants associated with preeclampsia. Application of this insight to other populations and related pregnancy-induced syndromes, such as HELLP, and acknowledgment of the features shared between chromosomal loci associated with preeclampsia in different populations provide the rationale for new strategies for the identification of susceptibility genes and for new and more effective diagnostic strategies.

Expression microarray analysis and oligo array comparative genomic hybridization of acquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations.

Gemcitabine is a commonly used therapy for many solid tumors. Acquired resistance to this nucleoside analogue, however, diminishes the long-term effectiveness in a majority of patients. To better define the molecular background of gemcitabine resistance, a mouse colon tumor was selected during successive rounds of transplantation with continued treatment of gemcitabine. Expression microarray analysis was applied to determine which genes are consistently and highly overexpressed or underexpressed in the resistant versus the nonresistant tumor. For the statistical interpretation of the microarray data, a parametric model was implemented, which returns model-based differential gene expression (log-) ratios and their uncertainties. This defined a set of 13 genes, putatively responsible for the gemcitabine resistance in solid tumors. One of these, RRM1, was previously identified as an important marker for gemcitabine resistance in human cell lines. Five of the 13 genes, including RRM1, are located within a 3 Mb region at chromosome 7E1 of which four are highly overexpressed, suggesting a chromosomal amplification. Therefore, chromosomal copy number changes were measured, using oligo array comparative genomic hybridization. A narrow and high amplification area was identified on 7E1 that encompassed all five genes. In addition, reduced RNA expression of two other genes at 8E1 encoding COX4I1 and RPL13 could be explained by a decrease in chromosomal copy number on chromosome 8. In conclusion, the array comparative genomic hybridization biologically validates our statistical approach and shows that gemcitabine is capable to select for chromosomally aberrant tumor cells, where changed gene expression levels lead to drug resistance.

Detection of the placental epigenetic signature of the maspin gene in maternal plasma.

The discovery of fetal DNA in the plasma of pregnant women has opened up new approaches for noninvasive prenatal diagnosis and monitoring. Up to now, the lack of a fetal DNA marker that can be universally detected in maternal plasma has limited the clinical application of this technology. We hypothesized that epigenetic differences between the placenta and maternal blood cells could be used for developing such a marker. By using bisulfite DNA sequencing, the methylation status of the maspin gene promoter in placental tissues and paired maternal blood cells from pregnant women was analyzed. The maspin gene promoter was found to be hypomethylated in placental tissues and densely methylated in maternal blood cells. Genotyping of a single nucleotide polymorphism within the unmethylated maspin sequences in maternal plasma demonstrated that these sequences were derived from the fetus. By using real-time quantitative methylation-specific PCR, unmethylated maspin sequences were detected in maternal plasma in all three trimesters of pregnancy and were cleared within 24 h after delivery. The maternal plasma concentration of unmethylated maspin sequences was elevated by a median of 5.7 times in preeclamptic pregnancies compared with nonpreeclamptic pregnancies. Hypomethylated maspin DNA is the first universal marker for fetal DNA in maternal plasma, thus allowing the measurement of fetal DNA concentrations in pregnancy-associated disorders, irrespective of fetal gender and genetic polymorphisms. Differential DNA methylation between the placenta and maternal blood cells may be exploited to develop further markers for noninvasive prenatal assessment.

MLC1: a novel protein in distal astroglial processes.

Megaloencephalic leukoencephalopathy with subcortical cysts (MLC) is a progressive cerebral white matter disease in children caused by mutations in the MLC1 gene. This disease is histopathologically characterized by myelin splitting and intramyelinic vacuole formation. MLC1 encodes a novel protein, MLC1, which is mainly expressed in the brain and leukocytes. The function is unknown, although a transport function has been suggested. In this article, we provide experimental data addressing the membrane topology and cellular localization of MLC1. We show that MLC1 contains an even number of transmembrane domains, supporting the possible transport function of MLC1. We demonstrate that MLC1 is specifically expressed in distal astroglial processes in perivascular, subependymal, and subpial regions. This localization suggests a role for MLC1 in a transport process across the blood-brain and brain-cerebrospinal fluid barriers. Astrocyte functions have long been debated. It is becoming increasingly clear that these cells are of fundamental importance in maintaining the structural and functional integrity of neural tissue. Elucidation of the function of MLC1 will contribute to a better understanding of not only the pathophysiology of the disease, but also the role of astrocytes in normal neural tissue.

Maternal segregation of the Dutch preeclampsia locus at 10q22 with a new member of the winged helix gene family.

Preeclampsia is a pregnancy-associated disease with maternal symptoms but placental origin. Epigenetic inheritance is involved in some populations. By sequence analysis of 17 genes in the 10q22 region with maternal effects, we narrowed the minimal critical region linked with preeclampsia in the Netherlands to 444 kb. All but one gene in this region, which lies within a female-specific recombination hotspot, encode DNA- or RNA-binding proteins. One gene, STOX1 (also called C10orf24), contained five different missense mutations, identical between affected sisters, cosegregating with the preeclamptic phenotype and following matrilineal inheritance. Four STOX1 transcripts are expressed in early placenta, including invasive extravillus trophoblast, generating three different isoforms. All contain a winged helix domain related to the forkhead (FOX) family. The largest STOX1 isoform has exclusive nuclear or cytoplasmic expression, indicating activation and inactivation, respectively, of the PI3K-Akt-FOX pathway. Because all 38 FOX proteins and all 8 STOX1 homologs have either tyrosine or phenylalanine at position 153, the predominant Y153H variation is highly mutagenic by conservation criteria but subject to incomplete penetrance. STOX1 is a candidate for preeclampsia controlling polyploidization of extravillus trophoblast.

C2360, a nuclear protein expressed in human proliferative cytotrophoblasts, is a representative member of a novel protein family with a conserved coiled coil-helix-coiled coil-helix domain.

In this study, we describe the identification of nine novel genes isolated from a unique human first-trimester cDNA library generated from the placental bed. One of these clones, called C2360 and located on chromosome 10q22, was selected as it showed restricted expression in placental bed tissue as well as in JEG3 choriocarcinoma cells with absent expression in adult tissues. We show that the expression is restricted to first-trimester proliferative trophoblasts of the proximal column and show that C2360 is a nuclear protein. No detectable transactivation potential was observed for different domains of the protein. Secondary structure prediction showed that C2360 is a representative member of a eukaryotic family of proteins with a low conservation at the amino acid level, but with strong conservation at the structural level, sharing the general domain (coiled coil 1)-(helix 1)-(coiled coil 2)-(helix 2), or CHCH domain. Each alpha-helix within this domain contains two cysteine amino acids, and these intrahelical cysteines are separated by nine amino acids (C-X(9)-C motif). The fixed position within each helix indicated that both helices could form a hairpin structure stabilized by two interhelical disulfide bonds. Other proteins belonging to the family include estrogen-induced gene 2 and the ethanol-induced 6 protein. The conserved motif was found in yeast, plant, Drosophila, Caenorhabditis elegans, mouse, and human proteins, indicating that the ancestor of this protein family is of eukaryotic origin. These results indicate that C2360 is a representative member of a multifamily of proteins, sharing a protein domain that is conserved in eukaryotes.