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Home > Section on Clinical and Developmental Genomics

Genetic and Genomic Studies in Normal Development and Diseases

Owen M. Rennert, MD
  • Owen M. Rennert, MD, Head, Section on Clinical Genomics
  • Vanessa Baxendale, BS, Laboratory Manager
  • Catherine Boucheron, PhD, Postdoctoral Fellow
  • Wai Yee Chan, PhD, Adjunct Scientist
  • Albert Cheung, BS, MS, Graduate Student
  • Lawrence Cho, Summer Student
  • Jessica Clark, BS, Postbaccalaureate Fellow
  • Elizabeth Jiali Fang, Summer Student
  • Tin Lap Lee, PhD, Staff Scientist
  • Sohan Nagrani, BS, Postbaccalaureate Fellow
  • Alan Lap-Yin Pang, PhD, Biologist
  • Drasti Patel, Summer Student
  • Margarita Raygada, MS, PhD, Staff Genetic Counselor
  • Diana H. Taft, BS, Postbaccalaureate Fellow

The Laboratory of Clinical and Developmental Genomics focuses on using genetic and genomic technologies, acquired through basic studies, to solve clinical and fundamental biological problems. An additional mission is to train physicians and postdoctoral scientists in the application of these genomic approaches to the study of human disease. One of our recent investigations attempted to define the regulatory pathways of human spermatogenesis and oogenesis, employing methodologies such as methylation tiling array to study transcriptional regulation of genes such as mArd2, a novel testis-specific gene that was first cloned in this laboratory. We also studied the role of Lin 28, a gene that regulates developmental timing; these investigations have employed proteomic approaches to identify RNAs bound to this protein. Gene knockdown experiments are under way to study the function of Lin 28 in determining cell fate. We have also continued our studies to identify and screen for risk factors of complex disorders. In addition to fundamental and translational research, we are involved in clinical protocols to study patients with genetic and malformational disorders and recently initiated novel investigational approaches to study childhood autism. This spectrum of investigational approaches affords us the opportunity to provide clinical genetics training to our trainees and clinical fellows.

Transcriptional regulation of Ard1b, the gene encoding a testis-predominant isoform of the catalytic subunit of mouse N-alpha terminal acetyltransferase, and the role of protein N-alpha acetylation

We cloned a novel Ard1a (Arrest defective 1) gene homolog known as Ard1b that demonstrated testis specificity and was highly expressed during male meiosis in the mouse. We showed ARD1B is functionally equivalent to ARD1A in the reconstitution of N-alpha-acetyltransferase activity in vitro. The testis-specific expression of Ard1b indicates that its transcription is suppressed in somatic tissues. We subsequently found that Ard1b transcription is regulated epigenetically by DNA methylation: reactivation of Ard1b transcription occurs after treatment with 5-aza-deoxycytidine in mouse cells that do not express the gene. The two CpG islands located at the 5’ end of the Ard1b gene are hypermethylated in mouse somatic tissues, but hypomethylated in mouse testicular germ cells. We characterized the promoter region of the Ard1b gene. Reporter assays on different regions upstream of the gene led to the identification of two upstream genomic regions that may contain inhibitory and enhancer element(s) for Ard1b transcription. We found that the core promoter sequence for Ard1b is localized to a region spanning from −148 to at least +150 base pairs with respect to the transcriptional start site of the gene, a region that is hypermethylated in cells that do not express Ard1b. Within this region, we predicted the presence of binding sequences for several transcription factors. By gene over-expression and knockdown experiments, we confirmed the role of Specificity protein 1 (Sp1) in the activation of Ard1b transcription. Similar experiments are under way to confirm the involvement of the other transcription factors predicted to regulate Ard1b transcription. Next we will examine coordination of DNA methylation and the role of specific transcription factor binding in promotion of Ard1b transcription.

The availability of Ard1b gene knockout mice enables us to study the role of Ard1b in testicular development as well as the biological significance of protein N-alpha acetylation. With a human cell culture model, we are investigating the difference in biological function between ARD1 and ARD1B, as well as, the global effect of deficiency of protein N-alpha acetylation on cellular function.

Transcriptional regulation of Lin28

Lin28 is a heterochronic gene involved in the temporal control of cell fate determination in C. elegans. It was shown to exert an enhancing effect on the reprogramming of somatic cells to embryonic stem cell– (ESC) like state (i.e., iPS cells). In mammals, Lin28 is detected mostly in cells that possess proliferative/renewal capacity (e.g., embryonic stem cells, embryonal carcinoma cells, and mouse type A spermatogonia) but is absent from differentiated cells. The tissue-restricted and developmentally-regulated expression pattern of Lin28 suggests that its expression is subject to temporal and spatial regulation. Our preliminary data showed that Lin28 transcription is not regulated by DNA methylation; we hypothesized Lin28 transcription is primarily activated by the action of specific transcription factors that bind to its promoter. Reporter assays in mouse embryonal carcinoma cells P19 (Lin28-expressing) and mouse embryonic fibroblast cells NIH/3T3 (Lin28–non-expressing) led to localization of the core promoter of Lin28 gene—a region about 400 base-pairs upstream from its start codon.

Specific modules of transcription factor binding sites have been predicted within this region, and our gene over-expression studies have shown that at least Sp1 is able to activate Lin28 transcription in P19 cells. The involvement of other transcription factors in stimulating Lin28 expression and the mechanism of suppression of Lin28 expression in non-expressing cells are now under investigation.

Effect of vitamin A deficiency on Sertoli cells and on the epigenomics of male germ cells

Sertoli cells, located in the seminiferous tubules, are essential to provide an adequate and protected environment for germ cell development. Sertoli cells from vitamin A–deficient (VAD) animals lose the ability to adhere to culture dishes, suggesting disruption of the extracellular matrix and/or cell junctions. Using real-time PCR, we documented VAD-induced decreased expression of GAP-43, a protein involved in the formation of the GAP junctions. Moreover PCR array analysis suggested dysregulation of numerous genes of the extracellular matrix and/or cell junction.

Literature reports identify a relationship between GAP-43, thyroid hormones, and Sertoli cell differentiation. Hypothyroidism, which is accompanied by a decrease in GAP-43 expression, is associated with increased Sertoli cell proliferation and a rise in the number of undifferentiated cells. We compared the number of Sertoli cells in the VAD animals with controls, using the cytoplasmic marker vimentin; immunohistochemistry showed a VAD-related increase of the number of vimentin-positive cells. We probed the proliferation/differentiation status of these cells, using real-time PCR and Cyclin D2, a marker of proliferation; AMH, a marker of undifferentiation; and keratin 18, a marker of differentiation. These studies utilize pure populations of Sertoli cells from control and treated animals. We will also compare by the expression profile of the Sertoli cells from VAD and Control animals.

We also study the molecular consequences of VAD, in mice, on spermatogenesis. We use expression profiling to study the genetic mechanism of vitamin A deficiency–induced arrest of spermatogonial stem cell differentiation, as well as the potential regeneration of spermatogenesis following restoration of vitamin A in the diet. The results will expand our knowledge of the molecular mechanisms of germ cell development.

During the initial phase of the study we characterized the VAD experimental model by introducing the VAD diet from 7 weeks up to 28 weeks. At each time point, measurement of vitamin A status included serum RBP (Retinol-Binding Protein) and mRNA expression levels of retinoic acid (active metabolite of vitamin A) receptors in liver, brain, and testis. In parallel, we used histological techniques to characterize morphological changes in the testis. We compared the gene expression profile of control and VAD animals (18 weeks of deficiency) in an enriched population of spermatogonia. Our data confirmed the down-regulation of the retinoid pathways, and highlighted the importance in two other pathways—NF-κB and beta-catenin. Future studies, will examine the effect of VAD on the epigenome of germ cells and determine the genes responsible for VAD-induced male sterility.

Identification of functional long non-coding RNAs in male germ cell development

Mammalian cells produce thousands of non-coding RNAs (ncRNAs) of unknown function. These non–protein-coding portions of the genome were often considered “junk,” but present research has shown that ncRNAs can have a wide range of regulatory functions. Long ncRNAs (more than 200bp) have been shown to be involved in mouse ESCs pluripotency and differentiation. Whole-genome tiling arrays and Serial Analysis of Gene Expression (SAGE) that we conducted have demonstrated widespread transcription of long ncRNAs during male germ cell development. This project attempts to identify male germ cell–specific long ncRNA candidates so as to enhance our understanding of the regulatory functions of long ncRNAs during male gamete differentiation and development. An analysis of our established male germ cell SAGE data showed that we isolated 494, 152, and 201 stage-specific tag sequences with at least five tag counts from spermatogonial stem cells (spermatogonia), pachytene spermatocytes, and round spermatids, respectively. To assess long ncRNA expression, a computational algorithm was developed to "blast," map, and compare the RNA secondary structure of these candidates. To determine the location of the tags in the genome, the SAGE sequences were also compared with various ncRNA databases, such as NRED, RNAdb, fRNAdb, and NONCODE v2.0. We based the selection of long ncRNAs on the SAGE tag count and the distance to the poly-A tail. Given that SAGE normally cuts the RNA strand within a short distance from the poly-A tail, sequences matched to ncRNAs that were located farther from the poly-A tail could be a result of repetitive sequences in the genome. The higher the SAGE tag count, the more highly expressed a sequence is, so it is more likely to play a regulatory role. Therefore, higher priority was given to sequences with a higher count that were located closer to the poly-A tail.

In total, we identified 50, 35, and 24 potential long ncRNA candidates in spermatogonia, pachytene spermatocytes and round spermatids, respectively. Our classification was based on various genomic features, including promoter, intronic, intergenic, and anti-sense. The top ten candidates were selected from each stage for subsequent analysis. The expression and size was validated in male germ cell samples by Northern blot analysis. Some long ncRNAs were a function of the developmental age of the testis. Using RNA from 2-week, 1-month, 2-month, 3-month, 6-month, and 12-month mouse testes, six spermatogonia-specific candidates, six spermatocyte-specific candidates, and seven spermatid-specific candidates displayed a unique expression pattern. Some candidates also exhibited tissue-specific expression patterns; one spermatid-specific candidate was testis-specific, one spermatid-specific candidate was testis- and brain-specific, and one spermatid-specific candidate was testis-, brain-, and ovary-specific. Preliminary functional analysis in a P19 differentiation cell model suggested that some long ncRNAs decreased remarkably following induction of differentiation by retinoic acid. The reduction was more obvious in the comparison of testis from VAD and control animals. The levels of some ncRNAs were more than a thousand-fold higher than in control testis. These results suggest that long ncRNA may play an active role in male germ cell differentiation and development via retinoic acid–related regulatory pathways. Additional functional studies are under way to identify the roles of these long ncRNA. We are developing an in vitro–differentiated ESC-to-male gamete model from mouse R1 ESCs to study these phenomena at key stages of male germ cell differentiation.

Male germ cell informatics

We developed GermSAGE, the first sequence-based germ cell transcriptome database for male germ cell transcriptome analysis. GermSAGE is a comprehensive web-based database, generated from Serial Analysis of Gene Expression (SAGE), representing major stages in mouse male germ cell development; it used sequence tag coverage of 150k in each SAGE library. A total of 452,095 tags derived from type A spermatogonia, pachytene spermatocytes, and round spermatid are included. It is a web-based tool with customizable searching parameters for browsing, comparing, and searching male germ cell transcriptome data at different developmental stages. The user can overlay male germ cell transcriptome data with a variety of annotated tracks below the genome view window, and create a custom map by adding tracks to view various types of data and specific genomic landmarks. The browser offers a broad list of options. It includes: (1) mapping and sequencing tracks that contains information about the position, marker, and GC percentage of the annotated gene region; (2) genes and gene prediction tracks on gene annotation and predictions from various sources; (3) mRNA and EST tracks that contains information on transcripts and CAGE (Cap Analysis of Gene Expression) tags to identify potential transcription start sites and alternatively spliced RNA species; (4) expression and its regulation that includes expression data from various microarray platforms and regulatory information such as CpG islands and microRNA; (5) comparative genomics identifies the sequence conservation among species; and (6) variation and repeats. This information provides insights into gene regulation and facilitates the generation of hypotheses.

The data can be exported and visualized in a tabulated format, which permits flexible processing and analysis of downstream pipelines as well as interaction analysis. It will be useful for revealing regulatory networks, allow novel gene discovery, and can provide insight into molecular and cellular processes. As an example, cross-platform data comparison of the molecular signature of spermatogonial stem/progenitor cells in 6-day-old mouse testis yielded novel candidates involved in stem cell maintenance. The long-term scientific vision of GermSAGE is to provide a central platform to scan the dynamic genomic changes in male germ cell development and identify/predict gene expression patterns. Work is in progress to allow such dynamic data analysis. GermSAGE is freely available at http://germsage.nichd.nih.gov.

Developmental staging of male murine embryonic gonad by SAGE analysis

Despite the identification of key genes such as (sex-determining region Y) Sry integral to embryonic gonadal development, the genomic classification and identification of chromosomal activation of this process are still poorly understood. To better understand the genetic regulation of gonadal development, we performed SAGE to profile the genes and novel transcripts, and an average of 152,000 tags from male embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5, and E17.5 were analyzed. A total of 275,583 non-singleton tags that do not map to any annotated sequence were identified in the six gonad libraries, and 47,255 tags were mapped to 24,975 annotated sequences, among which 987 sequences were uncharacterized. Using an unsupervised pattern identification technique, we established molecular staging of male gonadal development. Based on the global expression matrix, two major clusters that distinguished developmental time point at E10.5 to E12.5 (early) and E13.5 to E17.5 (late) (the first-level dendrogram) were observed, which is concordant with the sequential nature of sexual differentiation. In addition, a fine subdivision of developmental time point was observed in both early and late stage cluster, where expression matrix data supported the resemblance of E11.5/ E12.5 compared with E10.5 in the early stage and E15.5/ E17.5 compared with E13.5 in the late stage. To correlate molecular staging based on clustering analysis to the actual biological transition during male gonad development, we compared the grouping of the developmental time points derived from cluster analysis with the phenotypic changes and major genetic programs during male gonad development. Interestingly, the grouping was very similar to the morphology in the developmental transition, which includes the Sertoli genetic program at E10.5/E11.5, the transition between E12.5 to E13.5 with the appearance of Leydig cells and regression of Müllerian ducts, and the substantial growth of the testis due to establishment of testicular cords, cell migration, and proliferation from E13.5 to E17.5. The early and late staging implicated in the cluster analysis also agreed with the genetic program in male gonad development; the late stage represents testis determination (E13.5 to E17.5) after transient expression of Sry in the supporting cell lineage during the commitment to the testis pathway (E10.5 to E12.5) in the XY gonads and establishment of gonadal sex after E12.5.

To determine whether particular chromosomal regions play a particularly important role in male gonad development, the transcriptome activity at the chromosomal level at various time points in male gonad development was analyzed by locating the transcription hotspots, using positional gene enrichment analysis revealed in terms of chromosomal bands. We observed that there was a progressive increase in gene expression activities from E10.5 to E17.5, with the highest at E12.5 to E13.5 when Sertoli and Leydig cells undergo active genetic programming. To further delineate the functional roles of the transcription hotspots, positional gene enrichment analysis on over-represented chromosomal regions was conducted. We compared the genes associated with the over-represented chromosomal region with the available mouse model list from the Jackson laboratory (http://jaxmice.jax.org) to see if the mouse would carry defects in gonad development. The results showed that a number of transgenic mice carry developmental defects in the gonads or gonadal tumors with defective hotspot genes. Taken together, this approach provided an alternative perspective by focusing on key chromosomal regions in male gonad development.

Epigenetic inactivation of microRNA in male germ cell cancer

MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that have been shown to be deregulated in many diseases including cancer. An intertwined connection between epigenetics and miRNAs was supported by the recent identification of a specific subgroup of miRNAs, called "epi-miRNAs," that can directly and indirectly modulate the activity of the epigenetic machinery. Using a genome-wide approach for studying differential methylation in a testicular germ cell tumor cell line, we previously identified a novel hypermethylated locus on chromosome 1. Genomic mapping revealed that the hypermethylated region overlapped with a microRNA candidate, miR-199a, and its upstream promoter region. Bisulfite sequencing and treatment with the DNA methyltransferase inhibitor 5-aza in Ntera-2 testis cancer cells confirmed the observation made in the tiling microarray.

To explore the role of miR-199s, we further examined its methylation and expression patterns in several testicular embryonal carcinoma cell lines (Ntera-2, 833K, NCCIT, Cates-1B, and Tera-1), a cultured normal testis cell (HT), and three normal testis tissues from healthy males. Bisulfite sequencing showed that the normal cultured testis cell was unmethylated, while for normal testis tissues it is slightly methylated. Except for Cates-1B, all examined testicular embryonal carcinoma cell lines were methylated. Expression of miR-199a, as revealed by real-time qPCR, showed that this miRNA was downregulated in all cancer cell lines as compared with either normal testis cell lines or tissues. In our study, we observed a negative correlation between methylation and expression of miR-199a, indicating that DNA methylation is a critical regulator of miR-199a. Downregulation of miR-199a was not restricted to testicular cancer cell lines but also occurred in primary tissues. We collected total RNA from 9 normal testis biopsies, 8 embryonal carcinomas, and 9 seminomas from patients and analyzed the miR-199a expression by qPCR. After normalization, miR-199a-5p was found to be significantly down-regulated in both embryonal carcinomas and seminomas. Down-regulation in seminomas is more consistent in the analyzed cases, probably due to the more homogeneous nature of these poorly differentiated carcinoma cells. To expand the clinical sample size of testicular cancers, we are analyzing the expression of miR-199a in tissue arrays with LNA-based in situ hybridization. The results from tissue array will help us understand whether miR-199a is significantly dysregulated in primary tumors.

To examine the biological functions of miR-199a, we established stably transfected miR-199a cells (NT2-199a) and investigated the ability of NT2-199a cells to form colonies in soft agar medium. Cells of NT2-199a formed colonies similar to NT2-GFP cells. We also demonstrated that miR-199a inhibits cell proliferation. These data suggest that miR-199a may act as an anti-metastatic but not tumor suppressor miRNA. Bioinformatic analysis on microarray expression data on Ntera-2 cells suggested that podocalyxin-like (PODXL) might be one of the targets of miR-199a; PODXL was significantly up-regulated in testicular cancer cell line. Transient transfection of miR-199a into Ntera-2 cells or stable expression of miR-199a suppressed the mRNA and protein level of PODXL. Furthermore, demethylation treatment of Ntera-2 cells by 5-aza restored expression of miR-199a and decreased PODXL expression. These data support the notion that miR-199a-5p is a negative regulator of PODXL. Sequence analysis revealed two seed sequences of miR-199a in the 3′-UTR of PODXL. One of them, denoted UTR-A, is a highly conserved site in mammals. We cloned the two flanking seed sequences into a luciferase reporter vector. Co-transfection of the luciferase reporter vector with miR-199a-5p significantly suppressed the activity of luciferase that carries the UTR-A site; miR-199a-5p has a minor effect on the non-conserved UTR-B site, and mutation of the seed sequence of UTR-A resulted in loss of suppression function of miR-199a-5p. These data indicate that miR-199a-5p suppresses PODXL through the conserved seed sequence in the 3’-UTR.

Studies of pediatrics patients with genetic and metabolic disorders

We provide care for patients with a variety of rare genetic disorders. In addition, we offer an opportunity for training in clinical genetics, dysmorphology, and metabolic genetics in the National Institute of Child Health and Human Development (NICHD) and other Institutes of the National Institutes of Health (NIH). We also spearhead the development of new research protocols on particular aspects of diagnosis and care for specific genetic diseases. Evaluations of patients with a broad spectrum of metabolic and genetic conditions are performed, genetic counseling services are offered to patients and their families to assess risk, and we give information on preventive measures and testing options. One of our clinical protocols attempts to identify differential gene expression profiles of LPS-activated adult peripheral blood versus cord blood neutrophils from both term and preterm infants by whole genome oligonucleotide microarray. The protocol is designed to investigate the priming effect of histologic chorioamnionitis (HCA), a severe infection/inflammation implicated in many neonatal diseases, on the immune response in neutrophils in terms of gene expression. Additional potential outcomes include an assessment of the effect of prenatal administration of steroids on the immune response in preterm neutrophils. This study will provide insight into the molecular basis for genetic regulation of neutrophil development.

We studied conditions such as chromosomal and Mendelian disorders of childhood and/or adult onset, congenital anomalies and/or birth defects, dysmorphic syndromes, familial cancer syndromes, multifactorial disorders, and metabolic abnormalities. If not eligible for another NICHD research protocol (specific for a disease or a treatment), patients with genetic/metabolic-related conditions may be evaluated under the auspices of this protocol to advance the clinical skills of physicians participating in NICHD clinical research and training programs and to provide stimuli for new clinical research initiatives. The purpose of this protocol is to support our Institute’s training and research missions by expanding the spectrum of diseases that may be studied by our trainees and staff and to offer additional diagnostic assistance to patients with rare heritable diseases. These clinical protocols provide the foundation for training of Intramural Research Training Award (IRTA) fellows, undergraduate and graduate students, medical students, residents, and clinical fellows in the care and management of patients, and their families, who have genetic diseases.

Publications

  • Lee TL, Pang AL, Rennert OM, Chan WY. Genomic landscape of developing male germ cells. Birth Defects Res C Embryo Today 2009 87:43-63.
  • Lee TL, Cheung HH, Claus J, Sastry C, Singh S, Vu L, Rennert O, Chan WY. GermSAGE: a comprehensive SAGE database for transcript discovery on male germ cell development. Nucleic Acids Res 2009 37:D891-7.
  • Lee TL, Li Y, Alba D, Vong QP, Wu SM, Baxendale V, Rennert OM, Lau YF, Chan WY. Developmental staging of male murine embryonic gonad by SAGE analysis. J Genet Genomics 2009 36:215-27.
  • Su DM, Zhang Q, Wang X, He P, Zhu YJ, Zhao JX, Rennert OM, Su YA. Two types of human malignant melanoma cell lines revealed by expression patterns of mitochondrial and survival-apoptosis genes: implications for malignant melanoma therapy. Mol Cancer Ther 2009 8:1292-1304.
  • Pang ALY, Peacock S, Johnson W, Bear DH, Rennert OM, Chan WY. Cloning, characterization and expression analysis of a novel acetyltransferase retrogene Ard1b in the mouse. Biol Reprod 2009 81:302-309.

Collaborators

  • Joan Han, MD, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
  • Constantine Stratakis, MD, D(med)Sci, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
  • Yan A. Su, MD, PhD, George Washington University, Washington, DC

Contact

For more information, email rennerto@mail.nih.gov or visit www.nichd.nih.gov/about/org/dir/scientists-emeriti.

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