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Genetic and Genomic Studies in Normal Development and Diseases

Owen M. Rennert, MD, Head, Section on Clinical Genomics
Margarita Raygada, PhD, CGC, Staff Clinician (Genetic Counselor)
Alan Pang, PhD, Staff Biologist
Albert Cheung, PhD, Postdoctoral Fellow
Dalton Liu, BS, MS, NIH/HongKong PhD Student
Alexandra Title, BS, Postbaccalaureate Intramural Research Training Award Fellow
Mark Ziats, BS, NIH/Cambridge MD/PhD Program
Vanessa Baxendale, BS, Laboratory Manager
Lucile Thouennon, BS, Research Technician
Shaoming Wu, PhD, Volunteer

Autism spectrum disorder (ASD) normally exhibits the onset of symptoms before three years of age and is characterized by severe impairment in reciprocal socialization and communication skills and by repetitive or restrictive behaviors. It is a heterogeneous condition of multiple etiologies; no precise clinical assessment tools currently allow accurate definition of the many variants, nor are there biological markers to distinguish the variants. A rise in the number of children identified with ASDs, from five to 72 cases per 10,000 children in the USA and Europe, and the absence of definitive forms of therapy have given rise to mounting public concern. Our studies focus on the relationship between transcriptional networks formed during normal human brain development and genes that have been associated with ASDs. The work highlights the temporal and spatial (brain region) relationship of gene pathways to human brain development. Additional studies utilize neuronal cultures derived from induced pluripotent stem cells obtained from patients with ASD for studies of gene expression and neurophysiological properties.

The human diseases of premature aging in humans are characterized by early onset of aging phenotypes that are now known to be caused by mutations of various genes. Werner's syndrome (WS) is an adult progeroid syndrome caused by mutations in the Werner syndrome, RecQ helicase-like (WRN) gene. WRN has been implicated in a variety of biochemical processes, including DNA replication, repair, recombination, telomere maintenance, and transcription. Loss of the WRN protein results in genomic instability and dysfunctional telomeres. Skin fibroblasts from WS patients demonstrate reduced replication potential and accelerated senescence in culture, possibly a result of dysfunctional telomeres.

We also study the biological role of RNA–binding proteins in cell fate decision. The decision between self-renewal and differentiation determines the fate of pluripotent cells in the development of an organism. Our current knowledge of the cellular control and maintenance of pluripotency is derived mainly from studies on the action of specific transcription factors (for example, Oct4, Nanog, and Sox2) that control the expression of their downstream target genes. The availability, stability, and efficiency of translation of RNA transcripts of pluripotency-related genes represent another level of regulation in the context of cell-fate decision. We are interested in the role of RNA–binding proteins in the maintenance of pluripotency. We use embryonic stem cells and embryonal carcinoma cells as model systems in which to study the biological functions of RNA–binding proteins (Lin28a and Pumilio/Nanos families) that are preferentially expressed in pluripotent cells. We are also interested in the regulatory mechanism that controls the cell type–specific expression patterns of these gene products.

It is apparent that, in addition to protein-coding transcripts, the mammalian genome expresses a large amount of non-protein–coding RNA transcripts. Among them, several long non-protein–coding RNAs (lncRNAs) and many microRNAs (miRNAs) have been found to regulate cellular physiology by tuning the expression of other genes at transcriptional and translational levels, respectively. In mammalian cells, alterations in the expression level of individual protein-coding transcripts by epigenetic modifiers, which promote either histone acetylation or DNA demethylation, have been documented. However, the global change in the full transcriptome (comprising protein-coding RNA transcripts and their non-protein–coding counterparts) under the influence of such modifiers has never been analyzed systematically.

Autism research

Improved strategies for early identification of specific phenotypic characteristics and biological markers (e.g., electrophysiological changes) of ASD would improve the effectiveness of treatment. The invasiveness of collecting primary neuronal tissue from patients could be circumvented by using induced pluripotent stem cells (iPSCs) that subsequently become neuronally differentiated. The successful reprogramming of human fibroblasts into an ES cell-like (iPSC) state in the Yamanaka laboratory (Takahashi et al., Cell 2007;131:861) and iPSCs' subsequent derivation from patients with ALS, Parkinson disease, and other disorders into cultured neural cells has served as the proof of principle. The breakthroughs made it possible for us to generate a cell-culture model of ASD by applying the technology for subsequent neural differentiation. We thus established fibroblast cultures from patients (subjects with autism) and non-affected controls; the cells were reprogrammed into an ES cell–like state. We clone the reprogrammed cell colonies, propagate them, and induce them to differentiate in vitro into neuronal cultures. Based on our underlying assumption that synaptic transmission is aberrant in autism, the patient-specific neuronal cultures are used for neuronal network analysis using the photoconductive-stimulation system described by Gutiérrez et al. (Eur J Neurosci 2009;30:2042). Briefly, spontaneous or pulse-stimulated activity of networks is measured by fluorescent-optical techniques and the structural basis of the patterns analyzed by fractal dimension analysis. We have the capacity to characterize the arrangement and complexity of the networks' axonal architecture. We confirmed the methodology with hippocampal cultures of a rat model carrying the neurolignin mutation R471C-NL3 (identified in a subgroup of patients with ASD). We are attempting to evaluate membrane excitation and signal transduction in neural cells derived from patients with idiopathic autism.

We produced induced pluripotent cell lines (iPs) from four male children with idiopathic autism, four normal male siblings of these patients, four unaffected controls, and four patients with tuberous sclerosis; multiple clones of iP cultures from each group were established as well as neural progenitor cells and mature neural cultures from the cell lines. We developed a computer algorithm that quantitatively measures connectivity and synchronicity of activation. We are currently demonstrating proof of principle using neural cultures from hippocampal cultures derived from the mouse model of Rett syndrome. Studies are presently under way using normal human neuronal cultures.

ASDs have a significant hereditary component, but the identified genetic loci are heterogeneous and complex. Consequently, there is a gap in understanding how diverse genomic aberrations can all result in the clinical ASD phenotype. Gene expression studies from autism brain tissue demonstrated aberrantly expressed protein-coding genes that may converge into common molecular pathways, potentially reconciling the strong heritability and shared clinical phenotypes with the disorder’s genomic heterogeneity. Regulation of gene expression is extremely complex and governed by many mechanisms, including noncoding RNAs. However, no study of ASD brain tissue has assessed changes in regulatory lncRNAs, which represent a large proportion of the human transcriptome and actively modulate mRNA expression. To determine whether aberrant expression of lncRNAs plays a role in the molecular pathogenesis of ASD, we profiled, by microarray, over 33,000 annotated lncRNAs and 30,000 mRNA transcripts from postmortem brain tissue of autistic and control prefrontal cortex and cerebellum. We detected over 200 differentially expressed lncRNAs in ASD, which were enriched for genomic regions containing genes related to neurodevelopment and psychiatric disease. Additionally, comparison of differences in expression of mRNAs between prefrontal cortex and cerebellum within individual donors revealed that ASD brains had more transcriptional homogeneity. Moreover, this was also true of the lncRNA transcriptome (1, 2, 3, 4). Our results suggest that future investigation of lncRNA expression in autistic brain may further elucidate the molecular pathogenesis of this disorder.

Premature aging syndromes

Previous studies on the pathogenesis of WS were limited to skin fibroblasts or virus-transformed lymphocytes. Animal models of the mutant WRN helicase cannot accurately recapitulate the WS phenotype observed in humans. Reprogramming of WS cells to iPSCs may provide a cell model for the study of the pathogenesis, especially for the differentiation of WS embryonic and adult stem cells into specific, affected cell types such as skin, bone, and muscle. To establish an iPSC model of WS, we reprogrammed patient fibroblasts with the transcriptional factors OCT4, SOX2, KLF4, and c-MYC. The efficiency of reprogramming for WS was much lower than for normal fibroblasts. Given that WS fibroblasts quickly reach senescence (in less than 20 population doublings), many cells failed to form colonies. To increase the efficiency of reprogramming, we tested several inhibitors, including vitamin C, the histone deacetylase (HDAC) inhibitor valproic acid, the MAP kinase inhibitor SB203580, and a cocktail of HDAC and TGF-β RI kinase inhibitors as well as small molecules reported by Sheng Ding’s group at the Gladstone Institute of Cardiovascular Disease, University of California San Francisco. Our preliminary data indicate that optimal efficiency was achieved with the HDAC and TGF-β RI kinase inhibitors; using these inhibitors, we observed the appearance of some colonies after 30 days of induction. The colonies could be expanded and expressed markers of embryonic stem cells (NANOG, OCT4, SOX2, SSEA3, SSEA4, TRA1-60, TRA1-81). By forming embryoid bodies in serum-containing medium, WS iPSCs could spontaneously differentiate into cells expressing markers of endoderm (α-fetoprotein), mesoderm (smooth muscle actin), and ectoderm (nestin).

In addition to the use of inhibitors, we explored the possibility of genetic modification for derivation of WS iPSCs. As cells enter senescence, p53 is the central guardian protein that regulates the downstream target p21. Therefore, suppression of p53 should allow us to derive iPSC with a higher efficiency. We found that depletion of p53 by shRNA could augment the number of iPSC colonies, suggesting that the accelerated senescence in the parent WS cells is a barrier to reprogramming. Furthermore, expression of hTERT has been found to extend the telomere of WS fibroblasts and thus increase the capacity for proliferation. To determine whether hTERT can enhance reprogramming efficiency in WS, we transfected hTERT retrovirus to WS fibroblasts. As expected, WS cells with hTERT expression can be reprogrammed easily into iPSCs indistinguishable from normal iPSCs. All derived WS iPSCs with either p53 RNAi and/or hTERT expression showed expression of pluripotency markers, morphology, proliferation potential, and differentiation capacity similar to those of unmodified WS iPSCs and normal iPSCs. Interestingly, reprogrammed WS iPSCs did not show activity of the senescence-associated marker β-galactosidase, in contrast to their parent cells, suggesting that the reprogrammed iPSCs were “rejuvenated” (5).

Currently we are differentiating WS iPSCs into mesenchymal stem cells (MSCs). The MSC is an adult stem cell that can be differentiated to adipocytes, osteocytes, chondrocytes, myocytes, and fibroblasts. iPSC–derived MSCs showed similar morphology to bone marrow–derived MSCs, expressing the positive MSC markers CD105, CD73, CD29, CD44, and CD166 but not the hematopoietic markers CD34, CD45, or CD14. We are currently investigating the MSCs' capacity for trilineage differentiation to adipocytes, osteocytes, and chondrocytes.

Studies of transcription factors regulating cell renewal/proliferation

Transcriptional regulation of Lin28a expression

Lin28a encodes an RNA–binding protein and is an important regulator of cell proliferation. Lin28a is highly expressed in embryonic stem cells, embryonal carcinoma cells, spermatogonial stem cells, and a subset of cancer cells. With the exception of cardiac and skeletal muscle, no Lin28a expression is present in adult tissues or various types of somatic cells. We found that the cell type–specific expression of Lin28a is regulated epigenetically. Specifically, non-Lin28a–expressing cells were treated separately with HDAC inhibitor and DNA methyltransferase inhibitor and then harvested for gene expression analysis. We found that Lin28a expression was reactivated only in cells receiving HDAC inhibitor treatment. We further demonstrated that the expression of Lin28a is accompanied by enrichment of transcription-activating histone modification in its promoter region, which leads to a relaxation of the chromatin conformation and increased subsequent accessibility of transcription factors to the Lin28a promoter.

Functional studies of the Lin28a protein

Using embryonal carcinoma cells as the model, we are testing whether the Lin28a protein enhances the stability of RNA transcripts whose translated products display a promoting effect on cell proliferation. We successfully silenced the expression of the Lin28a protein by RNA interference. Consistent with previous findings in embryonic stem cells, we also observed a reduction in the cell-proliferative rate in embryonal carcinoma cells when Lin28a expression was attenuated. The stability of the selected RNA transcripts will be examined in the presence of inhibitor of RNA biosynthesis.

Role of Pumilio and Nanos proteins in mouse embryonic stem cell proliferation

Pumilio and Nanos are evolutionarily conserved RNA–binding proteins that play an important role in embryogenesis and germline development in Drosophila. Specifically, the two proteins interact to repress the translation of target RNA transcripts. In mammals, two isoforms of the Pumilio protein (Pumilio-1 and Pumilio-2) and three isoforms of the Nanos protein (Nanos-1, Nanos-2, and Nanos-3) have been identified. We observed a preferential expression pattern of Pumilio and specific Nanos proteins in mouse embryonic stem cells and embryonal carcinoma cells compared with other cell types, suggesting a functional role of the proteins in pluripotency. To test the role of Pumilio and Nanos proteins in the maintenance of pluripotency, we plan to silence their expression in mouse embryonic stem cells and examine whether the proliferation of the cells is affected. We will also measure the expression level of pluripotency-related genes in Pumilio- or Nanos-silenced cells to evaluate the proteins' involvement in the maintenance of pluripotency.

Epigenetic regulation of global transcriptome output

We have begun to examine the effect of epigenetic modifiers on transcriptome output in mammalian cells. Our preliminary analysis in several mouse cell lines indicated that the expression of specific lncRNAs is affected by the histone acetylation level. We are expanding the analysis to examine the effect of various epigenetic modifiers on full transcriptome output. From this study, we expect to identify subsets of non–protein-coding RNA transcripts that are commonly or distinctively regulated by different epigenetic modifiers, and we plan to investigate their roles in the homeostasis of the cells.

Publications

Ziats M, Rennert OM. Identification of differentially expressed microRNAs across the developing human brain. Mol Psychiatry 2014;19:848-852.
Edmundson C, Ziats M, Rennert OM. Altered glial marker expression in autistic post-mortem prefrontal cortex and cerebellum. Mol Autism 2014;5:3-6.
Cheung HH, Liu X, Canterel-Thouennon L, Li L, Edmonson C, Rennert OM. Telomerase protects Werner syndrome lineage-specific stem cells from premature aging. Stem Cell Reports 2014;2:532-546.
Luk AC, Chan WY, Rennert OM, Lee TL. Long noncoding RNAs in spermatogenesis: insights from recent high-throughput transcriptome studies. Reproduction 2014;147:131-141.
Pang AL, Title AC, Rennert OM. Modulation of microRNA expression in human lung cancer cells by the G9a histone methyltransferase inhibitor BIX01294. Oncol Lett 2014;7:1819-1825.

Collaborators

Joan Han, MD, PhD, Program on Developmental Endocrinology and Genetics, NICHD, Bethesda, MD
Dax Hoffman, PhD, Program in Developmental Neuroscience, NICHD, Bethesda, MD
James Russell, DVM, PhD, Microscopy and Imaging Core Facility, NICHD, Bethesda, MD
Susan Swedo, MD, PhD, Pediatrics and Developmental Neuroscience Branch, NIMH, Bethesda, MD
Audrey Thurm, PhD, Pediatrics and Developmental Neuroscience Branch, NIMH, Bethesda, MD

Contact

For more information, email rennerto@mail.nih.gov.

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