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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 3 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 precise definition of the multiple variants, nor are there biological markers to distinguish these 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 increasing public concern.
The human diseases of premature aging in human are characterized by the early onset of aging phenotypes that are now known to be caused by mutations of various genes. Werner syndrome (WS) is an adult progeroid syndrome caused by mutations of the RecQ helicase WRN. 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 are also studying 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 the studies of the action of specific transcription factors (for example, Oct4, Nanog, and Sox2) that control the expression of their downstream target genes at transcriptional level. 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 studying the role of RNA–binding proteins in the maintenance of pluripotency. We use embryonic stem cells and embryonal carcinoma cells as the model systems to study the biological functions of RNA–binding proteins (Lin28a and Pumilio/Nanos families) that are preferentially expressed in pluripotent cells. Also, we are interested in elucidating 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, a 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, alteration of the expression level of individual protein-coding transcripts by epigenetic modifiers, which promote either histone acetylation or DNA demethylation, has been documented. However, the global change in full transcriptome (comprised of protein-coding RNA transcripts and their non-protein–coding counterparts) under the influence of these modifiers has never been analyzed systematically.
Improved strategies for early identification of specific phenotypic characteristics and biological markers (e.g., electrophysiological changes) of autism spectrum disorder (ASD) would improve the effectiveness of treatment. The invasive nature 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 by Yamanaka et al. in 2007 of human fibroblasts from patients with ALS, Parkinson disease, and other disorders into ES cell‐like (iPSC) state and their subsequent derivation into cultured neural cells has served as the proof of principle. These breakthroughs make it possible for us to generate a cell-culture model of ASD by applying the technology for subsequent neural differentiation. In this study, fibroblast cultures from patients (subjects with autism) and non-affected controls were established; 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, these patient-specific neuronal cultures are used for neuronal network analysis using the photoconductive-stimulation system described in Gutierrez et al. Briefly, spontaneous or pulse-stimulated activity of networks is measured by fluorescent-optical techniques, and the structural basis of these patterns analyzed by fractal dimension analysis. We have the capacity to characterize the arrangement and complexity of their axonal architecture. We confirmed this 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 have iPs cell lines from children with idiopathic autism (male patients N=4), normal male siblings of these patients (N=4), unaffected controls (N=4), patients with tuberous sclerosis (N=4); multiple clones of iPs cultures from each group are established. Neural progenitor cells and mature neural cultures were established from these cell lines. We developed a computer algorithm that quantitatively measures connectivity and synchronicity of activation. We are currently demonstrating roof 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 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 its genomic heterogeneity. However, the regulation of gene expression is extremely complex and governed by many mechanisms, including noncoding RNAs. Yet 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 play a role in the molecular pathogenesis of ASD, we profiled over 33,000 annotated lncRNAs and 30,000 mRNA transcripts from postmortem brain tissue of autistic and control prefrontal cortex and cerebellum by microarray. 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 mutant WRN 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. Because 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, valproic acid, SB203580, and a cocktail of HDAC and TGF-β RI kinase inhibitors as well as small molecules reported by Seng Ding's group. Our preliminary data indicate that optimal efficiency was achieved by using 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 also explored the possibility of genetic modification for derivation of WS iPSCs. p53 is the central guardian protein that regulates the downstream target p21 as cells enter senescence. Therefore, suppression of p53 would allow us to derive iPSC with a higher efficiency. We found that depletion of p53 by shRNA could increase the number of iPSC colonies, suggesting that the accelerated senescence in the parent WS cells is a barrier for reprogramming. Secondly, 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 increase reprogramming efficiency in WS, we transfected hTERT retrovirus to WS fibroblasts. As expected, WS cells with hTERT expression can be easily reprogrammed to iPSCs indistinguishable from normal iPSC. All derived WS iPSCs with either p53 RNAi and/or hTERT expression showed similar expression of pluripotency markers, morphology, proliferation potential, and differentiation capacity to 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 to 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, and are negative for the hematopoietic markers CD34, CD45, and CD14. We are currently investigating the MSC's 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 found 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 histone deacetylase inhibitor and DNA methyltransferase inhibitor and then harvested for gene expression analysis. We found that Lin28a expression was reactivated only in cells receiving histone deacetylase inhibitor treatment. We further demonstrated that the expression of Lin28a is accompanied by an enrichment of transcription-activating histone modification in its promoter region, which leads to a relaxation of the chromatin conformation and subsequently an increased accessibility of transcription factors to the Lin28a promoter.
Functional studies of Lin28a protein: Using embryonal carcinoma cells as the model, we are testing whether Lin28a protein acts to enhance the stability of RNA transcripts whose translated products display a promoting effect on cell proliferation. We successfully silenced the expression of Lin28a protein by RNA interference technique. Consistent with the previous findings in embryonic stem cells, we also observed a reduction of cell-proliferative rate in embryonal carcinoma cells when Lin28a expression is 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 with each other to repress the translation of target RNA transcripts. In mammals, two isoforms of Pumilio protein (Pumilio-1 and -2) and three isoforms of Nanos protein (Nanos-1, -2, and -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 when comparing with other cell types, suggesting a functional role of these 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 began 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 the different epigenetic modifiers, and we plan to investigate their roles in the homeostasis of the cells.
- Ziats M, Rennert OM. Expression profiling of autism candidate genes during human brain development implicates central immune signaling pathways. PloSOne 2011;6(9):e24691.
- Lee TL, Raygada M, Rennert OM. Integrative gene network analysis provides insights on novel regulatory relationships, genetic contributions and susceptible targets in autism spectrum disorders. Gene 2012;496(2):88-96.
- Lee TL, Xiao A, Rennert OM. Identification of novel long noncoding RNA transcripts in male germ cells. Methods Mol Biol 2012;825:104-114.
- Ziats M, Rennert OM. Aberrant expression of long noncoding RNAs in autistic brain. J Mol Neurosci 2012;E-pub ahead of print.
- Pang ALY, ClarkJ, Chan W-Y, Rennert OM. Expression of human NAA11 (ARD1B) gene is tissue-specific and is regulated by DNA methylation. Epigenetics 2011;6(11):1391-1399.