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Two regulatory elements with opposite effects modulate the activity of prrxl1 alternative promoters
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Poster Abstracts / Int. J. Devl Neuroscience 28 (2010) 655–719
(mean fold change: 2.7) in comparison to a group of control individuals; the expression of the CDKL5 gene was similar to that of controls (mean fold change: 0.95). We are currently investigating the molecular mechanisms that lead to this de-regulation of the expression of the MECP2 gene in the absence of exonic structural changes. We propose this de-regulation of expression may underlie disease in other RTT-like patients who test negative for MECP2 mutations.Keywords: Developmental delay; Epilepsy; Molecular genetics; Mental retardation doi:10.1016/j.ijdevneu.2010.07.125 [P1.86] Essential roles of a novel zinc finger protein in human cerebral cortical development Y. Yang 1,2,∗ , A.E. Baltus 1 , C. Marshall 1 , E. Wang 1 , D. Gleason 1 , C.A. Walsh 1 , et al 1 2
Harvard Medical School, USA MIT, USA
Though development of the cerebral cortex is of singular importance to human cognition, it remains very poorly understood. Microcephaly, or “small head,” has proved to be a useful disease model system for elucidating the steps essential for proper cortical development and cognitive function. While several genes causal for microcephaly have been identified, a consanguineous ArabIsraeli family was recently identified in which children with a novel homozygous mutation show a profoundly small (<4SD), disorganized cerebral cortex and cerebellum, along with abnormal neuronal migration, neuronal polarity, and evidence of cell death. Genetic studies using homozygosity mapping and microsatellite analyses have localized the mutation to a gene on chromosome 20. Unlike previously identified microcephaly genes that encode a structural protein, the newly identified mutation lies in a gene that encodes a zinc finger protein recently identified as a part of a chromatin remodeling complex. In vitro short interference knockdown and subsequent cell fate specific marker analysis performed on primary neuronal and cerebellar cultures show that knockdown of the zinc finger protein alters neuronal fate. Furthermore, in utero electroporation of short interference knockdown constructs in embryonic day 14.5 mice lead to neurons that harbored an abnormal dendritic branching morphology. A global knockdown model using genetrap mice containing a germline mutation of the zinc finger protein are shown to be embryonic lethal. Taken together, these data along with the human phentoype suggest that the novel zinc finger protein is essential for neurogenesis, neuronal polarity, and neuronal survival during cerebral cortical and cerebellar development. Such studies can contribute to a better understanding of the cellular and molecular mechanisms regulating normal cortical development and as well as cellular disease and death.Keywords: Microcephaly; Zinc finger; Neurogenesis doi:10.1016/j.ijdevneu.2010.07.126 [P1.87] Two regulatory elements with opposite effects modulate the activity of prrxl1 alternative promoters I. Regadas ∗ , F. Monteiro, S. Rebelo, D. Lima, C. Reguenga Universidade do Porto, Portugal The transcription factor Prrxl1/Drg11 has emerged as a crucial molecule in the establishment of the pain circuitry, displaying a preponderant role in the survival of the small-diameter periph-
eral afferent neurons located in the Dorsal Root Ganglia (DRG) that synapse extensively with the superficial laminae of the the dorsal spinal cord. Despite Prrxl1s importance in the differentiation of both DRG and spinal cord nociceptive neurons, the molecular mechanisms that regulate its expression remain largely unknown. To address this issue, the highly conserved 5 -flanking region of Prrxl1 translation start point was analyzed by luciferase reporter assays, using the DRG-derived neuronal cell line (ND7/23) that endogenously expressed Prrxl1. Three distinct regions displaying promoter activity (named P1, P2 and P3) were identified, which are suggestive of alternative promoter usage as a mechanism of control of Prrxl1 expression. Each promoter seems to control the expression of specific Prrxl1 5 UTR mRNA variants with transcriptional start points located in their vicinity. These 5 UTR transcripts do not modify the Prrxl1 primary structure and appear to be differentially expressed at early stages of the spinal cord neurogenesis as revealed by RT-PCR analysis. Regarding the distal promoter region (P3), the basal transcriptional motifs TATA box and CCAAT box were identified and their relevance was assessed by site-directed mutagenesis and DNA/protein interaction methods such as DNAaffinity pull down and electrophoretic mobility shift assays. Most importantly, two adjacent regulatory elements were detected, one presenting capability to strongly reduce the combined activity of the three promoters and another one with the potential to inhibit the repressive trait of the former motif. Altogether, the present results led to the identification and characterization of Prrxl1 alternative promoters and regulatory elements that are implicated in the modulation of Prrxl1 expression.Keywords: Prrxl1; Alternative promoters; Nociceptive circuitry; 5 -UTR mRNA variants doi:10.1016/j.ijdevneu.2010.07.127 [P1.88] Analysis of histone deacetylase-mediated transcriptional control of forebrain neurogenesis via gene-array profiling K. Weissmueller ∗ , C. Scholl, S. Wolfl, K.L. Tucker University of Heidelberg, Germany Histone deacetylases (HDACs) are a family of histone-modifying enzymes controlling chromatin compaction and gene transcription, and have recently been shown to play crucial roles in the development and function of the central nervous system. We have previously shown that they control neurogenesis in both cortex and striatum (Shaked et al., 2008). We found that BMP2 signaling is inhibited by HDAC activity, and that inhibition of HDACs resulted in a suppression of striatal neurogenesis. To elucidate the mechanism by which HDACs influence neurogenesis, we have employed RNA microarray analysis to identify genes that are regulated by application of trichostatin A (TSA), an HDAC inhibitor, or BMP2, both leading to the suppression of neurogenesis in embryonic striatum. Candidate genes were verified by quantitative real-time RT-PCR. Several groups of genes could be identified as responsive to TSA and/or BMP2 treatment. After 6 h of TSA treatment, genes involved in chromatin regulation and detoxification showed transcriptional changes, whereas after 24 h genes involved in differentiation, cell adhesion, as well as genes encoding for membrane proteins, showed differential expression. BMP2 treatment for 6 h mainly affected genes involved in differentiation processes, whereas 24 h of BMP2 treatment showed a strong overlap with the effects of 24 h TSA treatment. BMP2 expression did not appear to be regulated by BMP2 treatment, however the decrease in BMP4 expression seen after application of TSA was even more pronounced upon BMP2 application.
Poster Abstracts / Int. J. Devl Neuroscience 28 (2010) 655–719
(mean fold change: 2.7) in comparison to a group of control individuals; the expression of the CDKL5 gene was similar to that of controls (mean fold change: 0.95). We are currently investigating the molecular mechanisms that lead to this de-regulation of the expression of the MECP2 gene in the absence of exonic structural changes. We propose this de-regulation of expression may underlie disease in other RTT-like patients who test negative for MECP2 mutations.Keywords: Developmental delay; Epilepsy; Molecular genetics; Mental retardation doi:10.1016/j.ijdevneu.2010.07.125 [P1.86] Essential roles of a novel zinc finger protein in human cerebral cortical development Y. Yang 1,2,∗ , A.E. Baltus 1 , C. Marshall 1 , E. Wang 1 , D. Gleason 1 , C.A. Walsh 1 , et al 1 2
Harvard Medical School, USA MIT, USA
Though development of the cerebral cortex is of singular importance to human cognition, it remains very poorly understood. Microcephaly, or “small head,” has proved to be a useful disease model system for elucidating the steps essential for proper cortical development and cognitive function. While several genes causal for microcephaly have been identified, a consanguineous ArabIsraeli family was recently identified in which children with a novel homozygous mutation show a profoundly small (<4SD), disorganized cerebral cortex and cerebellum, along with abnormal neuronal migration, neuronal polarity, and evidence of cell death. Genetic studies using homozygosity mapping and microsatellite analyses have localized the mutation to a gene on chromosome 20. Unlike previously identified microcephaly genes that encode a structural protein, the newly identified mutation lies in a gene that encodes a zinc finger protein recently identified as a part of a chromatin remodeling complex. In vitro short interference knockdown and subsequent cell fate specific marker analysis performed on primary neuronal and cerebellar cultures show that knockdown of the zinc finger protein alters neuronal fate. Furthermore, in utero electroporation of short interference knockdown constructs in embryonic day 14.5 mice lead to neurons that harbored an abnormal dendritic branching morphology. A global knockdown model using genetrap mice containing a germline mutation of the zinc finger protein are shown to be embryonic lethal. Taken together, these data along with the human phentoype suggest that the novel zinc finger protein is essential for neurogenesis, neuronal polarity, and neuronal survival during cerebral cortical and cerebellar development. Such studies can contribute to a better understanding of the cellular and molecular mechanisms regulating normal cortical development and as well as cellular disease and death.Keywords: Microcephaly; Zinc finger; Neurogenesis doi:10.1016/j.ijdevneu.2010.07.126 [P1.87] Two regulatory elements with opposite effects modulate the activity of prrxl1 alternative promoters I. Regadas ∗ , F. Monteiro, S. Rebelo, D. Lima, C. Reguenga Universidade do Porto, Portugal The transcription factor Prrxl1/Drg11 has emerged as a crucial molecule in the establishment of the pain circuitry, displaying a preponderant role in the survival of the small-diameter periph-
eral afferent neurons located in the Dorsal Root Ganglia (DRG) that synapse extensively with the superficial laminae of the the dorsal spinal cord. Despite Prrxl1s importance in the differentiation of both DRG and spinal cord nociceptive neurons, the molecular mechanisms that regulate its expression remain largely unknown. To address this issue, the highly conserved 5 -flanking region of Prrxl1 translation start point was analyzed by luciferase reporter assays, using the DRG-derived neuronal cell line (ND7/23) that endogenously expressed Prrxl1. Three distinct regions displaying promoter activity (named P1, P2 and P3) were identified, which are suggestive of alternative promoter usage as a mechanism of control of Prrxl1 expression. Each promoter seems to control the expression of specific Prrxl1 5 UTR mRNA variants with transcriptional start points located in their vicinity. These 5 UTR transcripts do not modify the Prrxl1 primary structure and appear to be differentially expressed at early stages of the spinal cord neurogenesis as revealed by RT-PCR analysis. Regarding the distal promoter region (P3), the basal transcriptional motifs TATA box and CCAAT box were identified and their relevance was assessed by site-directed mutagenesis and DNA/protein interaction methods such as DNAaffinity pull down and electrophoretic mobility shift assays. Most importantly, two adjacent regulatory elements were detected, one presenting capability to strongly reduce the combined activity of the three promoters and another one with the potential to inhibit the repressive trait of the former motif. Altogether, the present results led to the identification and characterization of Prrxl1 alternative promoters and regulatory elements that are implicated in the modulation of Prrxl1 expression.Keywords: Prrxl1; Alternative promoters; Nociceptive circuitry; 5 -UTR mRNA variants doi:10.1016/j.ijdevneu.2010.07.127 [P1.88] Analysis of histone deacetylase-mediated transcriptional control of forebrain neurogenesis via gene-array profiling K. Weissmueller ∗ , C. Scholl, S. Wolfl, K.L. Tucker University of Heidelberg, Germany Histone deacetylases (HDACs) are a family of histone-modifying enzymes controlling chromatin compaction and gene transcription, and have recently been shown to play crucial roles in the development and function of the central nervous system. We have previously shown that they control neurogenesis in both cortex and striatum (Shaked et al., 2008). We found that BMP2 signaling is inhibited by HDAC activity, and that inhibition of HDACs resulted in a suppression of striatal neurogenesis. To elucidate the mechanism by which HDACs influence neurogenesis, we have employed RNA microarray analysis to identify genes that are regulated by application of trichostatin A (TSA), an HDAC inhibitor, or BMP2, both leading to the suppression of neurogenesis in embryonic striatum. Candidate genes were verified by quantitative real-time RT-PCR. Several groups of genes could be identified as responsive to TSA and/or BMP2 treatment. After 6 h of TSA treatment, genes involved in chromatin regulation and detoxification showed transcriptional changes, whereas after 24 h genes involved in differentiation, cell adhesion, as well as genes encoding for membrane proteins, showed differential expression. BMP2 treatment for 6 h mainly affected genes involved in differentiation processes, whereas 24 h of BMP2 treatment showed a strong overlap with the effects of 24 h TSA treatment. BMP2 expression did not appear to be regulated by BMP2 treatment, however the decrease in BMP4 expression seen after application of TSA was even more pronounced upon BMP2 application.