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ABSTRACT: Imitation Switch (ISWI) proteins are catalytic subunits of chromatin remodeling complexes that alter nucleosome positioning by hydrolyzing ATP to regulate access to DNA. In mice there are two paralogs, SNF2-homolog (SNF2H) and SNF2-like (SNF2L), which participate in different complexes and have contrasting patterns of expression. Here we investigate the role of SNF2L in ovaries by characterizing a mouse bearing an inactivating deletion of exon 6 which disrupts the ATPase domain. Snf2l mutant mice produce significantly fewer eggs than control mice when superovulated. Gonadotropin stimulation leads to a significant deficit in secondary follicles and an increase in abnormal antral follicles. Mutant females also fail to induce Fibrinogen-like 2 (Fgl2) in response to human chorionic gonadotropin (hCG), while overexpression of SNF2L is sufficient to drive its expression in granulosa cells. SNF2L is also shown to directly interact with the nuclear receptor co-activator Flightless I (FLI-I) as shown by immunoprecipitation. These results begin to establish a role for SNF2L in the precise coordination of gene expression in granulosa cells during folliculogenesis, and its broader implications in fertility.
Biology of Reproduction 04/2013; · 4.01 Impact Factor
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ABSTRACT: The heterogeneous nature of congenital hydrocephalus has hampered our understanding of the molecular basis of this common clinical problem. However, disease gene identification and characterization of multiple transgenic mouse models has highlighted the importance of the subcommissural organ (SCO) and the ventricular ependymal (vel) cells. Here, we review how altered development and function of the SCO and vel cells contributes to hydrocephalus.
Physiology (Bethesda, Md.) 05/2009; 24:117-26. · 7.95 Impact Factor
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ABSTRACT: The Börjeson-Forssman-Lehmann syndrome is an X-linked mental retardation disorder caused by mutations in the PHF6 gene. The PHF6 gene contains 2 plant homeodomain zinc fingers, suggesting a role for the protein in chromatin remodeling. In this study, the authors report on a Finnish family with a classical Börjeson-Forssman-Lehmann syndrome phenotype caused by a G to T nucleotide substitution at position 266 within exon 4 within the PHF6 gene (c.266G>T). The resulting glycine to valine (p.G89V) change corresponds to a highly conserved residue within the first plant homeodomain zinc finger domain. This is a novel change that adds to the number of plant homeodomain zinc finger mutations identified, such that 23% of all Börjeson-Forssman-Lehmann syndrome mutations lie within this motif. Moreover, it highlights the functional importance of plant homeodomain zinc finger motifs to human disease and more specifically to PHF6 function.
Journal of child neurology 03/2009; 24(5):610-4. · 1.59 Impact Factor
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ABSTRACT: ATRX is an SWI/SNF-like chromatin remodeling protein that is mutated in several X-linked mental retardation syndromes, including the ATR-X syndrome. In mice, Atrx expression is widespread and attempts to understand its function in brain development are hampered by the lethality associated with ubiquitous or forebrain-restricted ablation of this gene. One way to circumvent this problem is to study its function in a region of the brain that is dispensable for long-term survival of the organism. The retina is a well-characterized tractable model of CNS development and in our review of 202 ATR-X syndrome patients, we found ocular defects present in approximately 25% of the cases, suggesting that studying Atrx in this tissue will provide insight into function. We report that Atrx is expressed in the neuroprogenitor pool in embryonic retina and in all cell types of the mature retina with the exception of rod photoreceptors. Conditional inactivation of Atrx in the retina during embryogenesis ultimately results in a loss of only two types of neurons, amacrine and horizontal cells. We show that this defect does not arise from a failure to specify these cells but rather a defect in interneuron differentiation and survival post-natally. The timing of cell loss is concomitant with light-dependent changes in synaptic organization in the retina and with a change in Atrx subnuclear localization within these interneurons. Moreover, these interneuron defects are associated with functional deficits as demonstrated by reduced b-wave amplitudes upon electroretinogram analysis. These results implicate a role for Atrx in interneuron survival and differentiation.
Human Molecular Genetics 01/2009; 18(5):966-77. · 7.64 Impact Factor
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ABSTRACT: ATRX is a SWI/SNF-like chromatin remodeling protein mutated in several X-linked mental retardation syndromes. Gene inactivation studies in mice demonstrate that ATRX is an essential protein and suggest that patient mutations likely retain partial activity. ATRX associates with the nuclear matrix, pericentromeric heterochromatin, and promyelocytic leukemia nuclear bodies (PML-NBs) in a speckled nuclear staining pattern. Here, we used GFP-ATRX fusion proteins to identify the specific domains of ATRX necessary for subnuclear targeting and the effect of patient mutations on this localization. We identified two functional nuclear localization signals (NLSs) and two domains that target ATRX to nuclear speckles. One of the latter domains is responsible for targeting ATRX to PML-NBs. Surprisingly, this domain encompassed motifs IV-VI of the SNF2 domain suggesting that in addition to chromatin remodeling, it may also have a role in subnuclear targeting. More importantly, four different patient mutations within this domain resulted in an approximately 80% reduction in the number of transfected cells with ATRX nuclear speckles and PML colocalization. These results demonstrate that patient mutations have a dramatic effect on subnuclear targeting to PML-NBs. Moreover, these findings support the hypothesis that ATRX patient mutations represent functional hypomorphs and suggest that loss of proper targeting to PML-NBs is an important contributor to the pathogenesis of the ATR-X syndrome.
European Journal of HumanGenetics 03/2008; 16(2):192-201. · 4.40 Impact Factor
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ABSTRACT: Mutations in genes whose products modify chromatin structure have been recognized as a cause of X-linked mental retardation (XLMR). These genes encode proteins that regulate DNA methylation (MeCP2), modify histones (RSK2 and JARID1C), and remodel nucleosomes through ATP hydrolysis (ATRX). Thus, genes encoding other chromatin modifying proteins should also be considered as disease candidate genes. In this work, we have characterized the SNF2L gene, encoding an ATP-dependent chromatin remodeling protein of the ISWI family, and sequenced the gene in patients from 12 XLMR families linked to Xq25-26.
We used an in silico and RT-PCR approach to fully characterize specific SNF2L isoforms. Mutation screening was performed in 12 patients from individual families with syndromic or non-syndromic XLMR. We sequenced each of the 25 exons encompassing the entire coding region, complete 5' and 3' untranslated regions, and consensus splice-sites.
The SNF2L gene spans 77 kb and is encoded by 25 exons that undergo alternate splicing to generate several distinct transcripts. Specific isoforms are generated through the alternate use of exons 1 and 13, and by the use of alternate donor splice sites within exon 24. Alternate splicing within exon 24 removes a NLS sequence and alters the subcellular distribution of the SNF2L protein. We identified 3 single nucleotide polymorphisms but no mutations in our 12 patients.
Our results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function. SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist. Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.
BMC Medical Genetics 02/2008; 9:11. · 2.33 Impact Factor
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ABSTRACT: Chromatin has emerged as an important regulator of gene expression, interposed between cell signaling pathways and transcriptional machinery. It participates in transmitting extra- and intra-cellular signals that coordinate ovarian events: ovarian follicle development, the meiotic maturation of the oocyte that precedes ovulation, and the ovulatory process and consequent luteinization. Recent evidence from model organisms and mammals suggests that chromatin signaling is achieved, in part, by imitation switch (ISWI) ATP-dependent chromatin-remodeling complexes. This review highlights a role for complexes containing the ISWI ATPase sucrose nonfermenting-2h (Snf2h) in proliferation in somatic and germ cells and also in meiosis in germ cells. Moreover, complexes containing the Snf2l ATPase dictate the differentiation of somatic cells and act in the induction of the terminal phases of meiosis in the oocyte.
Trends in Endocrinology and Metabolism 08/2007; 18(5):215-24. · 8.11 Impact Factor
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ABSTRACT: Luteinization is a complex process, stimulated by gonadotropins, that promotes ovulation and development of the corpus luteum through terminal differentiation of granulosa cells. The pronounced expression of the mammalian imitation switch (ISWI) genes, SNF2H and SNF2L, in adult ovaries prompted us to investigate the role of these chromatin remodeling proteins during follicular development and luteinization. SNF2H expression is highest during growth of preovulatory follicles and becomes less prevalent during luteinization. In contrast, both SNF2L transcript and SNF2L protein levels are rapidly increased in granulosa cells of the mouse ovary 8 h after human chorionic gonadotropin treatment, and continue to be expressed 36 h later within the functional corpus luteum. We demonstrate a physical interaction between SNF2L and the progesterone receptor A isoform, which regulates progesterone receptor-responsive genes required for ovulation. Moreover, chromatin immunoprecipitation demonstrated that, after gonadotropin stimulation, SNF2L is associated with the proximal promoter of the steroidogenic acute regulatory protein (StAR) gene, a classic marker of luteinization in granulosa cells. Interaction of SNF2L with the StAR promoter is required for StAR expression, because small interfering RNA knockdown of SNF2L prevents the activation of the StAR gene. Our results provide the first indication that ISWI chromatin remodeling proteins are responsive to the LH surge and that this response is required for the activation of the StAR gene and the overall development of a functional luteal cell.
Molecular Endocrinology 11/2006; 20(10):2406-17. · 4.54 Impact Factor
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David J Picketts
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ABSTRACT: Congenital hydrocephalus affects 0.1-0.3% of live births, with a high mortality rate (approximately 50%) in the absence of surgical intervention. Although the insertion of shunts alleviates the symptoms of the majority of congenital cases, the molecular basis of hydrocephalus and the mechanisms of cerebrospinal fluid (CSF) circulation remain largely unknown. Two important players are the subcommissural organ/Reissner's fiber (SCO/RF) complex and the ventricular ependymal (vel) cells that together facilitate the flow of the CSF through the narrow canals of the ventricular system. In this issue of the JCI, Lang et al. demonstrate that overexpression of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC1) receptor gene results in abnormal development of the SCO and vel cells, leading to congenital hydrocephalus (see the related article beginning on page 1924). The ligand for the PAC1 receptor is the neuropeptide PACAP, which uncovers what the authors believe to be a novel role for this signaling cascade in the regulation of CSF circulation.
Journal of Clinical Investigation 08/2006; 116(7):1828-32. · 15.39 Impact Factor
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ABSTRACT: Mutations in genes encoding chromatin-remodeling proteins, such as the ATRX gene, underlie a number of genetic disorders including several X-linked mental retardation syndromes; however, the role of these proteins in normal CNS development is unknown. Here, we used a conditional gene-targeting approach to inactivate Atrx, specifically in the forebrain of mice. Loss of ATRX protein caused widespread hypocellularity in the neocortex and hippocampus and a pronounced reduction in forebrain size. Neuronal "birthdating" confirmed that fewer neurons reached the superficial cortical layers, despite normal progenitor cell proliferation. The loss of cortical mass resulted from a 12-fold increase in neuronal apoptosis during early stages of corticogenesis in the mutant animals. Moreover, cortical progenitors isolated from Atrx-null mice undergo enhanced apoptosis upon differentiation. Taken together, our results indicate that ATRX is a critical mediator of cell survival during early neuronal differentiation. Thus, increased neuronal loss may contribute to the severe mental retardation observed in human patients.
Journal of Clinical Investigation 03/2005; 115(2):258-67. · 15.39 Impact Factor
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ABSTRACT: Mammalian genomes encode two imitation switch family chromatin remodeling proteins, SNF2H and SNF2L. In the mouse, SNF2H is expressed ubiquitously, whereas SNF2L expression is limited to the brain and gonadal tissue. This pattern of SNF2L expression suggests a critical role for SNF2L in neuronal physiology. Indeed, SNF2L was shown to promote neurite outgrowth as well as regulate the human engrailed homeotic genes, important regulators of brain development. Here we identify a novel splice variant of human SNF2L we call SNF2L+13, which contains a nonconserved in-frame exon within the conserved catalytic core domain of SNF2L. SNF2L+13 retains the ability to incorporate into multiprotein complexes; however, it is devoid of enzymatic activity. Most interestingly, unlike mouse SNF2L, human SNF2L is expressed ubiquitously, and regulation is mediated by isoform variation. The human SNF2L+13 null variant is predominant in non-neuronal tissue, whereas the human wild type active SNF2L isoform is expressed in neurons. Thus, like the mouse, active human SNF2L is limited to neurons and a few other tissues.
Journal of Biological Chemistry 11/2004; 279(43):45130-8. · 4.77 Impact Factor
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ABSTRACT: Death domain-associated protein (Daxx) is a multi-functional protein that modulates both apoptosis and transcription. Within the nucleus, Daxx is a component of the promyelocytic leukemia protein (PML) nuclear bodies (NBs) and interacts with a number of transcription factors, yet its precise role in transcription remains elusive. To further define the function of Daxx, we have isolated its interacting proteins in the nucleus using epitope-tagged affinity purification and identified X-linked mental retardation and alpha-thalassaemia syndrome protein (ATRX), a putative member of the SNF2 family of ATP-dependent chromatin remodeling proteins that is mutated in several X-linked mental retardation disorders. We show that substantial amounts of endogenous Daxx and ATRX exist in a nuclear complex. Daxx binds to ATRX through its paired amphipathic alpha helices domains. ATRX has ATPase activity that is stimulated by mononucleosomes, and patient mutations in the ATPase domain attenuate this activity. ATRX strongly represses transcription when tethered to a promoter. Daxx does not affect the ATPase activity of ATRX, however, it alleviates its transcription repression activity. In addition, ATRX is found in the PML-NBs, and this localization is mediated by Daxx. These results show that the ATRX.Daxx complex is a novel ATP-dependent chromatin-remodeling complex, with ATRX being the core ATPase subunit and Daxx being the targeting subunit. Moreover, the localization of ATRX to the PML-NBs supports the notion that these structures may play an important role in transcription regulation.
Journal of Biological Chemistry 06/2004; 279(19):20369-77. · 4.77 Impact Factor
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ABSTRACT: The modification of chromatin structure is an important regulatory mechanism for developmental gene expression. Differential expression of the mammalian ISWI genes, SNF2H and SNF2L, has suggested that they possess distinct developmental roles. Here we describe the purification and characterization of the first human SNF2L-containing complex. The subunit composition suggests that it represents the human ortholog of the Drosophila nucleosome-remodeling factor (NURF) complex. Human NURF (hNURF) is enriched in brain, and we demonstrate that it regulates human Engrailed, a homeodomain protein that regulates neuronal development in the mid-hindbrain. Furthermore, we show that hNURF potentiates neurite outgrowth in cell culture. Taken together, our data suggess a role for an ISWI complex in neuronal growth.
The EMBO Journal 12/2003; 22(22):6089-100. · 9.20 Impact Factor
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ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy associated with deletions of a subtelomeric repeat (D4Z4). A reduction in D4Z4 copy number coincides with increased expression of neighboring 4q35 genes, implying a normal repressive role for the repeats. Here we examine the effect of increasing D4Z4 repeat number on reporter gene activity in C2C12 cells. Repeat size had only a minor cis-effect on reporter gene activity but greatly compromised myotube formation. This latter trans-effect did not result from expression of a gene within the repeat (DUX4) but likely results from squelching of the D4Z4 recognition complex.
FEBS Letters 03/2003; 537(1-3):133-8. · 3.54 Impact Factor
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ABSTRACT: Several X-linked mental retardation syndromes are caused by mutations in the ATRX gene. Common clinical features associated with ATRX mutations include severe mental retardation, characteristic facial anomalies and variable degrees of urogenital defects and alpha-thalassemia. Although the ATRX protein is a member of the SWI/SNF family of chromatin remodeling proteins, little is known about the biochemical activity of the ATRX protein or its in vivo function during development. Here we demonstrate that ATRX is part of a large multiprotein complex similar in size to the SWI/SNF complex. Furthermore, we have generated transgenic mice that overexpress ATRX as an initial model for studying the function of this protein during development. Misexpression of ATRX was associated with growth retardation, neural tube defects and a high incidence of embryonic death. Moreover, brains from E10.5 transgenic embryos displayed abnormal growth and organization of the ventricular zone that was highly convoluted in the most severely affected embryos. Transgenic mice that survived to birth exhibited a high incidence of perinatal death, as well as seizures, mild craniofacial anomalies and abnormal behavior. Our findings indicate that ATRX dosage is crucial for normal development and organization of the cortex, and emphasize the relevance of our model for the study of ATRX function and disease pathogenesis.
Human Molecular Genetics 03/2002; 11(3):253-61. · 7.64 Impact Factor
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Mammalian Genome 04/1998; 9(5):400-403. · 2.89 Impact Factor
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ABSTRACT: The ATR-X syndrome is an X-linked disorder comprising severe psychomotor retardation, characteristic facial features, genital abnormalities, and a-thalassemia. We have shown that ATR-X results from diverse mutations of XH2, a member of a subgroup of the helicase superfamily that includes proteins involved in a wide range of cellular functions, including DNA recombination and repair (RAD16, RAD54, and ERCC6) and regulation of transcription (SWI2/SNF2, MOTs, and brahma). The complex ATR-X phenotype suggests that XH2, when mutated, down-regulates expression of several genes, including the α-globin genes, indicating that it could be a global transcriptional regulator. In addition to its role in the ATR-X syndrome, XH2 may be a good candidate for other forms of X-linked mental retardation mapping to Xg13.
Cell.
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Departments of Cellular and Molecular Medicine.
